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Sample records for maldi tof ms

  1. MALDI-TOF MS/MS measurements of PMMA

    NARCIS (Netherlands)

    Becer, C.R.; Baumgaertel, A.; Gottschaldt, M.; Schubert, U.S.

    2008-01-01

    The polymer poly(Me methacrylate) (PMMA) was analyzed using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique. The MALDI-TOF MS app. was coupled with a collision-induced dissocn. (CID) unit. The performance of the MALDI-TOF/TOF MS method in

  2. The identification of anaerobic bacteria using MALDI-TOF MS

    NARCIS (Netherlands)

    Veloo, A. C. M.; Welling, G. W.; Degener, J. E.

    Matrix Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has gained more and more popularity for the identification of bacteria. Several studies show that bacterial diagnosticis is being revolutionized by the application of MALDI-TOF MS. For anaerobic bacteria,

  3. MALDI-TOF MS in the Microbiology Laboratory: Current Trends.

    Science.gov (United States)

    Schubert, Sören; Kostrzewa, Markus

    2017-01-01

    Within less than a decade matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a gold standard for microbial identification in clinical microbiology laboratories. Besides identification of microorganisms the typing of single strains as well as the antibiotic and antimycotic resistance testing has come into focus in order to speed up the microbiological diagnostic. However, the full potential of MALDI-TOF MS has not been tapped yet and future technological advancements will certainly expedite this method towards novel applications and enhancement of current practice. So, the following chapter shall be rather a brainstorming and forecast of how MALDI-TOF MS will develop to influence clinical diagnostics and microbial research in the future. It shall open up the stage for further discussions and does not claim for overall validity.

  4. [Utility of MALDI-TOF MS for the identification of anaerobic bacteria].

    Science.gov (United States)

    Zárate, Mariela S; Romano, Vanesa; Nievas, Jimena; Smayevsky, Jorgelina

    2014-01-01

    The analysis by MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry) has become a reference method for the identification of microorganisms in Clinical Microbiology. However, data on some groups of microorganisms are still controversial. The aim of this study is to determine the utility of MALDI-TOF MS for the identification of clinical isolates of anaerobic bacteria. One-hundred and six anaerobic bacteria isolates were analyzed by MALDI-TOF MS and by conventional biochemical tests. In those cases where identification by conventional methodology was not applicable or in the face of discordance between sequencing methodologies, 16 S rRNA gene sequence analysis was performed. The conventional method and MALDI-TOF MS agreed at genus and species level by 95.3 %. Concordance in gram-negative bacilli was 91.4% and 100% among gram-positive bacilli; there was also concordance both in the 8 isolates studied in gram-positive cocci and in the single gram-negative cocci included. The data obtained in this study demonstrate that MALDI-TOF MS offers the possibility of adequate identification of anaerobic bacteria. Copyright © 2014 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

  5. Detection of Rickettsia spp in Ticks by MALDI-TOF MS

    Science.gov (United States)

    Yssouf, Amina; Almeras, Lionel; Terras, Jérôme; Socolovschi, Cristina; Raoult, Didier; Parola, Philippe

    2015-01-01

    Background Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to be an effective tool for the rapid identification of arthropods, including tick vectors of human diseases. Methodology/Principal Findings The objective of the present study was to evaluate the use of MALDI-TOF MS to identify tick species, and to determine the presence of rickettsia pathogens in the infected Ticks. Rhipicephalus sanguineus and Dermacentor marginatus Ticks infected or not by R. conorii conorii or R. slovaca, respectively, were used as experimental models. The MS profiles generated from protein extracts prepared from tick legs exhibited mass peaks that distinguished the infected and uninfected Ticks, and successfully discriminated the Rickettsia spp. A blind test was performed using Ticks that were laboratory-reared, collected in the field or removed from patients and infected or not by Rickettsia spp. A query against our in-lab arthropod MS reference database revealed that the species and infection status of all Ticks were correctly identified at the species and infection status levels. Conclusions/Significance Taken together, the present work demonstrates the utility of MALDI-TOF MS for a dual identification of tick species and intracellular bacteria. Therefore, MALDI-TOF MS is a relevant tool for the accurate detection of Rickettsia spp in Ticks for both field monitoring and entomological diagnosis. The present work offers new perspectives for the monitoring of other vector borne diseases that present public health concerns. PMID:25659152

  6. Towards Spectral Library-free MALDI-TOF MS Bacterial Identification.

    Science.gov (United States)

    Cheng, Ding; Qiao, Liang; Horvatovich, Péter

    2018-05-11

    Bacterial identification is of great importance in clinical diagnosis, environmental monitoring and food safety control. Among various strategies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has drawn significant interests, and has been clinically used. Nevertheless, current bioinformatics solutions use spectral libraries for the identification of bacterial strains. Spectral library generation requires acquisition of MALDI-TOF spectra from monoculture bacterial colonies, which is time-consuming and not possible for many species and strains. We propose a strategy for bacterial typing by MALDI-TOF using protein sequences from public database, i.e. UniProt. Ten genes were identified to encode proteins most often observed by MALD-TOF from bacteria through 500 times repeated a 10-fold double cross-validation procedure, using 403 MALDI-TOF spectra corresponding to 14 genera, 81 species and 403 strains, and the protein sequences of 1276 species in UniProt. The 10 genes were then used to annotate peaks on MALDI-TOF spectra of bacteria for bacterial identification. With the approach, bacteria can be identified at the genus level by searching against a database containing the protein sequences of 42 genera of bacteria from UniProt. Our approach identified 84.1% of the 403 spectra correctly at the genus level. Source code of the algorithm is available at https://github.com/dipcarbon/BacteriaMSLF.

  7. Identification of Algerian Field-Caught Phlebotomine Sand Fly Vectors by MALDI-TOF MS.

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    Ismail Lafri

    2016-01-01

    Full Text Available Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine.Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M'sila where P. (Phlebotomus papatasi was the only sand fly species detected.The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and

  8. Identification of Algerian Field-Caught Phlebotomine Sand Fly Vectors by MALDI-TOF MS.

    Science.gov (United States)

    Lafri, Ismail; Almeras, Lionel; Bitam, Idir; Caputo, Aurelia; Yssouf, Amina; Forestier, Claire-Lise; Izri, Arezki; Raoult, Didier; Parola, Philippe

    2016-01-01

    Phlebotomine sand flies are known to transmit Leishmania parasites, bacteria and viruses that affect humans and animals in many countries worldwide. Precise sand fly identification is essential to prevent phlebotomine-borne diseases. Over the past two decades, progress in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an accurate tool for arthropod identification. The objective of the present study was to investigate the usefulness of MALDI-TOF MS as a tool for identifying field-caught phlebotomine. Sand flies were captured in four sites in north Algeria. A subset was morphologically and genetically identified. Six species were found in these areas and a total of 28 stored frozen specimens were used for the creation of the reference spectrum database. The relevance of this original method for sand fly identification was validated by two successive blind tests including the morphological identification of 80 new specimens which were stored at -80°C, and 292 unknown specimens, including engorged specimens, which were preserved under different conditions. Intra-species reproducibility and inter-species specificity of the protein profiles were obtained, allowing us to distinguish specimens at the gender level. Querying of the sand fly database using the MS spectra from the blind test groups revealed concordant results between morphological and MALDI-TOF MS identification. However, MS identification results were less efficient for specimens which were engorged or stored in alcohol. Identification of 362 phlebotomine sand flies, captured at four Algerian sites, by MALDI-TOF MS, revealed that the subgenus Larroussius was predominant at all the study sites, except for in M'sila where P. (Phlebotomus) papatasi was the only sand fly species detected. The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF

  9. Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS

    DEFF Research Database (Denmark)

    Kudirkiene, Egle; Welker, Martin; Knudsen, Nanna Reumert

    2015-01-01

    phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspecies.......3±7.5%). This result may be attributed to the highly clonal population structure of SEE, as opposed to the diversity of SEZ seen in horses. Importantly strains with atypical colony appearance both within SEE and SEZ did not affect correct identification of the strains by MALDI-TOF MS. Atypical colony variants...... are often associated with a higher persistence or virulence of S. equi, thus their correct identification using the current method strengthens its potential use in routine clinical diagnostics. In conclusion, reliable identification of S. equi subspecies was achieved by combining a MALDI-TOF MS method...

  10. Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS

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    Lista Florigio

    2011-12-01

    Full Text Available Abstract Background The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. Results In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. Conclusions MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

  11. Application of MALDI-TOF MS for requalification of a Candida clinical isolates culture collection

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    Reginaldo Lima-Neto

    2014-06-01

    Full Text Available Microbial culture collections underpin biotechnology applications and are important resources for clinical microbiology by supplying reference strains and/or performing microbial identifications as a service. Proteomic profiles by MALDI-TOF MS have been used for Candida spp. identification in clinical laboratories and demonstrated to be a fast and reliable technique for the routine identification of pathogenic yeasts. The main aim of this study was to apply MALDI-TOF MS combined with classical phenotypic and molecular approaches to identify Candida clinical isolates preserved from 1 up to 52 years in a Brazilian culture collection and assess its value for the identification of yeasts preserved in this type of collections. Forty Candida spp. clinical isolates were identified by morphological and biochemical analyses. Identifications were also performed by the new proteomic approach based on MALDI-TOF MS. Results demonstrated 15% discordance when compared with morphological and biochemical analyses. Discordant isolates were analysed by ITS sequencing, which confirmed the MALDI-TOF MS identifications and these strains were renamed in the culture collection catalogue. In conclusion, proteomic profiles by MALDI-TOF MS represents a rapid and reliable method for identifying clinical Candida species preserved in culture collections and may present clear benefits when compared with the performance of existing daily routine methods applied at health centres and hospitals.

  12. [Application of MALDI-TOF-MS in gene testing for non-syndromic hearing loss].

    Science.gov (United States)

    Zeng, Yun; Jiang, Dan; Feng, Da-fei; Jin, Dong-dong; Wu, Xiao-hui; Ding, Yan-li; Zou, Jing

    2013-12-01

    To investigate the feasibility of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) , according to the genetic test of non-syndromic hearing loss (NSHL), and check using the direct sequencing. Peripheral blood was collected from 454 NSHL patients. DNA samples were extracted and 20 loci of the four common disease-causing genes were analysed by MALDI-TOF-MS, including GJB2 (35delG, 167delT, 176_191del16, 235delC, 299_300delAT ), GJB3 (538C→T, 547G→A), SLC26A4 (281C→T, 589G→A, IVS7-2A→G, 1174A→T, 1226G→A, 1229C→T, IVS15+5G→A, 1975G→C, 2027T→A, 2162C→T, 2168A→G), and mitochondrial 12S rRNA (1494C→T, 1555A→G). Direct sequencing was also used to analyse the aforementioned 20 loci in order to validate the accuracy of MALDI-TOF-MS. Among the 454 patients, 166 cases (36.56%) of disease-causing mutations were detected, which included 69 cases (21.15%) of GJB2 gene mutation, four cases (0.88%) of GJB3 gene mutation, 64 cases (14.10%) of SLC26A4 gene mutation, and three cases (0.66%) of mitochondrial 12S rRNA gene mutation. Moreover, the results obtained from direct sequencing and MALDI-TOF-MS were consistent, and the results showed that the two methods were consistent. The MALDI-TOF-MS detection method was designed based on the hearing loss-related mutation hotspots seen in the Chinese population, and it has a high detection rate for NSHL related mutations. In comparison to the conventional detection methods, MALDI-TOF-MS has the following advantages: more detection sites, greater coverage, accurate, high throughput and low cost. Therefore, this method is capable of satisfying the needs of clinical detection for hearing impairment and it is suitable for large-scale implementation.

  13. A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS.

    Science.gov (United States)

    Veloo, A C M; Jean-Pierre, H; Justesen, U S; Morris, T; Urban, E; Wybo, I; Shah, H N; Friedrich, A W; Morris, T; Shah, H N; Jean-Pierre, H; Justesen, U S; Nagy, E; Urban, E; Kostrzewa, M; Veloo, A; Friedrich, A W

    2017-12-01

    Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network for the Rapid Identification of Anaerobes (ENRIA). On arrival the strains were cultured and identified using MALDI-TOF MS. The spectra derived were compared with two different Biotyper MALDI-TOF MS databases, the db5627 and the db6903. The results obtained using the db5627 shows a reasonable variation between the different laboratories. However, when a more optimized database is used, the variation is less pronounced. In this study we show that an optimized database not only results in a higher number of strains which can be identified using MALDI-TOF MS, but also corrects for differences in performance between laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Assessment of heat treatment of dairy products by MALDI-TOF-MS.

    Science.gov (United States)

    Meltretter, Jasmin; Birlouez-Aragon, Inès; Becker, Cord-Michael; Pischetsrieder, Monika

    2009-12-01

    The formation of the Amadori product from lactose (protein lactosylation) is a major parameter to evaluate the quality of processed milk. Here, MALDI-TOF-MS was used for the relative quantification of lactose-adducts in heated milk. Milk was heated at a temperature of 70, 80, and 100 degrees C between 0 and 300 min, diluted, and subjected directly to MALDI-TOF-MS. The lactosylation rate of alpha-lactalbumin increased with increasing reaction temperature and time. The results correlated well with established markers for heat treatment of milk (concentration of total soluble protein, soluble alpha-lactalbumin and beta-lactoglobulin at pH 4.6, and fluorescence of advanced Maillard products and soluble tryptophan index; r=0.969-0.997). The method was also applied to examine commercially available dairy products. In severely heated products, protein pre-purification by immobilized metal affinity chromatography improved spectra quality. Relative quantification of protein lactosylation by MALDI-TOF-MS proved to be a very fast and reliable method to monitor early Maillard reaction during milk processing.

  15. Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

    Science.gov (United States)

    Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

    2012-01-01

    We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate–protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate–spacer–carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate. PMID:22012665

  16. MALDI-TOF MS as a tool to identify foodborne yeasts and yeast-like fungi.

    Science.gov (United States)

    Quintilla, Raquel; Kolecka, Anna; Casaregola, Serge; Daniel, Heide M; Houbraken, Jos; Kostrzewa, Markus; Boekhout, Teun; Groenewald, Marizeth

    2018-02-02

    Since food spoilage by yeasts causes high economic losses, fast and accurate identifications of yeasts associated with food and food-related products are important for the food industry. In this study the efficiency of the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify food related yeasts was evaluated. A CBS in-house MALDI-TOF MS database was created and later challenged with a blinded test set of 146 yeast strains obtained from food and food related products. Ninety eight percent of the strains were correctly identified with log score values>1.7. One strain, Mrakia frigida, gained a correct identification with a score value1.7. Ambiguous identifications were observed due to two incorrect reference mass spectra's found in the commercial database BDAL v.4.0, namely Candida sake DSM 70763 which was re-identified as Candida oleophila, and Candida inconspicua DSM 70631 which was re-identified as Pichia membranifaciens. MALDI-TOF MS can distinguish between most of the species, but for some species complexes, such as the Kazachstania telluris and Mrakia frigida complexes, MALDI-TOF MS showed limited resolution and identification of sibling species was sometimes problematic. Despite this, we showed that the MALDI-TOF MS is applicable for routine identification and validation of foodborne yeasts, but a further update of the commercial reference databases is needed. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. [Applications of MALDI-TOF-MS in clinical microbiology laboratory].

    Science.gov (United States)

    Carbonnelle, Etienne; Nassif, Xavier

    2011-10-01

    For twenty years, mass spectrometry (MS) has emerged as a particularly powerful tool for analysis and characterization of proteins in research. It is only recently that this technology, especially MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization Time-Of-Flight) has entered the field of routine microbiology. This method has proven to be reliable and safe for the identification of bacteria, yeasts, filamentous fungi and dermatophytes. MALDI-TOF-MS is a rapid, precise and cost-effective method for identification, compared to conventional phenotypic techniques or molecular biology. Its ability to analyse whole microorganisms with few sample preparation has greatly reduced the time to identification (1-2 min). Furthermore, this technology can be used to identify bacteria directly from clinical samples as blood culture bottles or urines. Future applications will be developed in order to provide direct information concerning virulence or resistance protein markers. © 2011 médecine/sciences – Inserm / SRMS.

  18. A simple and effective method for detecting precipitated proteins in MALDI-TOF MS.

    Science.gov (United States)

    Oshikane, Hiroyuki; Watabe, Masahiko; Nakaki, Toshio

    2018-04-01

    MALDI-TOF MS has developed rapidly into an essential analytical tool for the life sciences. Cinnamic acid derivatives are generally employed in routine molecular weight determinations of intact proteins using MALDI-TOF MS. However, a protein of interest may precipitate when mixed with matrix solution, perhaps preventing MS detection. We herein provide a simple approach to enable the MS detection of such precipitated protein species by means of a "direct deposition method" -- loading the precipitant directly onto the sample plate. It is thus expected to improve routine MS analysis of intact proteins. Copyright © 2018. Published by Elsevier Inc.

  19. Enhanced MALDI-TOF MS Analysis of Phosphopeptides Using an Optimized DHAP/DAHC Matrix

    Science.gov (United States)

    Hou, Junjie; Xie, Zhensheng; Xue, Peng; Cui, Ziyou; Chen, Xiulan; Li, Jing; Cai, Tanxi; Wu, Peng; Yang, Fuquan

    2010-01-01

    Selecting an appropriate matrix solution is one of the most effective means of increasing the ionization efficiency of phosphopeptides in matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In this study, we systematically assessed matrix combinations of 2, 6-dihydroxyacetophenone (DHAP) and diammonium hydrogen citrate (DAHC), and demonstrated that the low ratio DHAP/DAHC matrix was more effective in enhancing the ionization of phosphopeptides. Low femtomole level of phosphopeptides from the tryptic digests of α-casein and β-casein was readily detected by MALDI-TOF-MS in both positive and negative ion mode without desalination or phosphopeptide enrichment. Compared with the DHB/PA matrix, the optimized DHAP/DAHC matrix yielded superior sample homogeneity and higher phosphopeptide measurement sensitivity, particularly when multiple phosphorylated peptides were assessed. Finally, the DHAP/DAHC matrix was applied to identify phosphorylation sites from α-casein and β-casein and to characterize two phosphorylation sites from the human histone H1 treated with Cyclin-Dependent Kinase-1 (CDK1) by MALDI-TOF/TOF MS. PMID:20339515

  20. Enhanced MALDI-TOF MS Analysis of Phosphopeptides Using an Optimized DHAP/DAHC Matrix

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    Junjie Hou

    2010-01-01

    Full Text Available Selecting an appropriate matrix solution is one of the most effective means of increasing the ionization efficiency of phosphopeptides in matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS. In this study, we systematically assessed matrix combinations of 2, 6-dihydroxyacetophenone (DHAP and diammonium hydrogen citrate (DAHC, and demonstrated that the low ratio DHAP/DAHC matrix was more effective in enhancing the ionization of phosphopeptides. Low femtomole level of phosphopeptides from the tryptic digests of α-casein and β-casein was readily detected by MALDI-TOF-MS in both positive and negative ion mode without desalination or phosphopeptide enrichment. Compared with the DHB/PA matrix, the optimized DHAP/DAHC matrix yielded superior sample homogeneity and higher phosphopeptide measurement sensitivity, particularly when multiple phosphorylated peptides were assessed. Finally, the DHAP/DAHC matrix was applied to identify phosphorylation sites from α-casein and β-casein and to characterize two phosphorylation sites from the human histone H1 treated with Cyclin-Dependent Kinase-1 (CDK1 by MALDI-TOF/TOF MS.

  1. Polyphasic Approach Including MALDI-TOF MS/MS Analysis for Identification and Characterisation of Fusarium verticillioides in Brazilian Corn Kernels

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    Susane Chang

    2016-02-01

    Full Text Available Fusarium verticillioides is considered one of the most important global sources of fumonisins contamination in food and feed. Corn is one of the main commodities produced in the Northeastern Region of Brazil. The present study investigated potential mycotoxigenic fungal strains belonging to the F. verticillioides species isolated from corn kernels in 3 different Regions of the Brazilian State of Pernambuco. A polyphasic approach including classical taxonomy, molecular biology, MALDI-TOF MS and MALDI-TOF MS/MS for the identification and characterisation of the F. verticillioides strains was used. Sixty F. verticillioides strains were isolated and successfully identified by classical morphology, proteomic profiles of MALDI-TOF MS, and by molecular biology using the species-specific primers VERT-1 and VERT-2. FUM1 gene was further detected for all the 60 F. verticillioides by using the primers VERTF-1 and VERTF-2 and through the amplification profiles of the ISSR regions using the primers (GTG5 and (GACA4. Results obtained from molecular analysis shown a low genetic variability among these isolates from the different geographical regions. All of the 60 F. verticillioides isolates assessed by MALDI-TOF MS/MS presented ion peaks with the molecular mass of the fumonisin B1 (721.83 g/mol and B2 (705.83 g/mol.

  2. Ellagitannin composition of blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS.

    Science.gov (United States)

    Hager, Tiffany J; Howard, Luke R; Liyanage, Rohana; Lay, Jackson O; Prior, Ronald L

    2008-02-13

    Blackberries ( Rubus sp.) were evaluated by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the ellagitannins present in flesh, torus (receptacle tissue), and seeds. Most ellagitannins were present (or detectable) only in seed tissues. Ellagitannins identified by HPLC-ESI-MS in the seeds included pedunculagin, casuarictin/potentillin, castalagin/vescalagin, lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D. For several of the ellagitannins, isomeric separation was also obtained. The MALDI-TOF-MS analysis was primarily utilized to evaluate and identify high molecular mass (>1000 Da) ellagitannins. The MALDI analysis verified the presence of the ellagitannins identified by HPLC-ESI-MS including lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D, but the analysis also indicated the presence of several other compounds that were most likely ellagitannins based on the patterns observed in the masses (i.e., loss or addition of a gallic acid moiety to a known ellagitannin). This study determined the presence of several possible isomeric forms of ellagitannins previously unidentified in fruit and presents a possible analytical HPLC method for the analysis of the major ellagitannins present in the fruit.

  3. Complementary b/y fragment ion pairs from post-source decay of metastable YahO for calibration of MALDI-TOF-TOF-MS/MS

    Science.gov (United States)

    Complementary b/y fragment ion pairs from post-source decay (PSD) of metastable YahO protein ion were evaluated for use in the calibration of MALDI-TOF-TOF for tandem mass spectrometry (MS/MS). The yahO gene from pathogenic Escherichia coli O157:H7 strain EDL933 was cloned into a pBAD18 plasmid vect...

  4. Applications of MALDI-TOF MS in Microbiological identification

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    Soner Yilmaz

    2014-10-01

    Full Text Available MALDI-TOF MS (Matriks assisted laser desorption ionization time of flight mass spectrometry is a new metohod for identification of microorganisms nowadays. This method is based revealing of microorganisms protein profile with ionization of protein structure and these ionized mass pass through the electrical field. Profiles which were obtained from microorganisms compare with database of system thus identification is made by this way. Ribosomal proteins are used in identification which are less affected by enviromental conditions. Fresh culture should preferably use in MALDI-TOF MS identification. Ribosomal proteins can be deteriorate in old cultures. The correct identification rates are changing between 84,1% to 95,2% in routine bacterial isolates. The correct identification rates in yeasts are changing between 85% to 100%. It makes identification in positive blood culture bottles without the need of subculture, also makes identification on urine samples without the need of culture which has greater than 105 microorganisms in a microliter. When it compared with conventional and molecular identification methods, it is more effective on per sample costs and elapsed time on working [TAF Prev Med Bull 2014; 13(5.000: 421-426

  5. New Insights for Diagnosis of Pineapple Fusariosis by MALDI-TOF MS Technique.

    Science.gov (United States)

    Santos, Cledir; Ventura, José Aires; Lima, Nelson

    2016-08-01

    Fusarium is one of the most economically important fungal genus, since it includes many pathogenic species which cause a wide range of plant diseases. Morphological or molecular biology identification of Fusarium species is a limiting step in the fast diagnosis and treatment of plant disease caused by these fungi. Mass spectrometry by matrix-assisted laser/desorption ionisation-time-of-flight (MALDI-TOF)-based fingerprinting approach was applied to the fungal growth monitoring and direct detection of strain Fusarium guttiforme E-480 inoculated in both pineapple cultivars Pérola and Imperial side shoots, that are susceptible and resistant, respectively, to this fungal strain. MALDI-TOF MS technique was capable to detect fungal molecular mass peaks in the susceptible pineapple stem side shoot tissue. It is assumed that these molecular masses are mainly constituted by ribosomal proteins. MALDI-TOF-based fingerprinting approach has herein been demonstrated to be sensitive and accurate for the direct detection of F. guttiforme E-480 molecular masses on both susceptible and resistant pineapple side stem free of any pre-treatment. According to the results obtained, the changing on molecular mass peaks of infected susceptible pineapple tissue together with the possibility of fungal molecular masses analysis into this pineapple tissue can be a good indication for an early diagnosis by MALDI-TOF MS of pineapple fusariosis.

  6. [Evaluation of mass spectrometry: MALDI-TOF MS for fast and reliable yeast identification].

    Science.gov (United States)

    Relloso, María S; Nievas, Jimena; Fares Taie, Santiago; Farquharson, Victoria; Mujica, María T; Romano, Vanesa; Zarate, Mariela S; Smayevsky, Jorgelina

    2015-01-01

    The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  7. Detection and identification of bio-threats using MALDI-TOF-MS

    NARCIS (Netherlands)

    Paauw, A.

    2012-01-01

    MALDI-TOF-MS emerged as a new diagnostic tool in established clinical laboratories. Advantages compared to conventional techniques are that it is a fast, cost-effective, accurate method, which is suitable for high-throughput identification of bacteria by less skilled laboratory personnel because

  8. The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci

    DEFF Research Database (Denmark)

    Veloo, A C M; de Vries, E D; Jean-Pierre, H

    2016-01-01

    Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted......Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix......-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable...... if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under...

  9. Simplifying the Preparation of Pollen Grains for MALDI-TOF MS Classification

    Directory of Open Access Journals (Sweden)

    Franziska Lauer

    2017-03-01

    Full Text Available Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS is a well-implemented analytical technique for the investigation of complex biological samples. In MS, the sample preparation strategy is decisive for the success of the measurements. Here, sample preparation processes and target materials for the investigation of different pollen grains are compared. A reduced and optimized sample preparation process prior to MALDI-TOF measurement is presented using conductive carbon tape as target. The application of conductive tape yields in enhanced absolute signal intensities and mass spectral pattern information, which leads to a clear separation in subsequent pattern analysis. The results will be used to improve the taxonomic differentiation and identification, and might be useful for the development of a simple routine method to identify pollen based on mass spectrometry.

  10. MALDI-TOF MS typing of a nosocomial methicillin-resistant Staphylococcus aureus outbreak in a neonatal intensive care unit.

    Science.gov (United States)

    Steensels, Deborah; Deplano, Ariane; Denis, Olivier; Simon, Anne; Verroken, Alexia

    2017-08-01

    The early detection of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak is decisive to control its spread and rapidly initiate adequate infection control measures. Therefore, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods have a high discriminatory power but their availability remains restricted. In this study, we aimed to challenge matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a typing tool of a nosocomial MRSA outbreak in a neonatal intensive care unit. Over a 2-year period, 15 MRSA isolates were recovered from patients (n = 14) and health care workers (n = 1) at the neonatal intensive care unit. Five reference strains were included for comparison. Identification was performed by MALDI-TOF MS and susceptibility profiles determined by automated broth microdilution. Typing analysis by MALDI-TOF MS included mean spectrum profiles and subsequent dendrogram creation using BioNumerics software. Results were compared with spa typing and pulsed-field gel electrophoresis (PFGE). Our study showed good concordance (93%) between PFGE, spa typing, and MALDI-TOF MS for the outbreak-related MRSA strains. MALDI-TOF MS typing showed excellent typeability and discriminatory power but showed poor reproducibility. This study is one of the first to document the potential usefulness of MALDI-TOF MS with standardized data analysis as a typing tool for investigating a nosocomial MRSA outbreak. A concordance of 93% compared to reference typing techniques was observed. However, because of poor reproducibility, long-term follow-up of prospective isolated strains is not practical for routine use. Further studies are needed to confirm our observations.

  11. Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

    Science.gov (United States)

    Ricchi, M; Mazzarelli, A; Piscini, A; Di Caro, A; Cannas, A; Leo, S; Russo, S; Arrigoni, N

    2017-03-01

    The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex. © 2016 The Society for Applied Microbiology.

  12. Experimental design for optimizing MALDI-TOF-MS analysis of palladium complexes

    Directory of Open Access Journals (Sweden)

    Rakić-Kostić Tijana M.

    2017-01-01

    Full Text Available This paper presents optimization of matrix-assisted laser desorption/ionization (MALDI time-of-flight (TOF mass spectrometer (MS instrumental parameters for the analysis of chloro(2,2'',2"-terpyridinepalladium(II chloride dihydrate complex applying design of experiments methodology (DoE. This complex is of interest for potential use in the cancer therapy. DoE methodology was proved to succeed in optimization of many complex analytical problems. However, it has been poorly used for MALDI-TOF-MS optimization up to now. The theoretical mathematical relationships which explain the influence of important experimental factors (laser energy, grid voltage and number of laser shots on the selected responses (signal to noise – S/N ratio and the resolution – R of the leading peak is established. The optimal instrumental settings providing maximal S/N and R are identified and experimentally verified. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 172052 and Grant no. 172011

  13. Rapid Classification and Identification of Microcystis aeruginosa Strains Using MALDI-TOF MS and Polygenetic Analysis.

    Directory of Open Access Journals (Sweden)

    Li-Wei Sun

    Full Text Available Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS was used to establish a rapid, simple, and accurate method to differentiate among strains of Microcystis aeruginosa, one of the most prevalent types of bloom-forming cyanobacteria. M. aeruginosa NIES-843, for which a complete genome has been sequenced, was used to characterize ribosomal proteins as biomarkers and to optimize conditions for observing ribosomal proteins as major peaks in a given mass spectrum. Thirty-one of 52 ribosomal subunit proteins were detected and identified along the mass spectrum. Fifty-five strains of M. aeruginosa from different habitats were analyzed using MALDI-TOF MS; among these samples, different ribosomal protein types were observed. A polygenetic analysis was performed using an unweighted pair-group method with arithmetic means and different ribosomal protein types to classify the strains into five major clades. Two clades primarily contained toxic strains, and the other three clades contained exclusively non-toxic strains. This is the first study to differentiate cyanobacterial strains using MALDI-TOF MS.

  14. Rapid Classification and Identification of Microcystis aeruginosa Strains Using MALDI-TOF MS and Polygenetic Analysis.

    Science.gov (United States)

    Sun, Li-Wei; Jiang, Wen-Jing; Sato, Hiroaki; Kawachi, Masanobu; Lu, Xi-Wu

    2016-01-01

    Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was used to establish a rapid, simple, and accurate method to differentiate among strains of Microcystis aeruginosa, one of the most prevalent types of bloom-forming cyanobacteria. M. aeruginosa NIES-843, for which a complete genome has been sequenced, was used to characterize ribosomal proteins as biomarkers and to optimize conditions for observing ribosomal proteins as major peaks in a given mass spectrum. Thirty-one of 52 ribosomal subunit proteins were detected and identified along the mass spectrum. Fifty-five strains of M. aeruginosa from different habitats were analyzed using MALDI-TOF MS; among these samples, different ribosomal protein types were observed. A polygenetic analysis was performed using an unweighted pair-group method with arithmetic means and different ribosomal protein types to classify the strains into five major clades. Two clades primarily contained toxic strains, and the other three clades contained exclusively non-toxic strains. This is the first study to differentiate cyanobacterial strains using MALDI-TOF MS.

  15. MALDI-TOF MS identification of anaerobic bacteria: assessment of pre-analytical variables and specimen preparation techniques.

    Science.gov (United States)

    Hsu, Yen-Michael S; Burnham, Carey-Ann D

    2014-06-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a tool for identifying clinically relevant anaerobes. We evaluated the analytical performance characteristics of the Bruker Microflex with Biotyper 3.0 software system for identification of anaerobes and examined the impact of direct formic acid (FA) treatment and other pre-analytical factors on MALDI-TOF MS performance. A collection of 101 anaerobic bacteria were evaluated, including Clostridium spp., Propionibacterium spp., Fusobacterium spp., Bacteroides spp., and other anaerobic bacterial of clinical relevance. The results of our study indicate that an on-target extraction with 100% FA improves the rate of accurate identification without introducing misidentification (Panaerobes grown in suboptimal conditions, such as on selective culture media and following oxygen exposure. In conclusion, we report on a number of simple and cost-effective pre- and post-analytical modifications could enhance MALDI-TOF MS identification for anaerobic bacteria. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Characterisation of oligosaccharides in vegetables by HPLC and MALDI-TOF MS

    Czech Academy of Sciences Publication Activity Database

    Štikarovská, M.; Chmelík, Josef

    96(S), - (2002), s. S189-S191 ISSN 0009-2770. [Meeting of Chemistry & Life /2./. Brno, 10.09.2002-11.09.2002] Institutional research plan: CEZ:AV0Z4031919 Keywords : oligosaccharides * HPLC * MALDI-TOF-MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.336, year: 2002

  17. Automated protein identification by the combination of MALDI MS and MS/MS spectra from different instruments.

    Science.gov (United States)

    Levander, Fredrik; James, Peter

    2005-01-01

    The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification.

  18. Yeast identification by sequencing, biochemical kits, MALDI-TOF MS and rep-PCR DNA fingerprinting.

    Science.gov (United States)

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Chan, Jasper F W; Lau, Susanna K P; Kong, Fanrong; Xu, Yingchun; Woo, Patrick C Y

    2017-12-08

    No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI-TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI-TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI-TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI-TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Rapid Identification of Microorganisms from Positive Blood Culture by MALDI-TOF MS After Short-Term Incubation on Solid Medium.

    Science.gov (United States)

    Curtoni, Antonio; Cipriani, Raffaella; Marra, Elisa Simona; Barbui, Anna Maria; Cavallo, Rossana; Costa, Cristina

    2017-01-01

    Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a useful tool for rapid identification of microorganisms. Unfortunately, its direct application to positive blood culture is still lacking standardized procedures. In this study, we evaluated an easy- and rapid-to-perform protocol for MALDI-TOF MS direct identification of microorganisms from positive blood culture after a short-term incubation on solid medium. This protocol was used to evaluate direct identification of microorganisms from 162 positive monomicrobial blood cultures; at different incubation times (3, 5, 24 h), MALDI-TOF MS assay was performed from the growing microorganism patina. Overall, MALDI-TOF MS concordance with conventional methods at species level was 60.5, 80.2, and 93.8% at 3, 5, and 24 h, respectively. Considering only bacteria, the identification performances at species level were 64.1, 85.0, and 94.1% at 3, 5, and 24 h, respectively. This protocol applied to a commercially available MS typing system may represent, a fast and powerful diagnostic tool for pathogen direct identification and for a promptly and pathogen-driven antimicrobial therapy in selected cases.

  20. MALDI-TOF-mass spectrometry applications in clinical microbiology.

    Science.gov (United States)

    Seng, Piseth; Rolain, Jean-Marc; Fournier, Pierre Edouard; La Scola, Bernard; Drancourt, Michel; Raoult, Didier

    2010-11-01

    MALDI-TOF-mass spectrometry (MS) has been successfully adapted for the routine identification of microorganisms in clinical microbiology laboratories in the past 10 years. This revolutionary technique allows for easier and faster diagnosis of human pathogens than conventional phenotypic and molecular identification methods, with unquestionable reliability and cost-effectiveness. This article will review the application of MALDI-TOF-MS tools in routine clinical diagnosis, including the identification of bacteria at the species, subspecies, strain and lineage levels, and the identification of bacterial toxins and antibiotic-resistance type. We will also discuss the application of MALDI-TOF-MS tools in the identification of Archaea, eukaryotes and viruses. Pathogenic identification from colony-cultured, blood-cultured, urine and environmental samples is also reviewed.

  1. Evaluation of sample preparation protocols for spider venom profiling by MALDI-TOF MS.

    Science.gov (United States)

    Bočánek, Ondřej; Šedo, Ondrej; Pekár, Stano; Zdráhal, Zbyněk

    2017-07-01

    Spider venoms are highly complex mixtures containing biologically active substances with potential for use in biotechnology or pharmacology. Fingerprinting of venoms by Matrix-Assisted Laser Desorption-Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS) is a thriving technology, enabling the rapid detection of peptide/protein components that can provide comparative information. In this study, we evaluated the effects of sample preparation procedures on MALDI-TOF mass spectral quality to establish a protocol providing the most reliable analytical outputs. We adopted initial sample preparation conditions from studies already published in this field. Three different MALDI matrixes, three matrix solvents, two sample deposition methods, and different acid concentrations were tested. As a model sample, venom from Brachypelma albopilosa was used. The mass spectra were evaluated on the basis of absolute and relative signal intensities, and signal resolution. By conducting three series of analyses at three weekly intervals, the reproducibility of the mass spectra were assessed as a crucial factor in the selection for optimum conditions. A sample preparation protocol based on the use of an HCCA matrix dissolved in 50% acetonitrile with 2.5% TFA deposited onto the target by the dried-droplet method was found to provide the best results in terms of information yield and repeatability. We propose that this protocol should be followed as a standard procedure, enabling the comparative assessment of MALDI-TOF MS spider venom fingerprints. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Aspergillus ibericus : a new species of section nigri characterised by MALDI-TOF MS

    OpenAIRE

    Kallow, W.; Santos, Isabel; Erhard, M.; Serra, Rita; Venâncio, Armando; Lima, Nelson

    2006-01-01

    Strains from the new described species Aspergillus ibericus were characterised using MALDI-TOF MS and the results were compared with other related species of section Nigri. Fundação para a Ciência e a Tecnologia (FCT)

  3. Rapid identification of clinical members of Fusarium fujikuroi complex using MALDI-TOF MS

    NARCIS (Netherlands)

    Al-Hatmi, Abdullah Ms; Normand, Anne-Cécile; van Diepeningen, Anne D; Hendrickx, Marijke; de Hoog, G Sybren; Piarroux, Renaud

    2015-01-01

    AIM: To develop the matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) method for identification of Fusarium species within Fusarium fujikuroi complex for use in clinical microbiology laboratories. MATERIALS & METHODS: A total of 24 reference and 60 clinical and

  4. The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci

    NARCIS (Netherlands)

    Veloo, A. C. M.; de Vries, E D; Jean-Pierre, H.; Justesen, U. S.; Morris, T.; Urban, E.; Wybo, I.; van Winkelhoff, A. J.

    OBJECTIVES: Gram-positive anaerobic cocci (GPAC) account for 24-31% of the anaerobic bacteria isolated from human clinical specimens. At present GPAC are underrepresented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the MALDI-TOF

  5. Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi.

    Science.gov (United States)

    Normand, Anne-Cécile; Cassagne, Carole; Ranque, Stéphane; L'ollivier, Coralie; Fourquet, Patrick; Roesems, Sam; Hendrickx, Marijke; Piarroux, Renaud

    2013-04-08

    The poor reproducibility of matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) spectra limits the effectiveness of the MALDI-TOF MS-based identification of filamentous fungi with highly heterogeneous phenotypes in routine clinical laboratories. This study aimed to enhance the MALDI-TOF MS-based identification of filamentous fungi by assessing several architectures of reference spectrum libraries. We established reference spectrum libraries that included 30 filamentous fungus species with various architectures characterized by distinct combinations of the following: i) technical replicates, i.e., the number of analyzed deposits for each culture used to build a reference meta-spectrum (RMS); ii) biological replicates, i.e., the number of RMS derived from the distinct subculture of each strain; and iii) the number of distinct strains of a given species. We then compared the effectiveness of each library in the identification of 200 prospectively collected clinical isolates, including 38 species in 28 genera.Identification effectiveness was improved by increasing the number of both RMS per strain (plibrary markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi.

  6. Site-specific glycoprofiling of N-linked glycopeptides using MALDI-TOF MS: strong correlation between signal strength and glycoform quantities

    DEFF Research Database (Denmark)

    Thaysen-Andersen, Morten; Mysling, Simon; Højrup, Peter

    2009-01-01

    Site-specific glycoprofiling of N-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, but its quantitative accuracy lacks documentation. Thus, a systematic study of widely different glycopeptides was perf......Site-specific glycoprofiling of N-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, but its quantitative accuracy lacks documentation. Thus, a systematic study of widely different glycopeptides...... was performed to determine the relationship between the relative abundances of the individual glycoforms and the MALDI-TOF MS signal strength. Glycopeptides derived from glycoproteins containing neutral glycans (ribonuclease B, IgG, and ovalbumin) were initially profiled and yielded excellent and reproducible...... quantitation (correlation coefficient r = 0.9958, n = 5) when evaluated against a normal phase HPLC 2-AB glycan profile. Similarly, precise quantitation was observed for various forms of N-glycans (free, permethylated, and fluorescence-labeled) using MS. In addition, three different sialoglycopeptides from...

  7. A sample preparation method for recovering suppressed analyte ions in MALDI TOF MS

    NARCIS (Netherlands)

    Lou, X.; Waal, de B.F.M.; Milroy, L.G.; Dongen, van J.L.J.

    2015-01-01

    In matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS), analyte signals can be substantially suppressed by other compounds in the sample. In this technical note, we describe a modified thin-layer sample preparation method that significantly reduces the analyte

  8. MALDI-TOF MS Andromas strategy for the routine identification of bacteria, mycobacteria, yeasts, Aspergillus spp. and positive blood cultures.

    Science.gov (United States)

    Bille, E; Dauphin, B; Leto, J; Bougnoux, M-E; Beretti, J-L; Lotz, A; Suarez, S; Meyer, J; Join-Lambert, O; Descamps, P; Grall, N; Mory, F; Dubreuil, L; Berche, P; Nassif, X; Ferroni, A

    2012-11-01

    All organisms usually isolated in our laboratory are now routinely identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the Andromas software. The aim of this study was to describe the use of this strategy in a routine clinical microbiology laboratory. The microorganisms identified included bacteria, mycobacteria, yeasts and Aspergillus spp. isolated on solid media or extracted directly from blood cultures. MALDI-TOF MS was performed on 2665 bacteria isolated on solid media, corresponding to all bacteria isolated during this period except Escherichia coli grown on chromogenic media. All acquisitions were performed without extraction. After a single acquisition, 93.1% of bacteria grown on solid media were correctly identified. When the first acquisition was not contributory, a second acquisition was performed either the same day or the next day. After two acquisitions, the rate of bacteria identified increased to 99.2%. The failures reported on 21 strains were due to an unknown profile attributed to new species (9) or an insufficient quality of the spectrum (12). MALDI-TOF MS has been applied to 162 positive blood cultures. The identification rate was 91.4%. All mycobacteria isolated during this period (22) were correctly identified by MALDI-TOF MS without any extraction. For 96.3% and 92.2% of yeasts and Aspergillus spp., respectively, the identification was obtained with a single acquisition. After a second acquisition, the overall identification rate was 98.8% for yeasts (160/162) and 98.4% (63/64) for Aspergillus spp. In conclusion, the MALDI-TOF MS strategy used in this work allows a rapid and efficient identification of all microorganisms isolated routinely. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

  9. Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles.

    Science.gov (United States)

    Calvano, C D; Aresta, A; Iacovone, M; De Benedetto, G E; Zambonin, C G; Battaglia, M; Ditonno, P; Rutigliano, M; Bettocchi, C

    2010-03-11

    Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between

  10. Evaluation of MALDI-TOF MS (Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry) for routine identification of anaerobic bacteria.

    Science.gov (United States)

    Rodríguez-Sánchez, Belén; Alcalá, Luis; Marín, Mercedes; Ruiz, Adrián; Alonso, Elena; Bouza, Emilio

    2016-12-01

    Information regarding the use of MALDI-TOF MS as an alternative to conventional laboratory methods for the rapid and reliable identification of bacterial isolates is still limited. In this study, MALDI-TOF MS was evaluated on 295 anaerobic isolates previously identified by 16S rRNA gene sequencing and with biochemical tests (Rapid ID 32A system, BioMérieux). In total, 85.8% of the isolates were identified by MALDI-TOF MS at the species level vs 49.8% using the Rapid ID 32A system (p anaerobic isolates in the microbiology laboratory. Its implementation will reduce the turnaround time for a final identification and the number of isolates that require 16S rRNA sequencing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Whole-Cell MALDI-TOF MS Versus 16S rRNA Gene Analysis for Identification and Dereplication of Recurrent Bacterial Isolates

    Directory of Open Access Journals (Sweden)

    Michal Strejcek

    2018-06-01

    Full Text Available Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS, which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well-established bacterial identification and classification methods based on nearly complete 16S rRNA gene sequence analyses: a phylotype-based approach, using a closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4–10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision

  12. Comparison between MALDI-TOF MS and FilmArray Blood Culture Identification panel for rapid identification of yeast from positive blood culture.

    Science.gov (United States)

    Paolucci, M; Foschi, C; Tamburini, M V; Ambretti, S; Lazzarotto, T; Landini, M P

    2014-09-01

    In this study we evaluated MALDI-TOF MS and FilmArray methods for the rapid identification of yeast from positive blood cultures. FilmArray correctly identified 20/22 of yeast species, while MALDI-TOF MS identified 9/22. FilmArray is a reliable and rapid identification system for the direct identification of yeasts from positive blood cultures. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Comparison among four proposed direct blood culture microbial identification methods using MALDI-TOF MS

    Directory of Open Access Journals (Sweden)

    Ali M. Bazzi

    2017-05-01

    Full Text Available Summary: Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF mass spectrometry facilitates rapid and accurate identification of pathogens, which is critical for sepsis patients.In this study, we assessed the accuracy in identification of both Gram-negative and Gram-positive bacteria, except for Streptococcus viridans, using four rapid blood culture methods with Vitek MALDI-TOF-MS. We compared our proposed lysis centrifugation followed by washing and 30% acetic acid treatment method (method 2 with two other lysis centrifugation methods (washing and 30% formic acid treatment (method 1; 100% ethanol treatment (method 3, and picking colonies from 90 to 180 min subculture plates (method 4. Methods 1 and 2 identified all organisms down to species level with 100% accuracy, except for Streptococcus viridans, Streptococcus pyogenes, Enterobacter cloacae and Proteus vulgaris. The latter two were identified to genus level with 100% accuracy. Each method exhibited excellent accuracy and precision in terms of identification to genus level with certain limitations. Keywords: MALDI-TOF, Gram-negative, Gram-positive, Sepsis, Blood culture

  14. Identification of Apis mellifera gut microbiota with MALDI TOF MS Biotyper

    Directory of Open Access Journals (Sweden)

    Jaroslav Gasper

    2017-05-01

    Full Text Available The honey bee, Apis mellifera, is critically important for the pollination of many economically important crops. Continued colony losses have called for a deeper understanding of both symbiotic and pathogenic microbial interactions, particularly as they relate to food storage and the pollination environment. Therefore, the aim of this study was to explore and characterize the bacteria colonizing the alimentary tract of the native honey bees using MALDI TOF MS Biotyper. Content of the intestinal tract was cultured for isolation of Gram-negative, Gram-positive microorganisms and yeasts. Then, the identification of isolates with MALDI-TOF MS Biotyper was done. Results showed that the most abundant genera in bees’ samples were Lactobacillus, Pseudomonas and Serratia. Altogether, 12 genera with 21 bacterial species and one yeast genus with two species were isolated. Bacteria were represented with Acidovorax facilis, Lactobacillus gasseri, L. amylovorus, L. kunkeei, L. fructivorans, Pseudomonas oryzihabitans, Ps. brenneri, Ps. indica, Micrococcus luteus, Serratia fonticola, Ser. marcescens, Ser. ureilytica, Hafnia alvei, Candida magnolia, Bacillus oleronius, B. horneckiae, Issatchenkia orientalis, Pantoea agglomerans, Enterobacter cloacae, Staphylococcus epidermidis, Staph. pasteuri, Shewanella profunda.  The results of the study shows that the microflora of the bees gut is heterogenic and depend of locality and resources of environment for bees.

  15. Shortcomings of the Commercial MALDI-TOF MS Database and Use of MLSA as an Arbiter in the Identification of Nocardia Species

    Science.gov (United States)

    Carrasco, Gema; de Dios Caballero, Juan; Garrido, Noelia; Valdezate, Sylvia; Cantón, Rafael; Sáez-Nieto, Juan A.

    2016-01-01

    Nocardia species are difficult to identify, a consequence of the ever increasing number of species known and their homogeneous genetic characteristics. 16S rRNA analysis has been the gold standard for identifying these organisms, but proteomic techniques such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) and housekeeping gene analysis, have also been explored. One hundred high (n = 25), intermediate (n = 20), and low (n = 55) prevalence (for Spain) Nocardia strains belonging to 30 species were identified via 16S rRNA and MALDI-TOF MS analysis. The manufacturer-provided database MALDI Biotyper library v4.0 (5.627 entries, Bruker Daltonik) was employed. In the high prevalence group (Nocardia farcinica, N. abscessus, N. cyriacigeorgica and N. nova), the 16S rRNA and MALDI-TOF MS methods provided the same identification for 76% of the strains examined. For the intermediate prevalence group (N. brasiliensis, N. carnea, N. otitidiscaviarum and N. transvalensis complex), this figure fell to 45%. In the low-prevalence group (22 species), these two methods were concordant only in six strains at the species level. Tetra-gene multi-locus sequencing analysis (MLSA) involving the concatemer gyrB-16S rRNA-hsp65-secA1 was used to arbitrate between discrepant identifications (n = 67). Overall, the MLSA confirmed the results provided at species level by 16S rRNA analysis in 34.3% of discrepancies, and those provided by MALDI-TOF MS in 13.4%. MALDI-TOF MS could be a strong candidate for the identification of Nocardia species, but only if its reference spectrum database improves, especially with respect to unusual, recently described species and species included in the described Nocardia complexes. PMID:27148228

  16. A new strategy for faster urinary biomarkers identification by Nano-LC-MALDI-TOF/TOF mass spectrometry

    Directory of Open Access Journals (Sweden)

    Le Meur Y

    2008-11-01

    Full Text Available Abstract Background LC-MALDI-TOF/TOF analysis is a potent tool in biomarkers discovery characterized by its high sensitivity and high throughput capacity. However, methods based on MALDI-TOF/TOF for biomarkers discovery still need optimization, in particular to reduce analysis time and to evaluate their reproducibility for peak intensities measurement. The aims of this methodological study were: (i to optimize and critically evaluate each step of urine biomarker discovery method based on Nano-LC coupled off-line to MALDI-TOF/TOF, taking full advantage of the dual decoupling between Nano-LC, MS and MS/MS to reduce the overall analysis time; (ii to evaluate the quantitative performance and reproducibility of nano-LC-MALDI analysis in biomarker discovery; and (iii to evaluate the robustness of biomarkers selection. Results A pool of urine sample spiked at increasing concentrations with a mixture of standard peptides was used as a specimen for biological samples with or without biomarkers. Extraction and nano-LC-MS variabilities were estimated by analyzing in triplicates and hexaplicates, respectively. The stability of chromatographic fractions immobilised with MALDI matrix on MALDI plates was evaluated by successive MS acquisitions after different storage times at different temperatures. Low coefficient of variation (CV%: 10–22% and high correlation (R2 > 0.96 values were obtained for the quantification of the spiked peptides, allowing quantification of these peptides in the low fentomole range, correct group discrimination and selection of "specific" markers using principal component analysis. Excellent peptide integrity and stable signal intensity were found when MALDI plates were stored for periods of up to 2 months at +4°C. This allowed storage of MALDI plates between LC separation and MS acquisition (first decoupling, and between MS and MSMS acquisitions while the selection of inter-group discriminative ions is done (second decoupling

  17. A new strategy for faster urinary biomarkers identification by Nano-LC-MALDI-TOF/TOF mass spectrometry

    Science.gov (United States)

    Benkali, K; Marquet, P; Rérolle, JP; Le Meur, Y; Gastinel, LN

    2008-01-01

    Background LC-MALDI-TOF/TOF analysis is a potent tool in biomarkers discovery characterized by its high sensitivity and high throughput capacity. However, methods based on MALDI-TOF/TOF for biomarkers discovery still need optimization, in particular to reduce analysis time and to evaluate their reproducibility for peak intensities measurement. The aims of this methodological study were: (i) to optimize and critically evaluate each step of urine biomarker discovery method based on Nano-LC coupled off-line to MALDI-TOF/TOF, taking full advantage of the dual decoupling between Nano-LC, MS and MS/MS to reduce the overall analysis time; (ii) to evaluate the quantitative performance and reproducibility of nano-LC-MALDI analysis in biomarker discovery; and (iii) to evaluate the robustness of biomarkers selection. Results A pool of urine sample spiked at increasing concentrations with a mixture of standard peptides was used as a specimen for biological samples with or without biomarkers. Extraction and nano-LC-MS variabilities were estimated by analyzing in triplicates and hexaplicates, respectively. The stability of chromatographic fractions immobilised with MALDI matrix on MALDI plates was evaluated by successive MS acquisitions after different storage times at different temperatures. Low coefficient of variation (CV%: 10–22%) and high correlation (R2 > 0.96) values were obtained for the quantification of the spiked peptides, allowing quantification of these peptides in the low fentomole range, correct group discrimination and selection of "specific" markers using principal component analysis. Excellent peptide integrity and stable signal intensity were found when MALDI plates were stored for periods of up to 2 months at +4°C. This allowed storage of MALDI plates between LC separation and MS acquisition (first decoupling), and between MS and MSMS acquisitions while the selection of inter-group discriminative ions is done (second decoupling). Finally the recording of

  18. MALDI-TOF MS coupled with collision-induced dissociation (CID) measurements of poly(methyl methacrylate)

    NARCIS (Netherlands)

    Baumgaertel, A.; Becer, C.R.; Gottschaldt, M.; Schubert, U.S.

    2008-01-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was chosen for an in-detail analysis of poly(methyl methacrylate) (PMMA) in order to determine the possible fragmentation mechanism with the help of collision-induced dissociation (CID). All experiments were

  19. Flavonoids as matrices for MALDI-TOF mass spectrometric analysis of transition metal complexes

    Science.gov (United States)

    Petkovic, Marijana; Petrovic, Biljana; Savic, Jasmina; Bugarcic, Zivadin D.; Dimitric-Markovic, Jasmina; Momic, Tatjana; Vasic, Vesna

    2010-02-01

    Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a suitable method for the analysis of inorganic and organic compounds and biomolecules. This makes MALDI-TOF MS convenient for monitoring the interaction of metallo-drugs with biomolecules. Results presented in this manuscript demonstrate that flavonoids such as apigenin, kaempferol and luteolin are suitable for MALDI-TOF MS analysis of Pt(II), Pd(II), Pt(IV) and Ru(III) complexes, giving different signal-to-noise ratios of the analyte peak. The MALDI-TOF mass spectra of inorganic complexes acquired with these flavonoid matrices are easy to interpret and have some advantages over the application of other commonly used matrices: a low number of matrix peaks are detectable and the coordinative metal-ligand bond is, in most cases, preserved. On the other hand, flavonoids do not act as typical matrices, as their excess is not required for the acquisition of MALDI-TOF mass spectra of inorganic complexes.

  20. Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches.

    Science.gov (United States)

    Alibi, S; Ferjani, A; Gaillot, O; Marzouk, M; Courcol, R; Boukadida, J

    2015-09-01

    We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods. Copyright © 2015. Published by Elsevier SAS.

  1. The influence of culture conditions on the identification of Mycobacterium species by MALDI-TOF MS profiling.

    Science.gov (United States)

    Balážová, Tereza; Makovcová, Jitka; Šedo, Ondrej; Slaný, Michal; Faldyna, Martin; Zdráhal, Zbyněk

    2014-04-01

    Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a simple reliable approach for rapid bacterial identification based on specific peptide/protein fingerprints. However, cell-wall characteristics of mycobacterial species, and their well known stability, complicate MALDI-TOF MS profiling analysis. In this study, we tested two recently published protocols for inactivation and disruption of mycobacteria, and we also examined the influence of different culture conditions (four culture media and five cultivation times) on mass spectral quality and the discriminatory power of the method. We found a significant influence of sample pretreatment method and culture medium on species identification and differentiation for a total of 10 strains belonging to Mycobacterium phlei and Mycobacterium smegmatis. Optimum culture conditions yielding the highest identification success rate against the BioTyper database (Bruker Daltonics) and permitting the possibility of automatic acquisition of mass spectra were found to be distinct for the two mycobacterial species examined. Similarly, individual changes in growth conditions had diverse effects on the two species. For these reasons, thorough control over cultivation conditions should always be employed to maximize the performance and discriminatory power of MALDI-TOF MS profiling, and cultivation conditions must be optimized separately for individual groups of mycobacterial species/strains. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  2. Identification of Apis mellifera gut microbiota with MALDI TOF MS Biotyper

    OpenAIRE

    Jaroslav Gasper; Margarita Terentjeva; Attila Kántor; Eva Ivanišová; Maciej Kluz; Miroslava Kačániová

    2017-01-01

    The honey bee, Apis mellifera, is critically important for the pollination of many economically important crops. Continued colony losses have called for a deeper understanding of both symbiotic and pathogenic microbial interactions, particularly as they relate to food storage and the pollination environment. Therefore, the aim of this study was to explore and characterize the bacteria colonizing the alimentary tract of the native honey bees using MALDI TOF MS Biotyper. Content of the intestin...

  3. A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS

    DEFF Research Database (Denmark)

    Veloo, A; Jean-Pierre, H; Justesen, U S

    2017-01-01

    Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network...

  4. Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

    Science.gov (United States)

    Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

    2012-01-01

    A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

  5. Differentiation of isomeric N-glycan structures by normal-phase liquid chromatography-MALDI-TOF/TOF tandem mass spectrometry.

    Science.gov (United States)

    Maslen, Sarah; Sadowski, Pawel; Adam, Alex; Lilley, Kathryn; Stephens, Elaine

    2006-12-15

    The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins.

  6. Shortcomings of of the commercial MALDI-TOF MS database and use of MLSA as an arbiter in the identification of Nocardia species

    Directory of Open Access Journals (Sweden)

    Gema eCarrasco

    2016-04-01

    Full Text Available Nocardia species are difficult to identify, a consequence of the ever increasing number of species known and their homogeneous genetic characteristics. 16S rRNA analysis has been the gold standard for identifying these organisms, but proteomic techniques such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS and housekeeping gene analysis, have also been explored. One hundred high (n=25, intermediate (n=20 and low (n=55 prevalence (for Spain Nocardia strains belonging to 30 species were identified via 16S rRNA and MALDI-TOF MS analysis. The manufacturer-provided database MALDI Biotyper library v4.0 (5.627 entries, Bruker Daltonik was employed. In the high prevalence group (N. farcinica, N. abscessus, N. cyriacigeorgica and N. nova, the 16S rRNA and MALDI-TOF MS methods provided the same identification for 76% of the strains examined. For the intermediate prevalence group (N. brasiliensis, N. carnea, N. otitidiscaviarum and N. transvalensis complex, this figure fell to 45%. In the low-prevalence group (22 species, these two methods were concordant only in six strains at the species level. Tetra-gene multi-locus sequencing analysis (MLSA involving the concatemer gyrB-16S rRNA-hsp65-secA1 was used to arbitrate between discrepant identifications (n=67. Overall, the MLSA confirmed the results provided at species level by 16S rRNA analysis in 34.3% of discrepancies, and those provided by MALDI-TOF MS in 13.4%. MALDI-TOF MS could be a strong candidate for the identification of Nocardia species, but only if its reference spectrum database improves, especially with respect to unusual, recently described species and species included in the described Nocardia complexes.

  7. MALDI-TOF MS and CE-LIF Fingerprinting of Plant Cell Wall Polysaccharide Digests as a Screening Tool for Arabidopsis Cell Wall Mutants

    NARCIS (Netherlands)

    Westphal, Y.; Schols, H.A.; Voragen, A.G.J.; Gruppen, H.

    2010-01-01

    Cell wall materials derived from leaves and hypocotyls of Arabidopsis mutant and wild type plants have been incubated with a mixture of pure and well-defined pectinases, hemicellulases, and cellulases. The resulting oligosaccharides have been subjected to MALDI-TOF MS and CE-LIF analysis. MALDI-TOF

  8. A simpler method of preprocessing MALDI-TOF MS data for differential biomarker analysis: stem cell and melanoma cancer studies

    Directory of Open Access Journals (Sweden)

    Tong Dong L

    2011-09-01

    Full Text Available Abstract Introduction Raw spectral data from matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF with MS profiling techniques usually contains complex information not readily providing biological insight into disease. The association of identified features within raw data to a known peptide is extremely difficult. Data preprocessing to remove uncertainty characteristics in the data is normally required before performing any further analysis. This study proposes an alternative yet simple solution to preprocess raw MALDI-TOF-MS data for identification of candidate marker ions. Two in-house MALDI-TOF-MS data sets from two different sample sources (melanoma serum and cord blood plasma are used in our study. Method Raw MS spectral profiles were preprocessed using the proposed approach to identify peak regions in the spectra. The preprocessed data was then analysed using bespoke machine learning algorithms for data reduction and ion selection. Using the selected ions, an ANN-based predictive model was constructed to examine the predictive power of these ions for classification. Results Our model identified 10 candidate marker ions for both data sets. These ion panels achieved over 90% classification accuracy on blind validation data. Receiver operating characteristics analysis was performed and the area under the curve for melanoma and cord blood classifiers was 0.991 and 0.986, respectively. Conclusion The results suggest that our data preprocessing technique removes unwanted characteristics of the raw data, while preserving the predictive components of the data. Ion identification analysis can be carried out using MALDI-TOF-MS data with the proposed data preprocessing technique coupled with bespoke algorithms for data reduction and ion selection.

  9. Feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) networking in university hospitals in Brussels.

    Science.gov (United States)

    Martiny, D; Cremagnani, P; Gaillard, A; Miendje Deyi, V Y; Mascart, G; Ebraert, A; Attalibi, S; Dediste, A; Vandenberg, O

    2014-05-01

    The mutualisation of analytical platforms might be used to address rising healthcare costs. Our study aimed to evaluate the feasibility of networking a unique matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) system for common use in several university hospitals in Brussels, Belgium. During a one-month period, 1,055 successive bacterial isolates from the Brugmann University Hospital were identified on-site using conventional techniques; these same isolates were also identified using a MALDI-TOF MS system at the Porte de Hal Laboratory by sending target plates and identification projects via transportation and the INFECTIO_MALDI software (Infopartner, Nancy, France), respectively. The occurrence of transmission problems (MS networking always provided a faster identification result than conventional techniques, except when chromogenic culture media and oxidase tests were used (p MS networking could lead to substantial annual cost savings. MALDI-TOF MS networking presents many advantages, and few conventional techniques (optochin and oxidase tests) are required to ensure the same quality in patient care from the distant laboratory. Nevertheless, such networking should not be considered unless there is a reorganisation of workflow, efficient communication between teams, qualified technologists and a reliable IT department and helpdesk to manage potential connectivity problems.

  10. Multicenter evaluation of the Sepsityper™ extraction kit and MALDI-TOF MS for direct identification of positive blood culture isolates using the BD BACTEC™ FX and VersaTREK(®) diagnostic blood culture systems.

    Science.gov (United States)

    Schieffer, K M; Tan, K E; Stamper, P D; Somogyi, A; Andrea, S B; Wakefield, T; Romagnoli, M; Chapin, K C; Wolk, D M; Carroll, K C

    2014-04-01

    (i) Evaluation of delayed time to blood culture extraction by the Sepsityper kit and impact of shipping pellets off-site for MALDI-TOF MS analysis. (ii) Comparison of Sepsityper and laboratory-developed extraction methods from a literature review. Using two blood culture systems (BD BACTEC and VersaTREK), we extracted 411 positive blood cultures using the Sepsityper kit to mimic a potential protocol for institutions without a MALDI-TOF MS. Extracted pellets were shipped and analysed on the Bruker UltraflexIII. Successful extraction of 358 (87·1%) samples was determined by the presence of detectable proteins. MALDI-TOF MS correctly identified 332 (80·8%) samples. Delayed time to extraction did not affect Sepsityper extraction or MALDI-TOF MS accuracy. The extracted pellets remain stable and provide accurate results by MALDI-TOF MS when shipped at room temperature to off-site reference laboratories. This is the first study to show that institutions without a MALDI-TOF MS can take advantage of this innovative technology by shipping a volume of blood to an off-site laboratory for extraction and MALDI-TOF MS analysis. We also performed a literature review to compare various extraction methods. © 2014 The Society for Applied Microbiology.

  11. A comparison of Api 20A vs MALDI-TOF MS for routine identification of clinically significant anaerobic bacterial strains to the species level.

    Science.gov (United States)

    Kierzkowska, Marta; Majewska, Anna; Kuthan, Robert T; Sawicka-Grzelak, Anna; Młynarczyk, Grażyna

    2013-02-15

    Adequate identification of anaerobic bacteria still presents a challenge for laboratories conducting microbiological diagnostics. The aim of this study was to compare the use of Api 20A and MALDI-TOF MS techniques for identification of obligate anaerobes. The results indicate that MALDI-TOF MS ensures a rapid and accurate identification of the species isolated from patients. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Comparison of biomarker based Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and conventional methods in the identification of clinically relevant bacteria and yeast.

    Science.gov (United States)

    Kassim, Ali; Pflüger, Valentin; Premji, Zul; Daubenberger, Claudia; Revathi, Gunturu

    2017-05-25

    MALDI-TOF MS is an analytical method that has recently become integral in the identification of microorganisms in clinical laboratories. It relies on databases that majorly employ pattern recognition or fingerprinting. Biomarker based databases have also been developed and there is optimism that these may be superior to pattern recognition based databases. This study compared the performance of ribosomal biomarker based MALDI-TOF MS and conventional methods in the identification of selected bacteria and yeast. The study was a cross sectional study identifying clinically relevant bacteria and yeast isolated from varied clinical specimens submitted to a clinical laboratory. The identification of bacteria using conventional Vitek 2™ automated system, serotyping and MALDI-TOF MS was performed as per standard operating procedures. Comparison of sensitivities were then carried out using Pearson Chi-Square test and p-value of bacteria and Gram positive bacteria to the species level. For the Gram positive bacteria, significant difference was observed in the identification of Coagulase negative Staphylococci (p = 0.000) and Enterococcus (p = 0.008). Significant difference was also observed between serotyping and MALDI-TOF MS (p = 0.005) and this was attributed to the lack of identification of Shigella species by MALDI-TOF MS. There was no significant difference observed in the identification of yeast however some species of Candida were unidentified by MALDI-TOF MS. Biomarker based MALDI-TOF MS had good performance in a clinical laboratory setting with high sensitivities in the identification of clinically relevant microorganisms.

  13. Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Vidal-Acuña, M Reyes; Ruiz-Pérez de Pipaón, Maite; Torres-Sánchez, María José; Aznar, Javier

    2017-12-08

    An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For

  14. MALDI-TOF-MS with PLS Modeling Enables Strain Typing of the Bacterial Plant Pathogen Xanthomonas axonopodis

    Science.gov (United States)

    Sindt, Nathan M.; Robison, Faith; Brick, Mark A.; Schwartz, Howard F.; Heuberger, Adam L.; Prenni, Jessica E.

    2018-02-01

    Matrix-assisted desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) is a fast and effective tool for microbial species identification. However, current approaches are limited to species-level identification even when genetic differences are known. Here, we present a novel workflow that applies the statistical method of partial least squares discriminant analysis (PLS-DA) to MALDI-TOF-MS protein fingerprint data of Xanthomonas axonopodis, an important bacterial plant pathogen of fruit and vegetable crops. Mass spectra of 32 X. axonopodis strains were used to create a mass spectral library and PLS-DA was employed to model the closely related strains. A robust workflow was designed to optimize the PLS-DA model by assessing the model performance over a range of signal-to-noise ratios (s/n) and mass filter (MF) thresholds. The optimized parameters were observed to be s/n = 3 and MF = 0.7. The model correctly classified 83% of spectra withheld from the model as a test set. A new decision rule was developed, termed the rolled-up Maximum Decision Rule (ruMDR), and this method improved identification rates to 92%. These results demonstrate that MALDI-TOF-MS protein fingerprints of bacterial isolates can be utilized to enable identification at the strain level. Furthermore, the open-source framework of this workflow allows for broad implementation across various instrument platforms as well as integration with alternative modeling and classification algorithms.

  15. Bacterial flora analysis of coliforms in sewage, river water, and ground water using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Suzuki, Yoshihiro; Niina, Kouki; Matsuwaki, Tomonori; Nukazawa, Kei; Iguchi, Atsushi

    2018-01-28

    The aim of this study was to rapidly and effectively analyze coliforms, which are the most fundamental indicators of water quality for fecal pollution, using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Coliform bacteria were isolated from municipal sewage, river water, and groundwater. For each sample, 100 isolates were determined by MALDI-TOF MS. In addition, these same 100 isolates were also identified via 16S rRNA gene sequence analysis. Obtained MALDI-TOF MS data were compared with the 16S rRNA sequencing analysis, and the validity of MALDI-TOF MS for classification of coliform bacteria was examined. The concordance rate of bacterial identification for the 100 isolates obtained by MALDI-TOF MS analysis and 16S rRNA gene sequence analysis for sewage, river water, and ground water were 96%, 74%, and 62% at the genus level, respectively. Among the sewage, river water, and ground water samples, the coliform bacterial flora were distinct. The dominant genus of coliforms in sewage, river water, and groundwater were Klebsiella spp., Enterobacter spp., and Serratia spp., respectively. We determined that MALDI-TOF MS is a rapid and accurate tool that can be used to identify coliforms. Therefore, without using conventional 16S rRNA sequencing, it is possible to rapidly and effectively classify coliforms in water using MALDI-TOF MS.

  16. Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture.

    Science.gov (United States)

    Verroken, A; Defourny, L; Lechgar, L; Magnette, A; Delmée, M; Glupczynski, Y

    2015-02-01

    Speeding up the turn-around time of positive blood culture identifications is essential in order to optimize the treatment of septic patients. Several sample preparation techniques have been developed allowing direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of positive blood cultures. Yet, the hands-on time restrains their routine workflow. In this study, we evaluated an approach whereby MALDI-TOF MS identification without any additional steps was carried out on short subcultured colonies from positive blood bottles with the objective of allowing results reporting on the day of positivity detection. Over a 7-month period in 2012, positive blood cultures detected by 9 am with an automated system were inoculated onto a Columbia blood agar and processed after a 5-h incubation on a MALDI-TOF MicroFlex platform (Bruker Daltonik GmbH). Single-spotted colonies were covered with 1 μl formic acid and 1 μl matrix solution. The results were compared to the validated identification techniques. A total of 925 positive blood culture bottles (representing 470 bacteremic episodes) were included. Concordant identification was obtained in 727 (81.1 %) of the 896 monomicrobial blood cultures, with failure being mostly observed with anaerobes and yeasts. In 17 episodes of polymicrobic bacteremia, the identification of one of the two isolates was achieved in 24/29 (82.7 %) positive cultures. Routine implementation of MALDI-TOF MS identification on young positive blood subcultures provides correct results to the clinician in more than 80 % of the bacteremic episodes and allows access to identification results on the day of blood culture positivity detection, potentially accelerating the implementation of targeted clinical treatments.

  17. MALDI-TOF mass spectrometry in the clinical mycology laboratory: identification of fungi and beyond.

    Science.gov (United States)

    Posteraro, Brunella; De Carolis, Elena; Vella, Antonietta; Sanguinetti, Maurizio

    2013-04-01

    MALDI-TOF mass spectrometry (MS) is becoming essential in most clinical microbiology laboratories throughout the world. Its successful use is mainly attributable to the low operational costs, the universality and flexibility of detection, as well as the specificity and speed of analysis. Based on characteristic protein spectra obtained from intact cells - by means of simple, rapid and reproducible preanalytical and analytical protocols - MALDI-TOF MS allows a highly discriminatory identification of yeasts and filamentous fungi starting from colonies. Whenever used early, direct identification of yeasts from positive blood cultures has the potential to greatly shorten turnaround times and to improve laboratory diagnosis of fungemia. More recently, but still at an infancy stage, MALDI-TOF MS is used to perform strain typing and to determine antifungal drug susceptibility. In this article, the authors discuss how the MALDI-TOF MS technology is destined to become a powerful tool for routine mycological diagnostics.

  18. False results caused by solvent impurity in tetrahydrofuran for maldi tof ms analysis of amines

    NARCIS (Netherlands)

    Lou, X.; Leenders, C.M.A.; van Onzen, Thuur; Bovee, R.A.A.; Van Dongen, J.L.J.; Vekemans, J.A.J.M.; Meijer, E. W.

    Tetrahydrofuran (THF) is one of the most frequently used solvents in the MALDI TOF MS analysis of synthetic compounds. However, it should be used with caution because a trace amount of 4-hydroxybutanal (HBA) might be generated and accumulated in THF during storage. Since only a tiny amount of

  19. An Improved In-house MALDI-TOF MS Protocol for Direct Cost-Effective Identification of Pathogens from Blood Cultures

    Directory of Open Access Journals (Sweden)

    Menglan Zhou

    2017-09-01

    Full Text Available Background: Bloodstream infection is a major cause of morbidity and mortality in hospitalized patients worldwide. Delays in the identification of microorganisms often leads to a poor prognosis. The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS directly to blood culture (BC broth can potentially identify bloodstream infections earlier, and facilitate timely management.Methods: We developed an “in-house” (IH protocol for direct MALDI-TOF MS based identification of organisms in positive BCs. The IH protocol was initially evaluated and improved with spiked BC samples, and its performance was compared with the commercial Sepsityper™ kit using both traditional and modified cut-off values. We then studied in parallel the performance of the IH protocol and the colony MS identifications in positive clinical BC samples using only modified cut-off values. All discrepancies were investigated by “gold standard” of gene sequencing.Results: In 54 spiked BC samples, the IH method showed comparable results with Sepsityper™ after applying modified cut-off values. Specifically, accurate species and genus level identification was achieved in 88.7 and 3.9% of all the clinical monomicrobial BCs (284/301, 94.4%, respectively. The IH protocol exhibited superior performance for Gram negative bacteria than for Gram positive bacteria (92.8 vs. 82.4%. For anaerobes and yeasts, accurate species identification was achieved in 80.0 and 90.0% of the cases, respectively. For polymicrobial cultures (17/301, 5.6%, MALDI-TOF MS correctly identified a single species present in all the polymicrobial BCs under the Standard mode, while using the MIXED method, two species were correctly identified in 52.9% of the samples. Comparisons based on BC bottle type, showed that the BACTEC™ Lytic/10 Anaerobic/F culture vials performed the best.Conclusion: Our study provides a novel and effective sample preparation method

  20. An Improved In-house MALDI-TOF MS Protocol for Direct Cost-Effective Identification of Pathogens from Blood Cultures.

    Science.gov (United States)

    Zhou, Menglan; Yang, Qiwen; Kudinha, Timothy; Sun, Liying; Zhang, Rui; Liu, Chang; Yu, Shuying; Xiao, Meng; Kong, Fanrong; Zhao, Yupei; Xu, Ying-Chun

    2017-01-01

    Background: Bloodstream infection is a major cause of morbidity and mortality in hospitalized patients worldwide. Delays in the identification of microorganisms often leads to a poor prognosis. The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) directly to blood culture (BC) broth can potentially identify bloodstream infections earlier, and facilitate timely management. Methods: We developed an "in-house" (IH) protocol for direct MALDI-TOF MS based identification of organisms in positive BCs. The IH protocol was initially evaluated and improved with spiked BC samples, and its performance was compared with the commercial Sepsityper™ kit using both traditional and modified cut-off values. We then studied in parallel the performance of the IH protocol and the colony MS identifications in positive clinical BC samples using only modified cut-off values. All discrepancies were investigated by "gold standard" of gene sequencing. Results: In 54 spiked BC samples, the IH method showed comparable results with Sepsityper™ after applying modified cut-off values. Specifically, accurate species and genus level identification was achieved in 88.7 and 3.9% of all the clinical monomicrobial BCs (284/301, 94.4%), respectively. The IH protocol exhibited superior performance for Gram negative bacteria than for Gram positive bacteria (92.8 vs. 82.4%). For anaerobes and yeasts, accurate species identification was achieved in 80.0 and 90.0% of the cases, respectively. For polymicrobial cultures (17/301, 5.6%), MALDI-TOF MS correctly identified a single species present in all the polymicrobial BCs under the Standard mode, while using the MIXED method, two species were correctly identified in 52.9% of the samples. Comparisons based on BC bottle type, showed that the BACTEC™ Lytic/10 Anaerobic/F culture vials performed the best. Conclusion: Our study provides a novel and effective sample preparation method for MALDI-TOF MS

  1. Rapid first-line discrimination of methicillin resistant Staphylococcus aureus strains using MALDI-TOF MS

    DEFF Research Database (Denmark)

    Østergaard, Claus; Grønvall Kjær Hansen, Sanne; Møller, Jens K

    2015-01-01

    /z-values (peaks) and used a concept of arranging these peaks into pairs or small clusters within a small mass range, allowing for quality control of the spectra obtained. Using this concept we could reproducibly characterise and arrange the isolates into 26 MALDI-TOF groups, which strongly correlated with spa...... used for this purpose. These methods are all relatively time-consuming and not performed routinely in all laboratories. The aim of this study is to examine whether MALDI-TOF MS can be used as a fast, simple and easily implemented method for first-line discrimination of MRSA isolates. Mass spectra from...... 600 clinical MRSA isolates were included in the study, representing 89 spa types, associated with 16 different known clonal complexes. All spectra were obtained directly from colony material obtained from overnight cultures without prior protein extraction. We identified 43 useful discriminatory m...

  2. MALDI-TOF MS versus VITEK 2 ANC card for identification of anaerobic bacteria.

    Science.gov (United States)

    Li, Yang; Gu, Bing; Liu, Genyan; Xia, Wenying; Fan, Kun; Mei, Yaning; Huang, Peijun; Pan, Shiyang

    2014-05-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid and inexpensive technique that has initiated a revolution in the clinical microbiology laboratory for identification of pathogens. The Vitek 2 anaerobe and Corynebacterium (ANC) identification card is a newly developed method for identification of corynebacteria and anaerobic species. The aim of this study was to evaluate the effectiveness of the ANC card and MALDI-TOF MS techniques for identification of clinical anaerobic isolates. Five reference strains and a total of 50 anaerobic bacteria clinical isolates comprising ten different genera and 14 species were identified and analyzed by the ANC card together with Vitek 2 identification system and Vitek MS together with version 2.0 database respectively. 16S rRNA gene sequencing was used as reference method for accuracy in the identification. Vitek 2 ANC card and Vitek MS provided comparable results at species level for the five reference strains. Of 50 clinical strains, the Vitek MS provided identification for 46 strains (92%) to the species level, 47 (94%) to genus level, one (2%) low discrimination, two (4%) no identification and one (2%) misidentification. The Vitek 2 ANC card provided identification for 43 strains (86%) correct to the species level, 47 (94%) correct to the genus level, three (6%) low discrimination, three (6%) no identification and one (2%) misidentification. Both Vitek MS and Vitek 2 ANC card can be used for accurate routine clinical anaerobe identification. Comparing to the Vitek 2 ANC card, Vitek MS is easier, faster and more economic for each test. The databases currently available for both systems should be updated and further developed to enhance performance.

  3. Cocoa content influences chocolate molecular profile investigated by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Bonatto, Cínthia C; Silva, Luciano P

    2015-06-01

    Chocolate authentication is a key aspect of quality control and safety. Matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of cells, tissues, and even food. The present study evaluated if MALDI-TOF MS analysis on low molecular mass profile may classify chocolate samples according to the cocoa content. The molecular profiles of seven processed commercial chocolate samples were compared by using MALDI-TOF MS. Some ions detected exclusively in chocolate samples corresponded to the metabolites of cocoa or other constituents. This method showed the presence of three distinct clusters according to confectionery and sensorial features of the chocolates and was used to establish a mass spectra database. Also, novel chocolate samples were evaluated in order to check the validity of the method and to challenge the database created with the mass spectra of the primary samples. Thus, the method was shown to be reliable for clustering unknown samples into the main chocolate categories. Simple sample preparation of the MALDI-TOF MS approach described will allow the surveillance and monitoring of constituents during the molecular profiling of chocolates. © 2014 Society of Chemical Industry.

  4. MALDI-TOF MS Profiling-Advances in Species Identification of Pests, Parasites, and Vectors

    Directory of Open Access Journals (Sweden)

    Jayaseelan Murugaiyan

    2017-05-01

    Full Text Available Invertebrate pests and parasites of humans, animals, and plants continue to cause serious diseases and remain as a high treat to agricultural productivity and storage. The rapid and accurate species identification of the pests and parasites are needed for understanding epidemiology, monitoring outbreaks, and designing control measures. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS profiling has emerged as a rapid, cost effective, and high throughput technique of microbial species identification in modern diagnostic laboratories. The development of soft ionization techniques and the release of commercial pattern matching software platforms has resulted in the exponential growth of applications in higher organisms including parasitology. The present review discusses the proof-of-principle experiments and various methods of MALDI MS profiling in rapid species identification of both laboratory and field isolates of pests, parasites and vectors.

  5. MALDI-TOF MS Profiling-Advances in Species Identification of Pests, Parasites, and Vectors.

    Science.gov (United States)

    Murugaiyan, Jayaseelan; Roesler, Uwe

    2017-01-01

    Invertebrate pests and parasites of humans, animals, and plants continue to cause serious diseases and remain as a high treat to agricultural productivity and storage. The rapid and accurate species identification of the pests and parasites are needed for understanding epidemiology, monitoring outbreaks, and designing control measures. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as a rapid, cost effective, and high throughput technique of microbial species identification in modern diagnostic laboratories. The development of soft ionization techniques and the release of commercial pattern matching software platforms has resulted in the exponential growth of applications in higher organisms including parasitology. The present review discusses the proof-of-principle experiments and various methods of MALDI MS profiling in rapid species identification of both laboratory and field isolates of pests, parasites and vectors.

  6. Rapid detection of AAC(6')-Ib-cr production using a MALDI-TOF MS strategy.

    Science.gov (United States)

    Pardo, C-A; Tan, R N; Hennequin, C; Beyrouthy, R; Bonnet, R; Robin, F

    2016-12-01

    Plasmid-mediated quinolone resistance mechanisms have become increasingly prevalent among Enterobacteriaceae strains since the 1990s. Among these mechanisms, AAC(6')-Ib-cr is the most difficult to detect. Different detection methods have been developed, but they require expensive procedures such as Sanger sequencing, pyrosequencing, polymerase chain reaction (PCR) restriction, or the time-consuming phenotypic method of Wachino. In this study, we describe a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method which can be easily implemented in clinical laboratories that use the MALDI-TOF technique for bacterial identification. We tested 113 strains of Enterobacteriaceae, of which 64 harbored the aac(6')-Ib-cr gene. We compared two MALDI-TOF strategies, which differed by their norfloxacin concentration (0.03 vs. 0.5 g/L), and the method of Wachino with the PCR and sequencing strategy used as the reference. The MALDI-TOF strategy, performed with 0.03 g/L norfloxacin, and the method of Wachino yielded the same high performances (Se = 98 %, Sp = 100 %), but the turnaround time of the MALDI-TOF strategy was faster (<5 h), simpler, and inexpensive (<1 Euro). Our study shows that the MALDI-TOF strategy has the potential to become a major method for the detection of many different enzymatic resistance mechanisms.

  7. Discrimination of Aspergillus lentulus from Aspergillus fumigatus by Raman spectroscopy and MALDI-TOF MS.

    Science.gov (United States)

    Verwer, P E B; van Leeuwen, W B; Girard, V; Monnin, V; van Belkum, A; Staab, J F; Verbrugh, H A; Bakker-Woudenberg, I A J M; van de Sande, W W J

    2014-02-01

    In 2005, a new sibling species of Aspergillus fumigatus was discovered: Aspergillus lentulus. Both species can cause invasive fungal disease in immune-compromised patients. The species are morphologically very similar. Current techniques for identification are PCR-based or morphology-based. These techniques are labour-intense and not sufficiently discriminatory. Since A. lentulus is less susceptible to several antifungal agents, it is important to correctly identify the causative infectious agent in order to optimize antifungal therapy. In this study we determined whether Raman spectroscopy and/or MALDI-TOF MS were able to differentiate between A. lentulus and A. fumigatus. For 16 isolates of A. lentulus and 16 isolates of A. fumigatus, Raman spectra and peptide profiles were obtained using the Spectracell and MALDI-TOF MS (VITEK MS RUO, bioMérieux) respectively. In order to obtain reliable Raman spectra for A. fumigatus and A. lentulus, the culture medium needed to be adjusted to obtain colourless conidia. Only Raman spectra obtained from colourless conidia were reproducible and correctly identified 25 out of 32 (78 %) of the Aspergillus strains. For VITEK MS RUO, no medium adjustments were necessary. Pigmented conidia resulted in reproducible peptide profiles as well in this case. VITEK MS RUO correctly identified 100 % of the Aspergillus isolates, within a timeframe of approximately 54 h including culture. Of the two techniques studied here, VITEK MS RUO was superior to Raman spectroscopy in the discrimination of A. lentulus from A. fumigatus. VITEK MS RUO seems to be a successful technique in the daily identification of Aspergillus spp. within a limited timeframe.

  8. Comparative analysis of Gram's stain, PNA-FISH and Sepsityper with MALDI-TOF MS for the identification of yeast direct from positive blood cultures.

    Science.gov (United States)

    Gorton, Rebecca L; Ramnarain, P; Barker, K; Stone, N; Rattenbury, S; McHugh, T D; Kibbler, C C

    2014-10-01

    Fungaemia diagnosis could be improved by reducing the time to identification of yeast from blood cultures. This study aimed to evaluate three rapid methods for the identification of yeast direct from blood cultures; Gram's stain analysis, the AdvanDX Peptide Nucleic Acid in Situ Hybridisation Yeast Traffic Light system (PNA-FISH YTL) and Bruker Sepsityper alongside matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Fifty blood cultures spiked with a known single yeast strain were analysed by blinded operators experienced in each method. Identifications were compared with MALDI-TOF MS CHROMagar Candida culture and ITS rRNA sequence-based identifications. On first attempt, success rates of 96% (48/50) and 76% (36/50) were achieved using PNA-FISH YTL and Gram's stain respectively. MALDI-TOF MS demonstrated a success rate of 56% (28/50) when applying manufacturer's species log score thresholds and 76% (38/50) using in-house parameters, including lowering the species log score threshold to >1.5. In conclusion, PNA-FISH YTL demonstrated a high success rate successfully identifying yeast commonly encountered in fungaemia. Sepsityper(™) with MALDI-TOF MS was accurate but increased sensitivity is required. Due to the misidentification of commonly encountered yeast Gram's stain analysis demonstrated limited utility in this setting. © 2014 Blackwell Verlag GmbH.

  9. Rapid typing of Mannheimia haemolytica major genotypes 1 and 2 using MALDI-TOF mass spectrometry

    Science.gov (United States)

    Genotype 2 M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate ge...

  10. A novel cluster of Mycobacterium abscessus complex revealed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Suzuki, Hiromichi; Yoshida, Shiomi; Yoshida, Atsushi; Okuzumi, Katsuko; Fukusima, Atsuhito; Hishinuma, Akira

    2015-12-01

    Mycobacterium abscessus complex is a rapidly growing mycobacterium consisting of 3 subspecies, M. abscessus, Mycobacterium massiliense, and Mycobacterium bolletii. However, rapid and accurate species identification is difficult. We first evaluated a suitable protocol of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for distinguishing these subspecies. Then, we studied spectral signals by MALDI-TOF MS in 59 M. abscessus, 42 M. massiliense, and 2 M. bolletii. Among several specific spectral signals, 4 signals clearly differentiate M. massiliense from the other 2 subspecies, M. abscessus and M. bolletii. Moreover, 6 of the 42 M. massiliense isolates showed a spectral pattern similar to M. abscessus. These isolates correspond to the distinctive class of M. massiliense (cluster D) which is closer to M. abscessus by the previous variable number tandem repeat analysis. These results indicate that MALDI-TOF MS is not only useful for the identification of 3 subspecies of M. abscessus complex but also capable of distinguishing clusters of M. massiliense. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. [Applications of MALDI-TOF technology in clinical microbiology].

    Science.gov (United States)

    Suarez, S; Nassif, X; Ferroni, A

    2015-02-01

    Until now, the identification of micro-organisms has been based on the cultural and biochemical characteristics of bacterial and fungal species. Recently, Mass Spectrometry type Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS) was developed in clinical microbiology laboratories. This new technology allows identification of micro-organisms directly from colonies of bacteria and fungi within few minutes. In addition, it can be used to identify germs directly from positive blood culture bottles or directly from urine samples. Other ways are being explored to expand the use of MALDI-TOF in clinical microbiology laboratories. Indeed, some studies propose to detect bacterial antibiotic resistance while others compare strains within species for faster strain typing. The main objective of this review is to update data from the recent literature for different applications of MALDI-TOF technique in microbiological diagnostic routine. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  12. Rapid identification and source-tracking of Listeria monocytogenes using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Jadhav, Snehal; Gulati, Vandana; Fox, Edward M; Karpe, Avinash; Beale, David J; Sevior, Danielle; Bhave, Mrinal; Palombo, Enzo A

    2015-06-02

    Listeria monocytogenes is an important foodborne pathogen responsible for the sometimes fatal disease listeriosis. Public health concerns and stringent regulations associated with the presence of this pathogen in food and food processing environments underline the need for rapid and reliable detection and subtyping techniques. In the current study, the application of matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) as a single identification and source-tracking tool for a collection of L. monocytogenes isolates, obtained predominantly from dairy sources within Australia, was explored. The isolates were cultured on different growth media and analysed using MALDI-TOF MS at two incubation times (24 and 48 h). Whilst reliable genus-level identification was achieved from most media, identification at the species level was found to be dependent on culture conditions. Successful speciation was highest for isolates cultured on the chromogenic Agar Listeria Ottaviani Agosti agar (ALOA, 91% of isolates) and non-selective horse blood agar (HBA, 89%) for 24h. Chemometric statistical analysis of the MALDI-TOF MS data enabled source-tracking of L. monocytogenes isolates obtained from four different dairy sources. Strain-level discrimination was also observed to be influenced by culture conditions. In addition, t-test/analysis of variance (ANOVA) was used to identify potential biomarker peaks that differentiated the isolates according to their source of isolation. Source-tracking using MALDI-TOF MS was compared and correlated with the gold standard pulsed-field gel electrophoresis (PFGE) technique. The discriminatory index and the congruence between both techniques were compared using the Simpsons Diversity Index and adjusted Rand and Wallace coefficients. Overall, MALDI-TOF MS based source-tracking (using data obtained by culturing the isolates on HBA) and PFGE demonstrated good congruence with a Wallace coefficient of 0.71 and

  13. Application of MALDI-TOF MS fingerprinting as a quick tool for identification and clustering of foodborne pathogens isolated from food products.

    Science.gov (United States)

    Elbehiry, Ayman; Marzouk, Eman; Hamada, Mohamed; Al-Dubaib, Musaad; Alyamani, Essam; Moussa, Ihab M; AlRowaidhan, Anhar; Hemeg, Hassan A

    2017-10-01

    Foodborne pathogens can be associated with a wide variety of food products and it is very important to identify them to supply safe food and prevent foodborne infections. Since traditional techniques are timeconsuming and laborious, this study was designed for rapid identification and clustering of foodborne pathogens isolated from various restaurants in Al-Qassim region, Kingdom of Saudi Arabia (KSA) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sixty-nine bacterial and thirty-two fungal isolates isolated from 80 food samples were used in this study. Preliminary identification was carried out through culture and BD Phoenix™ methods. A confirmatory identification technique was then performed using MALDI-TOF MS. The BD Phoenix results revealed that 97% (67/69 isolates) of bacteria were correctly identified as 75% Enterobacter cloacae, 95.45% Campylobacter jejuni and 100% for Escherichia coli, Salmonella enterica, Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella pneumoniae. While 94.44% (29/32 isolates) of fungi were correctly identified as 77.77% Alternaria alternate, 88.88% Aspergillus niger and 100% for Aspergillus flavus, Penicillium digitatum, Candida albicans and Debaryomyces hansenii. However, all bacterial and fungal isolates were 100% properly identified by MALDI-TOF MS fingerprinting with a score value ≥2.00. A gel view illustrated that the spectral peaks for the identified isolates fluctuate between 3,000 and 10,000 Da. The results of main spectra library (MSP) dendrogram showed that the bacterial and fungal isolates matched with 19 and 9 reference strains stored in the Bruker taxonomy, respectively. Our results indicated that MALDI-TOF MS is a promising technique for fast and accurate identification of foodborne pathogens.

  14. Early Diagnosis of Irkut Virus Infection Using Magnetic Bead-Based Serum Peptide Profiling by MALDI-TOF MS in a Mouse Model

    Directory of Open Access Journals (Sweden)

    Nan Li

    2014-03-01

    Full Text Available Early diagnosis is important for the prompt post-exposure prophylaxis of lyssavirus infections. To diagnose Irkut virus (IRKV infection during incubation in mice, a novel method using magnetic bead-based serum peptide profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS has been established. For this test, serum peptides were concentrated by adsorption to and elution from the magnetic bead-based weak cation ion exchanger. Mass spectrograms obtained by MALDI-TOF MS were analyzed using ClinProTools bioinformatics software. Construction of the diagnostic model was performed using serum samples from mice infected with IRKV and rabies virus (RABV BD06, Flury-LEP, and SRV9 (as controls. The method accurately diagnosed sera 2, 4 and 8 days after IRKV and RABV infections. The sensitivity, specificity, and total accuracy of diagnosis were 86.7%, 95.2%, and 92.9%, respectively. However, IRKV could not be differentiated from RABV 1 day after infection. The results of the present study indicate that serum peptide profiling by MALDI-TOF MS is a promising technique for the early clinical diagnosis of lyssavirus infections and needs to be further tested in humans and farm animals.

  15. Enterococcus genus identification isolated from gastrointestinal tract of chickens after bees products application using MALDI TOF MS Biotyper

    Directory of Open Access Journals (Sweden)

    Miroslava Kačániová

    2013-10-01

    Full Text Available The general objective of this study was to examine the effect of bee product on the Enterococci colonization of chickens. Bee products were administered to both feed mixtures in various amounts in addition to the control group. First experimental group was with propolis in feed mixture with the addition of 200 mg propolis per 1 kg of compound and second group was with pollen with the addition of 250 mg pollen per 1 kg of compound. In this experiment, quantitative counts of Enterococci in ceca of 49-day-old chicken (Ross 308 using classical and MALDI TOF MS Biotyper method were investigated. Counts of Enterococci on Slanetz-Bartley agar were monitored. Enterococcus cells, isolated from gastrointestinal tract, were detected using MALDI TOF MS Biotyper. Counts of CFU of Enterococci were compared in experimental and control treatments, respectively. The lowest count was detected in the control experimental group. The highest count was detected in the first experimental group where was 200 mg of propolis added to 1 kg of feed mixture. Using MALDI TOF MS Biotyper, we identified the species range of the genera Enterococcus in the intestinal tract of broiler. Detected species from the genus Enterococcus were:      E. avium, E. casseliflavus, E cecorum, E. faecalis, E. faecium, E. gallinarum, E. hirae and E. malodoratus. In the experimental groups (caecal samples were most frequent species of E. avium E. faecium and E. gallinarum.

  16. ATR-FTIR Spectroscopy Highlights the Problem of Distinguishing Between Exophiala dermatitidis and E. phaeomuriformis Using MALDI-TOF MS

    NARCIS (Netherlands)

    Ergin, C.; Gok, Y.; Baygu, Y.; Gumral, R.; Ozhak-Baysan, B.; Dogen, A.; Ogunc, D.; Ilkit, M.; Seyedmousavi, S.

    2016-01-01

    The present study compared two chemical-based methods, namely, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, to understand the misidentification of Exophiala

  17. Direct identification of microorganisms from positive blood cultures by MALDI-TOF MS using an in-house saponin method.

    Science.gov (United States)

    Yonetani, Shota; Ohnishi, Hiroaki; Ohkusu, Kiyofumi; Matsumoto, Tetsuya; Watanabe, Takashi

    2016-11-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria. A MALDI Sepsityper kit is generally used to prepare samples obtained directly from culture bottles. However, the relatively high cost of this kit is a major obstacle to introducing this method into routine clinical use. In this study, the accuracies of three different preparation methods for rapid direct identification of bacteria from positive blood culture bottles by MALDI-TOF MS analysis were compared. In total, 195 positive bottles were included in this study. Overall, 78.5%, 68.7%, and 76.4% of bacteria were correctly identified to the genus level (score ≥1.7) directly from positive blood cultures using the Sepsityper, centrifugation, and saponin methods, respectively. The identification rates using the Sepsityper and saponin methods were significantly higher than that using the centrifugation method (Sepsityper vs. centrifugation, pdirectly from blood culture bottles, and could be a less expensive alternative to the Sepsityper method. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  18. Applications of MALDI-TOF MS to large-scale human mtDNA population-based studies

    Czech Academy of Sciences Publication Activity Database

    Cerezo, M.; Černý, Viktor; Carracedo, Á.; Salas, A.

    2009-01-01

    Roč. 30, č. 21 (2009), s. 3665-3673 ISSN 0173-0835 R&D Projects: GA ČR GA206/08/1587 Institutional research plan: CEZ:AV0Z80020508 Keywords : Haplogroup * High-throughput SNP genotyping * MALDI-TOF MS * Mitochondrial DNA * Multiplex assay Subject RIV: AC - Archeology, Anthropology, Ethnology Impact factor: 3.077, year: 2009 http://www3.interscience.wiley.com/journal/122665008/abstract?CRETRY=1&SRETRY=0

  19. [Detection of serum proteins in the patients of lung adenocarcinoma by the method of magnetic bead based sample fractionation and MALDI-TOF-MS].

    Science.gov (United States)

    Liu, Dan; Liu, Lun-Xu; Yuan, Quan; Li, Xiao-Liang; Huang, Na; Yang, Xiao-Dong

    2010-05-01

    To screen the serum proteins related to human lung adenocarcinoma using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) technology. The blood samples were collected from 10 patients of lung adenocarcinoma before and one week after the surgery, while 10 healthy subjects were used as control. The differential protein expression between the two groups and the change of those proteins after surgery were studied by ClinProt magnetic bead enrichment and MALDI-TOF-MS. Six protein peaks were identified, 2 of them were highly expressed protein biomarkers with relative molecular weights of 2661, 2991, and increased after the surgery, 4 of them were lowly expressed protein biomarkers with relative molecular weights of 4091, 4210, 4644, 5336, which continuously decreased after the surgery. ClinProt magnetic bead enrichment and MALDI-TOF-MS is a quick, easy and sensitive method of proteomics. The differential expressed proteins may be the latent tumor marker of lung adenocarcinoma. The alteration of those proteins after surgery might be helpful to assess the therapeutic effect and prognosis.

  20. Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting.

    Science.gov (United States)

    Andrés-Barrao, Cristina; Benagli, Cinzia; Chappuis, Malou; Ortega Pérez, Ruben; Tonolla, Mauro; Barja, François

    2013-03-01

    Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species. Copyright © 2012 Elsevier GmbH. All rights reserved.

  1. Evaluation of repetitive-PCR and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid strain typing of Bacillus coagulans.

    Science.gov (United States)

    Sato, Jun; Nakayama, Motokazu; Tomita, Ayumi; Sonoda, Takumi; Hasumi, Motomitsu; Miyamoto, Takahisa

    2017-01-01

    In order to establish rapid and accurate typing method for Bacillus coagulans strains which is important for controlling in some canned foods and tea-based beverages manufacturing because of the high-heat resistance of the spores and high tolerance of the vegetative cells to catechins and chemicals, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and repetitive-PCR (rep-PCR) were evaluated. For this purpose, 28 strains of B. coagulans obtained from various culture collections were tested. DNA sequence analyses of the genes encoding 16S rRNA and DNA gyrase classified the test strains into two and three groups, respectively, regardless of their phenotypes. Both MALDI-TOF MS and rep-PCR methods classified the test strains in great detail. Strains classified in each group showed similar phenotypes, such as carbohydrate utilization determined using API 50CH. In particular, the respective two pairs of strains which showed the same metabolic characteristic were classified into the same group by both MALDI-TOF MS and rep-PCR methods separating from the other strains. On the other hand, the other strains which have the different profiles of carbohydrate utilization were separated into different groups by these methods. These results suggested that the combination of MALDI-TOF MS and rep-PCR analyses was advantageous for the rapid and detailed typing of bacterial strains in respect to both phenotype and genotype.

  2. NMR, ESI/MS, and MALDI-TOF/MS analysis of pear juice polymeric proanthocyanidins with potent free radical scavenging activity.

    Science.gov (United States)

    Es-Safi, Nour-Eddine; Guyot, Sylvain; Ducrot, Paul-Henri

    2006-09-20

    The structure of a polymeric proanthocyanidin fraction isolated from pear juice was characterized by NMR, ESI/MS, and MALDI-TOF/MS analyses, and its antioxidant activity was investigated using the DPPH free radical scavenging method. The results obtained from 13C NMR analysis showed the predominance of signals representative of procyanidins. Typical signals in the chemical shift region between 70 and 90 ppm demonstrated the exclusive presence of epicatechin units. The results obtained through negative ESI/MS analysis showed singly and doubly charged ions corresponding to the molecular mass of procyanidins with a degree of polymerization up to 22. The spectra obtained through MALDI-TOF/MS analysis revealed the presence of two series of tannin oligomers. Supporting the observations from NMR spectroscopy, the first series consists of well-resolved tannin identified as procyanidin polymers units with chain lengths of up to 25. A second series of monogalloyl flavan-3-ols polymers with polymerization degree up to 25 were also detected. This is the first mass spectrometric evidence confirming the existence of galloylated procyanidin oligomers in pear fruits. Within each of these oligomers, various signals exist suggesting the presence of several oligomeric tannins. The antioxidant properties of the polymeric fraction were investigated through reduction of the DPPH free radical, and the results obtained showed that the polymeric fraction exhibited a higher antioxidant power compared to those of (+)-catechin and B3 procyanidin dimer.

  3. Microorganism Identification Based On MALDI-TOF-MS Fingerprints

    Science.gov (United States)

    Elssner, Thomas; Kostrzewa, Markus; Maier, Thomas; Kruppa, Gary

    Advances in MALDI-TOF mass spectrometry have enabled the ­development of a rapid, accurate and specific method for the identification of bacteria directly from colonies picked from culture plates, which we have named the MALDI Biotyper. The picked colonies are placed on a target plate, a drop of matrix solution is added, and a pattern of protein molecular weights and intensities, "the protein fingerprint" of the bacteria, is produced by the MALDI-TOF mass spectrometer. The obtained protein mass fingerprint representing a molecular signature of the microorganism is then matched against a database containing a library of previously measured protein mass fingerprints, and scores for the match to every library entry are produced. An ID is obtained if a score is returned over a pre-set threshold. The sensitivity of the techniques is such that only approximately 104 bacterial cells are needed, meaning that an overnight culture is sufficient, and the results are obtained in minutes after culture. The improvement in time to result over biochemical methods, and the capability to perform a non-targeted identification of bacteria and spores, potentially makes this method suitable for use in the detect-to-treat timeframe in a bioterrorism event. In the case of white-powder samples, the infectious spore is present in sufficient quantity in the powder so that the MALDI Biotyper result can be obtained directly from the white powder, without the need for culture. While spores produce very different patterns from the vegetative colonies of the corresponding bacteria, this problem is overcome by simply including protein fingerprints of the spores in the library. Results on spores can be returned within minutes, making the method suitable for use in the "detect-to-protect" timeframe.

  4. Capillary and gel electromigration techniques and MALDI-TOF MS – Suitable tools for identification of filamentous fungi

    Czech Academy of Sciences Publication Activity Database

    Horká, Marie; Kubesová, Anna; Šalplachta, Jiří; Zapletalová, E.; Horký, J.; Šlais, Karel

    2012-01-01

    Roč. 716, - (2012), s. 155-162 ISSN 0003-2670 R&D Projects: GA MV VG20102015023; GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : electormigration techniques * MALDI - TOF MS * Monilinia spp. Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.387, year: 2012

  5. Comparison among four proposed direct blood culture microbial identification methods using MALDI-TOF MS.

    Science.gov (United States)

    Bazzi, Ali M; Rabaan, Ali A; El Edaily, Zeyad; John, Susan; Fawarah, Mahmoud M; Al-Tawfiq, Jaffar A

    Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry facilitates rapid and accurate identification of pathogens, which is critical for sepsis patients. In this study, we assessed the accuracy in identification of both Gram-negative and Gram-positive bacteria, except for Streptococcus viridans, using four rapid blood culture methods with Vitek MALDI-TOF-MS. We compared our proposed lysis centrifugation followed by washing and 30% acetic acid treatment method (method 2) with two other lysis centrifugation methods (washing and 30% formic acid treatment (method 1); 100% ethanol treatment (method 3)), and picking colonies from 90 to 180min subculture plates (method 4). Methods 1 and 2 identified all organisms down to species level with 100% accuracy, except for Streptococcus viridans, Streptococcus pyogenes, Enterobacter cloacae and Proteus vulgaris. The latter two were identified to genus level with 100% accuracy. Each method exhibited excellent accuracy and precision in terms of identification to genus level with certain limitations. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

  6. Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections.

    Science.gov (United States)

    Pulcrano, Giovanna; Iula, Dora Vita; Vollaro, Antonio; Tucci, Alessandra; Cerullo, Monica; Esposito, Matilde; Rossano, Fabio; Catania, Maria Rosaria

    2013-09-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has recently become an effective instrument for rapid microbiological diagnostics and in particular for identification of micro-organisms directly in a positive blood culture. The aim of the study was to evaluate a collection of 82 stored yeast isolates from bloodstream infection, by MALDI-TOF MS; 21 isolates were identified also directly from positive blood cultures and in the presence of other co-infecting micro-organisms. Of the 82 isolates grown on plates, 64 (76%) were correctly identified by the Vitek II system and 82 (100%) by MALDI-TOF MS; when the two methods gave different results, the isolate was identified by PCR. MALDI-TOF MS was unreliable in identifying two isolates (Candida glabrata and Candida parapsilosis) directly from blood culture; however, direct analysis from positive blood culture samples was fast and effective for the identification of yeast, which is of great importance for early and adequate treatment. © 2013. Published by Elsevier B.V. All rights reserved.

  7. Reproducibility in protein profiling by MALDI-TOF mass spectrometry

    DEFF Research Database (Denmark)

    Albrethsen, Jakob

    2007-01-01

    , immunocapture, prestructured target surfaces, standardized matrix (co)crystallization, improved MALDI-TOF MS instrument components, internal standard peptides, quality-control samples, replicate measurements, and algorithms for normalization and peak detection. CONCLUSIONS: Further evaluation and optimization...

  8. Evaluation of repetitive-PCR and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS for rapid strain typing of Bacillus coagulans.

    Directory of Open Access Journals (Sweden)

    Jun Sato

    Full Text Available In order to establish rapid and accurate typing method for Bacillus coagulans strains which is important for controlling in some canned foods and tea-based beverages manufacturing because of the high-heat resistance of the spores and high tolerance of the vegetative cells to catechins and chemicals, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS and repetitive-PCR (rep-PCR were evaluated. For this purpose, 28 strains of B. coagulans obtained from various culture collections were tested. DNA sequence analyses of the genes encoding 16S rRNA and DNA gyrase classified the test strains into two and three groups, respectively, regardless of their phenotypes. Both MALDI-TOF MS and rep-PCR methods classified the test strains in great detail. Strains classified in each group showed similar phenotypes, such as carbohydrate utilization determined using API 50CH. In particular, the respective two pairs of strains which showed the same metabolic characteristic were classified into the same group by both MALDI-TOF MS and rep-PCR methods separating from the other strains. On the other hand, the other strains which have the different profiles of carbohydrate utilization were separated into different groups by these methods. These results suggested that the combination of MALDI-TOF MS and rep-PCR analyses was advantageous for the rapid and detailed typing of bacterial strains in respect to both phenotype and genotype.

  9. MALDI-TOF mass spectrometry as a potential tool for Trichomonas vaginalis identification.

    Science.gov (United States)

    Calderaro, Adriana; Piergianni, Maddalena; Montecchini, Sara; Buttrini, Mirko; Piccolo, Giovanna; Rossi, Sabina; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Chezzi, Carlo; De Conto, Flora

    2016-06-10

    Trichomonas vaginalis is a flagellated protozoan causing trichomoniasis, a sexually transmitted human infection, with around 276.4 million new cases estimated by World Health Organization. Culture is the gold standard method for the diagnosis of T. vaginalis infection. Recently, immunochromatographic assays as well as PCR assays for the detection of T. vaginalis antigen or DNA, respectively, have been also available. Although the well-known genome sequence of T. vaginalis has made possible the application of proteomic studies, few data are available about the overall proteomic expression profiling of T. vaginalis. The aim of this study was to investigate the potential application of MALDI-TOF MS as a new tool for the identification of T. vaginalis. Twenty-one isolates were analysed by MALDI-TOF MS after the creation of a Main Spectrum Profile (MSP) from a T. vaginalis reference strain (G3) and its subsequent supplementation in the Bruker Daltonics database, not including any profile of protozoa. This was achieved after the development of a new identification method created by modifying the range setting (6-10 kDa) for the MALDI-TOF MS analysis in order to exclude the overlapping of peaks derived from the culture media used in this study. Two MSP reference spectra were created in 2 different range: 3-15 kDa (standard range setting) and 6-10 kDa (new range setting). Both MSP spectra were deposited in the MALDI BioTyper database for further identification of additional T. vaginalis strains. All the 21 strains analysed in this study were correctly identified by using the new identification method. In this study it was demonstrated that changes in the MALDI-TOF MS standard parameters usually used to identify bacteria and fungi allowed the identification of the protozoan T. vaginalis. This study shows the usefulness of MALDI-TOF MS in the reliable identification of microorganism grown on complex liquid media such as the protozoan T. vaginalis, on the basis of the

  10. Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

    Science.gov (United States)

    Winter, Martin; Bretschneider, Tom; Kleiner, Carola; Ries, Robert; Hehn, Jörg P; Redemann, Norbert; Luippold, Andreas H; Bischoff, Daniel; Büttner, Frank H

    2018-07-01

    Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC 50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

  11. MALDI-TOF MS as a Tool To Detect a Nosocomial Outbreak of Extended-Spectrum-β-Lactamase- and ArmA Methyltransferase-Producing Enterobacter cloacae Clinical Isolates in Algeria.

    Science.gov (United States)

    Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi; Rolain, Jean-Marc

    2015-10-01

    Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6')-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. HPLC, NMR and MALDI-TOF MS analysis of condensed tannins from Lithocarpus glaber leaves with potent free radical scavenging activity.

    Science.gov (United States)

    Zhang, Liang Liang; Lin, Yi Ming

    2008-12-04

    Using acid-catalyzed degradation in the presence of cysteamine, the condensed tannins from Lithocarpus glaber leaves were characterized, following thiolysis, by means of reversed-phase HPLC, 13C-NMR and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. The thiolysis reaction products showed the presence of the procyanidin (PC) and prodelphinidin (PD) structures. The 13C-NMR spectrum revealed that the condensed tannins were comprised of PD (72.4%) and PC (27.6%), and with a greater content of cis configuration rather than the trans configuration of C2-C3. The MALDI-TOF MS analysis proved the presence of PD units, and the maximum degree of polymerization (DP) was an undecamer. The antioxidant activity of condensed tannins from L. glaber leaves was evaluated by using a free radical scavenging activity assay.

  13. Application of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.

    Science.gov (United States)

    De Carolis, Elena; Vella, Antonietta; Vaccaro, Luisa; Torelli, Riccardo; Spanu, Teresa; Fiori, Barbara; Posteraro, Brunella; Sanguinetti, Maurizio

    2014-09-12

    Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful technique for identification of microorganisms, changing the workflow of well-established laboratories so that its impact on microbiological diagnostics has been unparalleled. In comparison with conventional identification methods that rely on biochemical tests and require long incubation procedures, MALDI-TOF MS has the advantage of identifying bacteria and fungi directly from colonies grown on culture plates in a few minutes and with simple procedures. Numerous studies on different systems available demonstrate the reliability and accuracy of the method, and new frontiers have been explored besides microbial species level identification, such as direct identification of pathogens from positive blood cultures, subtyping, and drug susceptibility detection.

  14. MALDI-TOF MS performance compared to direct examination, culture, and 16S rDNA PCR for the rapid diagnosis of bone and joint infections.

    Science.gov (United States)

    Lallemand, E; Coiffier, G; Arvieux, C; Brillet, E; Guggenbuhl, P; Jolivet-Gougeon, A

    2016-05-01

    The rapid identification of bacterial species involved in bone and joint infections (BJI) is an important element to optimize the diagnosis and care of patients. The aim of this study was to evaluate the usefulness of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) for the rapid diagnosis of bone infections, directly on synovial fluid (SF) or on crushed osteoarticular samples (CS). From January to October 2013, we prospectively analyzed 111 osteoarticular samples (bone and joint samples, BJS) from 78 patients in care at the University Hospital of Rennes, France. The diagnosis procedure leading to the sample collection was linked to a suspicion of infection, inflammatory disease, arthritis, or for any bone or joint abnormalities. Standard bacteriological diagnosis and molecular biology analysis [16S rRNA polymerase chain reaction (PCR) and sequencing] were conducted. In addition, analysis by MALDI-TOF MS was performed directly on the osteoarticular samples, as soon as the amount allowed. Culture, which remains the gold standard for the diagnosis of BJI, has the highest sensitivity (85.9 %) and remains necessary to test antimicrobial susceptibility. The 16S rDNA PCR results were positive in the group with positive BJI (28.6 %) and negative in the group without infection. Direct examination remains insensitive (31.7 %) but more effective than MALDI-TOF MS directly on the sample (6.3 %). The specificity was 100 % in all cases, except for culture (74.5 %). Bacterial culture remains the gold standard, especially enrichment in blood bottles. Direct analysis of bone samples with MALDI-TOF MS is not useful, possibly due to the low inoculum of BJS.

  15. Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Can Precisely Discriminate the Lineages of Listeria monocytogenes and Species of Listeria.

    Science.gov (United States)

    Ojima-Kato, Teruyo; Yamamoto, Naomi; Takahashi, Hajime; Tamura, Hiroto

    2016-01-01

    The genetic lineages of Listeria monocytogenes and other species of the genus Listeria are correlated with pathogenesis in humans. Although matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a prevailing tool for rapid and reliable microbial identification, the precise discrimination of Listeria species and lineages remains a crucial issue in clinical settings and for food safety. In this study, we constructed an accurate and reliable MS database to discriminate the lineages of L. monocytogenes and the species of Listeria (L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi, and L. rocourtiae) based on the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) proteotyping method, which relies on both genetic information (genomics) and observed MS peaks in MALDI-TOF MS (proteomics). The specific set of eight biomarkers (ribosomal proteins L24, L6, L18, L15, S11, S9, L31 type B, and S16) yielded characteristic MS patterns for the lineages of L. monocytogenes and the different species of Listeria, and led to the construction of a MS database that was successful in discriminating between these organisms in MALDI-TOF MS fingerprinting analysis followed by advanced proteotyping software Strain Solution analysis. We also confirmed the constructed database on the proteotyping software Strain Solution by using 23 Listeria strains collected from natural sources.

  16. Bactec™ blood culture bottles allied to MALDI-TOF mass spectrometry: rapid etiologic diagnosis of bacterial endophthalmitis.

    Science.gov (United States)

    Tanaka, Tatiana; Oliveira, Luiza Manhezi de Freitas; Ferreira, Bruno Fortaleza de Aquino; Kato, Juliana Mika; Rossi, Flavia; Correa, Karoline de Lemes Giuntini; Pimentel, Sergio Luis Gianotti; Yamamoto, Joyce Hisae; Almeida Junior, João Nóbrega

    2017-07-01

    Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been used for direct identification of pathogens from blood-inoculated blood culture bottles (BCBs). We showed that MALDI-TOF MS is an useful technique for rapid identification of the causative agents of endophthalmitis from vitreous humor-inoculated BCBs with a simple protocol. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. HPLC-UV, MALDI-TOF-MS and ESI-MS/MS analysis of the mechlorethamine DNA crosslink at a cytosine-cytosine mismatch pair.

    Directory of Open Access Journals (Sweden)

    Pornchai Rojsitthisak

    Full Text Available Mechlorethamine [ClCH(2CH(2N(CH(3CH(2CH(2Cl], a nitrogen mustard alkylating agent, has been proven to form a DNA interstrand crosslink at a cytosine-cytosine (C-C mismatch pair using gel electrophoresis. However, the atomic connectivity of this unusual crosslink is unknown.HPLC-UV, MALDI-TOF-MS, and ESI-MS/MS were used to determine the atomic connectivity of the DNA C-C crosslink formed by mechlorethamine, MALDI-TOF-MS of the HPLC-purified reaction product of mechlorethamine with the DNA duplex d[CTCACACCGTGGTTC]•d[GAACCACCGTGTGAG] (underlined bases are a C-C mismatch pair indicated formation of an interstrand crosslink at m/z 9222.088 [M-2H+Na](+. Following enzymatic digestion of the crosslinked duplex by snake venom phosphodiesterase and calf intestinal phosphatase, ESI-MS/MS indicated the presence of dC-mech-dC [mech = CH(2CH(2N(CH(3CH(2CH(2] at m/z 269.2 [M](2+ (expected m/z 269.6, exact mass 539.27 and its hydrolytic product dC-mech-OH at m/z 329.6 [M](+ (expected m/z 329.2. Fragmentation of dC-mech-dC gave product ions at m/z 294.3 and 236.9 [M](+, which are both due to loss of the 4-amino group of cytosine (as ammonia, in addition to dC and dC+HN(CH(3CH = CH(2, respectively. The presence of m/z 269.2 [M](2+ and loss of ammonia exclude crosslink formation at cytosine N(4 or O(2 and indicate crosslinking through cytosine N(3 with formation of two quaternary ammonium ions.Our results provide an important addition to the literature, as the first example of the use of HPLC and MS for analysis of a DNA adduct at the N(3 position of cytosine.

  18. Matrix-assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a Reliable Tool to Identify Species of Catalase-negative Gram-positive Cocci not Belonging to the Streptococcus Genus.

    Science.gov (United States)

    Almuzara, Marisa; Barberis, Claudia; Velázquez, Viviana Rojas; Ramirez, Maria Soledad; Famiglietti, Angela; Vay, Carlos

    2016-01-01

    To evaluate the performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) by using 190 Catalase-negative Gram-Positive Cocci (GPC) clinical isolates. All isolates were identified by conventional phenotypic tests following the proposed scheme by Ruoff and Christensen and MALDI-TOF MS (Bruker Daltonics, BD, Bremen, Germany). Two different extraction methods (direct transfer formic acid method on spot and ethanol formic acid extraction method) and different cut-offs for genus/specie level identification were used. The score cut-offs recommended by the manufacturer (≥ 2.000 for species-level, 1.700 to 1.999 for genus level and genus level, ≥ 1.700 for species-level and score genus or species. MALDI-TOF MS identification was considered correct when the result obtained from MS database agreed with the phenotypic identification result. When both methods gave discordant results, the 16S rDNA or sodA genes sequencing was considered as the gold standard identification method. The results obtained by MS concordant with genes sequencing, although discordant with conventional phenotyping, were considered correct. MS results discordant with 16S or sod A identification were considered incorrect. Using the score cut-offs recommended by the manufacturer, 97.37% and 81.05% were correctly identified to genus and species level, respectively. On the other hand, using lower cut-off scores for identification, 97.89% and 94.21% isolates were correctly identified to genus and species level respectively by MALDI-TOF MS and no significant differences between the results obtained with two extraction methods were obtained. The results obtained suggest that MALDI-TOF MS has the potential of being an accurate tool for Catalase-negative GPC identification even for those species with difficult diagnosis as Helcococcus , Abiotrophia , Granulicatella , among others. Nevertheless, expansion of the library, especially including more strains with

  19. Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Dolatabadi, Somayeh; Kolecka, Anna; Versteeg, Matthijs; de Hoog, Sybren G; Boekhout, Teun

    2015-07-01

    This study addresses the usefulness of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS for reliable identification of the two most frequently occurring clinical species of Rhizopus, namely Rhizopus arrhizus with its two varieties, arrhizus and delemar, and Rhizopus microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by internal transcribed spacer (ITS) sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF ) showed two monophylic clades for Rhizopus arrhizus and Rhizopus microsporus (bootstrap values of 99 %). Cluster analysis confirmed the presence of two distinct clades within Rhizopus arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between Rhizopus arrhizus and Rhizopus microsporus and the presence of two varieties within the species Rhizopus arrhizus. An in-house database of 30 reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an in-house database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on principal component analysis, two MSP dendrograms were created and showed concordance with the multi-locus tree; thus, MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.

  20. MALDI-TOF MS analysis of labile Lolium perenne major allergens in mixes.

    Science.gov (United States)

    Irañeta, S G; Acosta, D M; Duran, R; Apicella, C; Orlando, U D; Seoane, M A; Alonso, A; Duschak, V G

    2008-08-01

    It is well known that allergen extracts used for specific therapy of allergic disorders are commonly stored as mixtures, causing an alteration of its stability. The aim of this report is to identify pollen allergens susceptible to degradation during storage of mixtures containing different sources of proteases in the absence of glycerol as a preserving agent. Mixes containing Lolium perenne (Lol p) pollen extract with either Aspergillus fumigatus or Periplaneta americana extracts were prepared and co-incubated for 90 days at 4 degrees C. Samples were taken off at fixed times and comparatively tested by in vitro and in vivo assays with atopic patients. Selected pollinic allergens were subjected to MALDI-TOF MS analysis. ELISA inhibition evidenced the loss of potency from ryegrass extract, and immunoblotting assays showed the degradation of specific pollinic allergens during storage of mixtures containing protease-rich sources. An in vivo intradermal skin assay confirmed the gradual loss of the biological activity of L. perenne pollen extract co-incubated with non-related protease-rich extracts in comparison with that of the control pollen extract. MALDI-TOF MS analysis allowed us to determine that Lol p 1 and Lol p 5 are susceptible to proteolysis whereas Lol p 4 was found to be resistant to degradation during storage. Lol p 1 and Lol p 5 degradation is responsible for the loss of the biological activity of L. perenne pollen extract when co-incubated with protease-rich fungal and cockroach extracts in the same vial for months in the absence of glycerol as a preserving agent. The integrity of these major allergens must be preserved to increase the vaccine stability and to assure efficacy when mixes are used for immunotherapy.

  1. Confirmation of Fructans biosynthesized in vitro from [1-13C]glucose in asparagus tissues using MALDI-TOF MS and ESI-MS.

    Science.gov (United States)

    Suzuki, Takashi; Maeda, Tomoo; Grant, Suzanne; Grant, Gordon; Sporns, Peter

    2013-05-15

    Accumulation of Fructans was confirmed in asparagus tissues that had been cultured for 2 days on media supplemented with glucose. It is very common that Fructans are biosynthesized from sucrose. We hypothesized however that Fructans could also be biosynthesized from glucose. Stem tissues of in vitro-cultured asparagus were subcultured for 72 h on a medium containing 0.5M of [1-(13)C]glucose. A medium containing 0.5M of normal ((12)C) glucose was used as control. Carbohydrates were extracted from the tissues and analyzed using HPLC, MALDI-TOF MS and ESI-MS. HPLC results indicated that the accumulation of short-chain Fructans was similar in both (13)C-labelled and control samples. Short-chain Fructans of DP=3-7 were detected using MALDI-TOF MS. The molecular mass of each oligomer in the (13)C-labelled sample was higher than the mass of the natural sample by 1 m/z unit per sugar moiety. The results of ESI-MS on the HPLC fractions of neokestose and 1-kestose showed that these oligomers (DP=3) were biosynthesized from exogenous glucose added to the medium. We conclude that not only exogenous sucrose but glucose can induce Fructan biosynthesis; fructans of both inulin type and inulin neoseries are also biosynthesized from glucose accumulated in asparagus tissues; the glucose molecules (or its metabolic products) were incorporated into Fructans as structural monomers. Copyright © 2013 Elsevier GmbH. All rights reserved.

  2. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.

    Science.gov (United States)

    Singhal, Neelja; Kumar, Manish; Kanaujia, Pawan K; Virdi, Jugsharan S

    2015-01-01

    Currently microorganisms are best identified using 16S rRNA and 18S rRNA gene sequencing. However, in recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. The technology has been readily imbibed by microbiologists who have reported usage of MALDI-TOF MS for a number of purposes like, microbial identification and strain typing, epidemiological studies, detection of biological warfare agents, detection of water- and food-borne pathogens, detection of antibiotic resistance and detection of blood and urinary tract pathogens etc. The limitation of the technology is that identification of new isolates is possible only if the spectral database contains peptide mass fingerprints of the type strains of specific genera/species/subspecies/strains. This review provides an overview of the status and recent applications of mass spectrometry for microbial identification. It also explores the usefulness of this exciting new technology for diagnosis of diseases caused by bacteria, viruses, and fungi.

  3. MALDI-TOF mass spectrometry for differentiation between Streptococcus pneumoniae and Streptococcus pseudopneumoniae.

    Science.gov (United States)

    van Prehn, Joffrey; van Veen, Suzanne Q; Schelfaut, Jacqueline J G; Wessels, Els

    2016-05-01

    We compared the Vitek MS and Microflex MALDI-TOF mass spectrometry platform for species differentiation within the Streptococcus mitis group with PCR assays targeted at lytA, Spn9802, and recA as reference standard. The Vitek MS correctly identified 10/11 Streptococcus pneumoniae, 13/13 Streptococcus pseudopneumoniae, and 12/13 S. mitis/oralis. The Microflex correctly identified 9/11 S. pneumoniae, 0/13 S. pseudopneumoniae, and 13/13 S. mitis/oralis. MALDI-TOF is a powerful tool for species determination within the mitis group. Diagnostic accuracy varies depending on platform and database used. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. A computational platform for MALDI-TOF mass spectrometry data: application to serum and plasma samples.

    Science.gov (United States)

    Mantini, Dante; Petrucci, Francesca; Pieragostino, Damiana; Del Boccio, Piero; Sacchetta, Paolo; Candiano, Giovanni; Ghiggeri, Gian Marco; Lugaresi, Alessandra; Federici, Giorgio; Di Ilio, Carmine; Urbani, Andrea

    2010-01-03

    Mass spectrometry (MS) is becoming the gold standard for biomarker discovery. Several MS-based bioinformatics methods have been proposed for this application, but the divergence of the findings by different research groups on the same MS data suggests that the definition of a reliable method has not been achieved yet. In this work, we propose an integrated software platform, MASCAP, intended for comparative biomarker detection from MALDI-TOF MS data. MASCAP integrates denoising and feature extraction algorithms, which have already shown to provide consistent peaks across mass spectra; furthermore, it relies on statistical analysis and graphical tools to compare the results between groups. The effectiveness in mass spectrum processing is demonstrated using MALDI-TOF data, as well as SELDI-TOF data. The usefulness in detecting potential protein biomarkers is shown comparing MALDI-TOF mass spectra collected from serum and plasma samples belonging to the same clinical population. The analysis approach implemented in MASCAP may simplify biomarker detection, by assisting the recognition of proteomic expression signatures of the disease. A MATLAB implementation of the software and the data used for its validation are available at http://www.unich.it/proteomica/bioinf. (c) 2009 Elsevier B.V. All rights reserved.

  5. Comparative analysis of storage conditions and homogenization methods for tick and flea species for identification by MALDI-TOF MS.

    Science.gov (United States)

    Nebbak, A; El Hamzaoui, B; Berenger, J-M; Bitam, I; Raoult, D; Almeras, L; Parola, P

    2017-12-01

    Ticks and fleas are vectors for numerous human and animal pathogens. Controlling them, which is important in combating such diseases, requires accurate identification, to distinguish between vector and non-vector species. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was applied to the rapid identification of arthropods. The growth of this promising tool, however, requires guidelines to be established. To this end, standardization protocols were applied to species of Rhipicephalus sanguineus (Ixodida: Ixodidae) Latreille and Ctenocephalides felis felis (Siphonaptera: Pulicidae) Bouché, including the automation of sample homogenization using two homogenizer devices, and varied sample preservation modes for a period of 1-6 months. The MS spectra were then compared with those obtained from manual pestle grinding, the standard homogenization method. Both automated methods generated intense, reproducible MS spectra from fresh specimens. Frozen storage methods appeared to represent the best preservation mode, for up to 6 months, while storage in ethanol is also possible, with some caveats for tick specimens. Carnoy's buffer, however, was shown to be less compatible with MS analysis for the purpose of identifying ticks or fleas. These standard protocols for MALDI-TOF MS arthropod identification should be complemented by additional MS spectrum quality controls, to generalize their use in monitoring arthropods of medical interest. © 2017 The Royal Entomological Society.

  6. MALDI-TOF MS Versus VITEK®2: Comparison of Systems for the Identification of Microorganisms Responsible for Bacteremia.

    Science.gov (United States)

    Febbraro, Filomena; Rodio, Donatella Maria; Puggioni, Gianluca; Antonelli, Guido; Pietropaolo, Valeria; Trancassini, Maria

    2016-12-01

    We evaluated the reliability and accuracy of the combined use of MALDI-TOF MS and classical ID VITEK 2 to identify monomicrobial infection in blood culture bottles. In total, 70 consecutive positive blood cultures were included in this study. Positive blood culture bottles were subjected to Gram staining and subcultured on solid media. Isolates grown from such culture media were used for classical ID using VITEK 2 system. In parallel, an aliquot was subjected to a lysing-centrifugation method and used for the identification with the MALDI-TOF system. Results evidenced the correct genus and species identification of 91.4 % of microorganisms responsible for bacteremia with an agreement to the species and the genus level. If compared with the standard method VITEK 2 , our simple and cost-effective sample preparation method would be very useful for rapid identification of microorganisms using blood culture bottles. In fact, the direct method showed rapid and reliable results, especially for the gram-negative group.

  7. Characterization of Proteins Present in Isolated Senile Plaques from Alzheimer's Diseased Brains by MALDI-TOF MS with MS/MS.

    Science.gov (United States)

    Kelley, Andrea R; Perry, George; Bach, Stephan B H

    2018-04-18

    The increase of insoluble senile plaques in the brain is a primary hallmark of Alzheimer's disease. The usefulness of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with tandem MS for the characterization of senile plaques from AD brains and the relevance of the components identified to furthering AD research using MS is discussed. Thirty-three components were reproducibly observed within tryptic aliquots of senile plaques from two different AD brains after sample preparation optimization. Additionally, this is one of the first accounts of LIFT being utilized for the direct sequencing of peptides from isolated senile plaques. While many of the species observed coisolated within senile plaques have been linked to AD etiology, if only speculatively, this is the first instance that many of them have been demonstrated to be a part of the plaques themselves. This work is the first step in determining the potential roles that the species may have in the aggregation or proliferation of the plaques.

  8. Species-Level Discrimination of Psychrotrophic Pathogenic and Spoilage Gram-Negative Raw Milk Isolates Using a Combined MALDI-TOF MS Proteomics-Bioinformatics-based Approach.

    Science.gov (United States)

    Vithanage, Nuwan R; Bhongir, Jeevana; Jadhav, Snehal R; Ranadheera, Chaminda S; Palombo, Enzo A; Yeager, Thomas R; Datta, Nivedita

    2017-06-02

    Identification of psychrotrophic pathogenic and spoilage Gram-negative bacteria using rapid and reliable techniques is important in commercial milk processing, as these bacteria can produce heat-resistant proteases and act as postprocessing contaminants in pasteurized milk. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a proven technology for identification of bacteria in food, however, may require optimization for identification of pathogenic and spoilage bacteria in milk and dairy products. The current study evaluated the effects of various culture conditions and sample preparation methods on assigning of raw milk isolates to the species level by MALDI-TOF MS. The results indicated that culture media, incubation conditions (temperature and time), and sample preparation significantly affected the identification rates of bacteria to the species level. Nevertheless, the development of spectral libraries of isolates grown on different media using a web tool for hierarchical clustering of peptide mass spectra (SPECLUST) followed by a ribosomal protein based bioinformatics approach significantly enhanced the assigning of bacteria, with at least one unique candidate biomarker peak identified for each species. Phyloproteomic relationships based on spectral profiles were compared to phylogenetic analysis using 16S rRNA gene sequences and demonstrated similar clustering patterns with significant discriminatory power. Thus, with appropriate optimization, MALDI-TOF MS is a valuable tool for species-level discrimination of pathogenic and milk spoilage bacteria.

  9. A rapid MALDI-TOF MS identification database at genospecies level for clinical and environmental Aeromonas strains.

    Directory of Open Access Journals (Sweden)

    Cinzia Benagli

    Full Text Available The genus Aeromonas has undergone a number of taxonomic and nomenclature revisions over the past 20 years, and new (subspecies and biogroups are continuously described. Standard identification methods such as biochemical characterization have deficiencies and do not allow clarification of the taxonomic position. This report describes the development of a matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS identification database for a rapid identification of clinical and environmental Aeromonas isolates.

  10. Gram-stain plus MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for a rapid diagnosis of urinary tract infection.

    Directory of Open Access Journals (Sweden)

    Almudena Burillo

    Full Text Available Microbiological confirmation of a urinary tract infection (UTI takes 24-48 h. In the meantime, patients are usually given empirical antibiotics, sometimes inappropriately. We assessed the feasibility of sequentially performing a Gram stain and MALDI-TOF MS mass spectrometry (MS on urine samples to anticipate clinically useful information. In May-June 2012, we randomly selected 1000 urine samples from patients with suspected UTI. All were Gram stained and those yielding bacteria of a single morphotype were processed for MALDI-TOF MS. Our sequential algorithm was correlated with the standard semiquantitative urine culture result as follows: Match, the information provided was anticipative of culture result; Minor error, the information provided was partially anticipative of culture result; Major error, the information provided was incorrect, potentially leading to inappropriate changes in antimicrobial therapy. A positive culture was obtained in 242/1000 samples. The Gram stain revealed a single morphotype in 207 samples, which were subjected to MALDI-TOF MS. The diagnostic performance of the Gram stain was: sensitivity (Se 81.3%, specificity (Sp 93.2%, positive predictive value (PPV 81.3%, negative predictive value (NPV 93.2%, positive likelihood ratio (+LR 11.91, negative likelihood ratio (-LR 0.20 and accuracy 90.0% while that of MALDI-TOF MS was: Se 79.2%, Sp 73.5, +LR 2.99, -LR 0.28 and accuracy 78.3%. The use of both techniques provided information anticipative of the culture result in 82.7% of cases, information with minor errors in 13.4% and information with major errors in 3.9%. Results were available within 1 h. Our serial algorithm provided information that was consistent or showed minor errors for 96.1% of urine samples from patients with suspected UTI. The clinical impacts of this rapid UTI diagnosis strategy need to be assessed through indicators of adequacy of treatment such as a reduced time to appropriate empirical treatment or

  11. Magnetic graphene composites as both an adsorbent for sample enrichment and a MALDI-TOF MS matrix for the detection of nitropolycyclic aromatic hydrocarbons in PM2.5.

    Science.gov (United States)

    Zhang, Jiangang; Zhang, Li; Li, Ruijin; Hu, Di; Ma, Nengxuan; Shuang, Shaomin; Cai, Zongwei; Dong, Chuan

    2015-03-07

    A simple and rapid method that uses synthesized magnetic graphene composites as both an adsorbent for enrichment and as a matrix in MALDI-TOF MS analysis was developed for the detection of nitropolycyclic hydrocarbons (nitro-PAHs) in PM2.5 samples. Three nitro-PAHs were detected down to sub pg μL(-1) levels based on calculations from an instrumental signal-to-noise better than 3, which shows the feasibility of using the new materials in MALDI-TOF MS as a potential powerful analytical approach for the analysis of nitro-PAHs in PM2.5 samples.

  12. 16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry.

    Science.gov (United States)

    Dec, Marta; Puchalski, Andrzej; Urban-Chmiel, Renata; Wernicki, Andrzej

    2016-06-13

    The objective of our study is to evaluate the potential use of Amplified 16S Ribosomal DNA Restriction Analysis (16S-ARDRA) and MALDI-TOF mass spectrometry (MS) as methods for species identification of Lactobacillus strains in poultry. A total of 80 Lactobacillus strains isolated from the cloaca of chicken, geese and turkeys were identified to the species level by MALDI-TOF MS (on-plate extraction method) and 16S-ARDRA. The two techniques produced comparable classification results, some of which were additionally confirmed by sequencing of 16S rDNA. MALDI-TOF MS enabled rapid species identification but produced more than one reliable identification result for 16.25 % of examined strains (mainly of the species L. johnsonii). For 30 % of isolates intermediate log(scores) of 1.70-1.99 were obtained, indicating correct genus identification but only presumptive species identification. The 16S-ARDRA protocol was based on digestion of 16S rDNA with the restriction enzymes MseI, HinfI, MboI and AluI. This technique was able to distinguish 17 of the 19 Lactobacillus reference species tested and enabled identification of all 80 wild isolates. L. salivarius dominated among the 15 recognized species, followed by L. johnsonii and L. ingluviei. The MALDI-TOF MS and 16S-ARDRA assays are valuable tools for the identification of avian lactobacilli to the species level. MALDI-TOF MS is a fast, simple and cost-effective technique, and despite generating a high percentage of results with a log(score) Lactobacillus bacteria from different habitats.

  13. Characterization of Bacteria in Ballast Water Using MALDI-TOF Mass Spectrometry

    Digital Repository Service at National Institute of Oceanography (India)

    Emami, K.; Askari, V.; Ullrich, M.; Mohinudeen, K.; Anil, A.C.; Khandeparker, L.; Burgess, J.G.; Mesbahi, E.

    To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since...

  14. Multicenter validation of the VITEK MS v2.0 MALDI-TOF mass spectrometry system for the identification of fastidious gram-negative bacteria.

    Science.gov (United States)

    Branda, John A; Rychert, Jenna; Burnham, Carey-Ann D; Bythrow, Maureen; Garner, Omai B; Ginocchio, Christine C; Jennemann, Rebecca; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Sercia, Linda F; Westblade, Lars F; Ferraro, Mary Jane

    2014-02-01

    The VITEK MS v2.0 MALDI-TOF mass spectrometry system's performance in identifying fastidious gram-negative bacteria was evaluated in a multicenter study. Compared with the reference method (DNA sequencing), the VITEK MS system provided an accurate, species-level identification for 96% of 226 isolates; an additional 1% were accurately identified to the genus level. © 2013.

  15. Novel, Improved Sample Preparation for Rapid, Direct Identification from Positive Blood Cultures Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

    OpenAIRE

    Schubert, Sören; Weinert, Kirsten; Wagner, Chris; Gunzl, Beatrix; Wieser, Andreas; Maier, Thomas; Kostrzewa, Markus

    2011-01-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for rapid and reliable identification of bacteria and yeast grown on agar plates. Moreover, MALDI-TOF MS also holds promise for bacterial identification from blood culture (BC) broths in hospital laboratories. The most important technical step for the identification of bacteria from positive BCs by MALDI-TOF MS is sample preparation to remove blood cells and host proteins. We present a m...

  16. Use of Maldi-Tof Mass spectrometry in direct microorganism identification in clinical laboratories

    Directory of Open Access Journals (Sweden)

    Tamara Brunelli

    2010-09-01

    Full Text Available Mass Spectrometry is an old technique that has recently been introduced in the clinical microbiology laboratory as Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS. MALDI is a soft ionization technique used in mass spectrometry that allows the analysis of biomolecules and large organic molecules which tend to be fragile and fragment when ionized.To obtain ions biological specimens are mixed with a matrix which specifically absorbs the ionization source (a laser beam. The high energy impact is followed by the formation of ions which are extract through an elastic field, focussed and detected as mass/charge (m/z spectrum.The differences between ions are seen with TOF, a revelation system that relates the time of flight of a ion to the charge/mass value: ion with a higher m/z have are slower (a bigger time of flight than ions with lower m/z. MALDI-TOF MS, in clinical microbiology laboratory, is used to identify bacteria and fungi directly from samples. The identification of microorganisms can be performed directly from body fluids (e.g. urine, blood culture, after centrifugation and recovery of microorganisms or from colonies (after cultivation. The rapidity of identification is of great importance in blood cultures. Positive cultures with one microorganism are processed in a different way than those with more than one microorganism. In positive monomicrobial cultures, after separation of microbs from blood cells,we can perform an immediate identification with MALDI-TOF MS that we can communicate to the clinician, and that gives indication to perform the correct antibiogram. Major problems are present when more than one microorganism are in the culture: in this case we have to use the method of subcultivation and then the identification with mass-spectrometry can be performed. MALDI-TOF MS is a rapid, reliable and low cost technique, that can identify a growing number of microorganisms. This technique can

  17. Identification of Brucella by MALDI-TOF mass spectrometry. Fast and reliable identification from agar plates and blood cultures.

    Directory of Open Access Journals (Sweden)

    Laura Ferreira

    Full Text Available BACKGROUND: MALDI-TOF mass spectrometry (MS is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany and their usefulness for identifying brucellae from culture plates and blood cultures. METHODOLOGY/PRINCIPAL FINDINGS: We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis, and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct. CONCLUSIONS/SIGNIFICANCE: MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles.

  18. Utilidad de la espectrometría de masas MALDI-TOF en la identificación de bacterias anaerobias

    Directory of Open Access Journals (Sweden)

    Mariela S Zárate

    Full Text Available El análisis de espectrometría de masas mediante la metodología hoy conocida como MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry se ha convertido en un recurso de referencia para la identificación de microorganismos en microbiología clínica. No obstante, los datos relativos a algunos grupos de microorganismos son todavía controvertidos. El objetivo del presente estudio fue determinar la utilidad del MALDI-TOF MS para la identificación de aislamientos clínicos de bacterias anaerobias. Se analizaron 106 aislamientos de bacterias anaerobias mediante MALDI-TOF MS y por pruebas bioquímicas convencionales. En aquellos casos en los que la identificación por metodología convencional no era aplicable o frente a una discordancia de resultados entre las metodologías citadas, se realizó la secuenciación del gen 16S del ARNr. El método convencional y el MALDI-TOF MS coincidieron a nivel de género y especie en un 95,3 % de los casos considerando la totalidad de los aislamientos estudiados. Al considerar solo el conjunto de los bacilos gram negativos, la coincidencia fue del 91,4 %; entre los bacilos gram positivos, fue del 100 %; los 8 aislados de cocos gram positivos estudiados coincidieron y también hubo coincidencia en el único coco gram negativo incluido. Los datos obtenidos en este estudio demuestran que el MALDI-TOF MS ofrece la posibilidad de llegar a una adecuada identificación de bacterias anaerobias.

  19. Novel, improved sample preparation for rapid, direct identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

    Science.gov (United States)

    Schubert, Sören; Weinert, Kirsten; Wagner, Chris; Gunzl, Beatrix; Wieser, Andreas; Maier, Thomas; Kostrzewa, Markus

    2011-11-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for rapid and reliable identification of bacteria and yeast grown on agar plates. Moreover, MALDI-TOF MS also holds promise for bacterial identification from blood culture (BC) broths in hospital laboratories. The most important technical step for the identification of bacteria from positive BCs by MALDI-TOF MS is sample preparation to remove blood cells and host proteins. We present a method for novel, rapid sample preparation using differential lysis of blood cells. We demonstrate the efficacy and ease of use of this sample preparation and subsequent MALDI-TOF MS identification, applying it to a total of 500 aerobic and anaerobic BCs reported to be positive by a Bactec 9240 system. In 86.5% of all BCs, the microorganism species were correctly identified. Moreover, in 18/27 mixed cultures at least one isolate was correctly identified. A novel method that adjusts the score value for MALDI-TOF MS results is proposed, further improving the proportion of correctly identified samples. The results of the present study show that the MALDI-TOF MS-based method allows rapid (directly from positive BCs and with high accuracy. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  20. MALDI-TOF mass spectrometry following short incubation on a solid medium is a valuable tool for rapid pathogen identification from positive blood cultures.

    Science.gov (United States)

    Kohlmann, Rebekka; Hoffmann, Alexander; Geis, Gabriele; Gatermann, Sören

    2015-01-01

    Rapid identification of the causative microorganism is a key element in appropriate antimicrobial therapy of bloodstream infections. Whereas traditional analysis of positive blood cultures requires subculture over at least 16-24h prior to pathogen identification by, e.g. matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), sample preparation procedures enabling direct MALDI-TOF MS, i.e. without preceding subculture, are associated with additional effort and costs. Hence, we integrated an alternative MALDI-TOF MS approach in diagnostic routine using a short incubation on a solid medium. Positive blood cultures were routinely plated on chocolate agar plates and incubated for 4h (37 °C, 5% CO2). Subsequently, MALDI-TOF MS using a Microflex LT instrument (Bruker Daltonics) and direct smear method was performed once per sample. For successful identification of bacteria at species level, score cut-off values were used as proposed by the manufacturer (≥ 2.0) and in a modified form (≥ 1.5 for MALDI-TOF MS results referring to Gram-positive cocci and ≥ 1.7 for MALDI-TOF MS results referring to bacteria other than Gram-positive cocci). Further data analysis also included an assessment of the clinical impact of the MALDI-TOF MS result. Applying the modified score cut-off values, our approach led to an overall correct species identification in 69.5% with misidentification in 3.4% (original cut-offs: 49.2% and 1.8%, respectively); for Gram-positive cocci, correct identification in 68.4% (100% for Staphylococcus aureus and enterococci, 80% for beta-hemolytic streptococci), for Gram-negative bacteria, correct identification in 97.6%. In polymicrobial blood cultures, in 72.7% one of the pathogens was correctly identified. Results were not reliable for Gram-positive rods and yeasts. The approach was easy to implement in diagnostic routine. In cases with available clinical data and successful pathogen identification, in 51.1% our

  1. Proteome-based bacterial identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS): A revolutionary shift in clinical diagnostic microbiology.

    Science.gov (United States)

    Nomura, Fumio

    2015-06-01

    Rapid and accurate identification of microorganisms, a prerequisite for appropriate patient care and infection control, is a critical function of any clinical microbiology laboratory. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a quick and reliable method for identification of microorganisms, including bacteria, yeast, molds, and mycobacteria. Indeed, there has been a revolutionary shift in clinical diagnostic microbiology. In the present review, the state of the art and advantages of MALDI-TOF MS-based bacterial identification are described. The potential of this innovative technology for use in strain typing and detection of antibiotic resistance is also discussed. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Verification of Ribosomal Proteins of Aspergillus fumigatus for Use as Biomarkers in MALDI-TOF MS Identification.

    Science.gov (United States)

    Nakamura, Sayaka; Sato, Hiroaki; Tanaka, Reiko; Yaguchi, Takashi

    2016-01-01

    We have previously proposed a rapid identification method for bacterial strains based on the profiles of their ribosomal subunit proteins (RSPs), observed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method can perform phylogenetic characterization based on the mass of housekeeping RSP biomarkers, ideally calculated from amino acid sequence information registered in public protein databases. With the aim of extending its field of application to medical mycology, this study investigates the actual state of information of RSPs of eukaryotic fungi registered in public protein databases through the characterization of ribosomal protein fractions extracted from genome-sequenced Aspergillus fumigatus strains Af293 and A1163 as a model. In this process, we have found that the public protein databases harbor problems. The RSP names are in confusion, so we have provisionally unified them using the yeast naming system. The most serious problem is that many incorrect sequences are registered in the public protein databases. Surprisingly, more than half of the sequences are incorrect, due chiefly to mis-annotation of exon/intron structures. These errors could be corrected by a combination of in silico inspection by sequence homology analysis and MALDI-TOF MS measurements. We were also able to confirm conserved post-translational modifications in eleven RSPs. After these verifications, the masses of 31 expressed RSPs under 20,000 Da could be accurately confirmed. These RSPs have a potential to be useful biomarkers for identifying clinical isolates of A. fumigatus .

  3. Preparative isoelectric focusing of microorganisms in cellulose-based separation medium and subsequent analysis by CIEF and MALDI-TOF MS

    Czech Academy of Sciences Publication Activity Database

    Horká, Marie; Šlais, Karel; Šalplachta, Jiří; Růžička, F.

    2017-01-01

    Roč. 990, OCT (2017), s. 185-193 ISSN 0003-2670 R&D Projects: GA ČR(CZ) GA16-03749S; GA MV(CZ) VI20172020069; GA MZd(CZ) NV16-29916A Institutional support: RVO:68081715 Keywords : preparative isoelectric focusing * colored microorganisms * isoelectric points * CIEF and MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 4.950, year: 2016

  4. Evaluación de la espectrometría de masas: MALDI-TOF MS para la identificación rápida y confiable de levaduras

    Directory of Open Access Journals (Sweden)

    María S Relloso

    2015-06-01

    Full Text Available La espectrometría de masas, conocida como matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, es una técnica utilizada en la identificación de microorganismos mediante la creación de un espectro basado en el perfil de proteínas, que es único para una especie dada. El objetivo del presente trabajo fue evaluar la identificación de aislamientos clínicos de levaduras por MALDI-TOF MS en un hospital universitario de Argentina y analizar 2 procedimientos para la extracción de proteínas: el recomendado por el fabricante del equipo y una técnica abreviada rápida. Utilizando el primero de estos procedimientos se analizaron 201 aislamientos identificados previamente por métodos convencionales y se obtuvo coincidencia en la identificación a nivel de especie en el 95,38 % de los aislamientos analizados. Con 100 de estos aislamientos se utilizó, además, el procedimiento abreviado para la extracción de proteínas; se obtuvo una identificación correcta a nivel de género y especie en el 98,0 % de ellos. La espectrometría de masas MALDI-TOF MS demostró ser una técnica rápida, sencilla y precisa para la identificación de levaduras.

  5. Unusual analyte-matrix adduct ions and mechanism of their formation in MALDI TOF MS of benzene-1,3,5-tricarboxamide and urea compounds

    NARCIS (Netherlands)

    Lou, X.; Fransen, M.; Stals, P.J.M.; Mes, T.; Bovee, R.; Dongen, van J.L.J.; Meijer, E.W.

    2013-01-01

    Analyte-matrix adducts are normally absent under typical matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) conditions. Interestingly, though, in the analysis of several types of organic compounds synthesized in our laboratory, analyte-matrix adduct ion peaks

  6. The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria

    NARCIS (Netherlands)

    Veloo, A. C. M.; Elgersma, P. E.; Friedrich, A. W.; Nagy, E.; van Winkelhoff, A. J.

    2014-01-01

    With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the

  7. Insight into Identification of Acinetobacter Species by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) in the Clinical Laboratory

    Science.gov (United States)

    Li, Xiuyuan; Tang, Yanyan; Lu, Xinxin

    2018-04-01

    Currently, the capability of identification for Acinetobacter species using MALDI-TOF MS still remains unclear in clinical laboratories due to certain elusory phenomena. Thus, we conducted this research to evaluate this technique and reveal the causes of misidentification. Briefly, a total of 788 Acinetobacter strains were collected and confirmed at the species level by 16S rDNA and rpoB sequencing, and subsequently compared to the identification by MALDI-TOF MS using direct smear and bacterial extraction pretreatments. Cluster analysis was performed based on the mass spectra and 16S rDNA to reflect the diversity among different species. Eventually, 19 Acinetobacter species were confirmed, including 6 species unavailable in Biotyper 3.0 database. Another novel species was observed, temporarily named A. corallinus. The accuracy of identification for Acinetobacter species using MALDI-TOF MS was 97.08% (765/788), regardless of which pretreatment was applied. The misidentification only occurred on 3 A. parvus strains and 20 strains of species unavailable in the database. The proportions of strains with identification score ≥ 2.000 using direct smear and bacterial extraction pretreatments were 86.04% (678/788) and 95.43% (752/788), χ 2 = 41.336, P clinical samples was deemed reliable. Misidentification occurred occasionally due to the insufficiency of the database rather than sample extraction failure. We suggest gene sequencing should be performed when the identification score is under 2.000 even when using bacterial extraction pretreatment. [Figure not available: see fulltext.

  8. Collagen-based proteinaceous binder-pigment interaction study under UV ageing conditions by MALDI-TOF-MS and principal component analysis.

    Science.gov (United States)

    Romero-Pastor, Julia; Navas, Natalia; Kuckova, Stepanka; Rodríguez-Navarro, Alejandro; Cardell, Carolina

    2012-03-01

    This study focuses on acquiring information on the degradation process of proteinaceous binders due to ultra violet (UV) radiation and possible interactions owing to the presence of historical mineral pigments. With this aim, three different paint model samples were prepared according to medieval recipes, using rabbit glue as proteinaceus binders. One of these model samples contained only the binder, and the other two were prepared by mixing each of the pigments (cinnabar or azurite) with the binder (glue tempera model samples). The model samples were studied by applying Principal Component Analysis (PCA) to their mass spectra obtained with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). The complementary use of Fourier Transform Infrared Spectroscopy to study conformational changes of secondary structure of the proteinaceous binder is also proposed. Ageing effects on the model samples after up to 3000 h of UV irradiation were periodically analyzed by the proposed approach. PCA on MS data proved capable of identifying significant changes in the model samples, and the results suggested different aging behavior based on the pigment present. This research represents the first attempt to use this approach (PCA on MALDI-TOF-MS data) in the field of Cultural Heritage and demonstrates the potential benefits in the study of proteinaceous artistic materials for purposes of conservation and restoration. Copyright © 2012 John Wiley & Sons, Ltd.

  9. Rapid and reliable discrimination between Shigella species and Escherichia coli using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Paauw, Armand; Jonker, Debby; Roeselers, Guus; Heng, Jonathan M E; Mars-Groenendijk, Roos H; Trip, Hein; Molhoek, E Margo; Jansen, Hugo-Jan; van der Plas, Jan; de Jong, Ad L; Majchrzykiewicz-Koehorst, Joanna A; Speksnijder, Arjen G C L

    2015-01-01

    E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli. Copyright © 2015 Elsevier GmbH. All rights reserved.

  10. Mapping the dark space of chemical reactions with extended nanomole synthesis and MALDI-TOF MS.

    Science.gov (United States)

    Lin, Shishi; Dikler, Sergei; Blincoe, William D; Ferguson, Ronald D; Sheridan, Robert P; Peng, Zhengwei; Conway, Donald V; Zawatzky, Kerstin; Wang, Heather; Cernak, Tim; Davies, Ian W; DiRocco, Daniel A; Sheng, Huaming; Welch, Christopher J; Dreher, Spencer D

    2018-05-24

    Understanding the practical limitations of chemical reactions is critically important for efficiently planning the synthesis of compounds in pharmaceutical, agrochemical and specialty chemical research and development. However, literature reports of the scope of new reactions are often cursory and biased toward successful results, severely limiting the ability to predict reaction outcomes for untested substrates. We herein illustrate strategies for carrying out large scale surveys of chemical reactivity using a material-sparing nanomole-scale automated synthesis platform with greatly expanded synthetic scope combined with ultra-high throughput (uHT) matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Copyright © 2018, American Association for the Advancement of Science.

  11. The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus, BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS.

    Science.gov (United States)

    Almuhayawi, Mohammed; Altun, Osman; Abdulmajeed, Adam Dilshad; Ullberg, Måns; Özenci, Volkan

    2015-01-01

    Detection and identification of anaerobic bacteria in blood cultures (BC) is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux), BACTEC-Plus and -Lytic (Becton Dickinson BioSciences) BC bottles in detection and time to detection (TTD) of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94%) than BacT/ALERT FN Plus (80/100, 80%) (panaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD) and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (panaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.

  12. Direct identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) from positive blood culture bottles: An opportunity to customize growth conditions for fastidious organisms causing bloodstream infections.

    Science.gov (United States)

    Sharma, Megha; Gautam, Vikas; Mahajan, Monika; Rana, Sudesh; Majumdar, Manasi; Ray, Pallab

    2017-10-01

    Culture-negative bacteraemia has been an enigmatic entity with respect to its aetiological agents. In an attempt to actively identify those positive blood cultures that escape isolation and detection on routine workflow, an additional step of MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) based detection was carried out directly from the flagged blood culture bottles. Blood samples from 200 blood culture bottles that beeped positive with automated (BACTEC) system and showed no growth of organism on routine culture media, were subjected to analysis by MALDI-TOF MS. Forty seven of the 200 (23.5%) bacterial aetiology could be established by bottle-based method. Based on these results, growth on culture medium could be achieved for the isolates by providing special growth conditions to the fastidious organisms. Direct identification by MALDI-TOF MS from BACTEC-positive bottles provided an opportunity to isolate those fastidious organisms that failed to grow on routine culture medium by providing them with necessary alterations in growth environment.

  13. Characterization of Enterococcus species isolated from marine recreational waters by MALDI-TOF MS and Rapid ID API® 20 Strep system.

    Science.gov (United States)

    Christ, Ana Paula Guarnieri; Ramos, Solange Rodrigues; Cayô, Rodrigo; Gales, Ana Cristina; Hachich, Elayse Maria; Sato, Maria Inês Zanoli

    2017-05-15

    MALDI-TOF Mass Spectrometry Biotyping has proven to be a reliable method for identifying bacteria at the species level based on the analysis of the ribosomal proteins mass fingerprint. We evaluate the usefulness of this method to identify Enterococcus species isolated from marine recreational water at Brazilian beaches. A total of 127 Enterococcus spp. isolates were identified to species level by bioMérieux's API® 20 Strep and MALDI-TOF systems. The biochemical test identified 117/127 isolates (92%), whereas MALDI identified 100% of the isolates, with an agreement of 63% between the methods. The 16S rRNA gene sequencing of isolates with discrepant results showed that MALDI-TOF and API® correctly identified 74% and 11% of these isolates, respectively. This discrepancy probably relies on the bias of the API® has to identify clinical isolates. MALDI-TOF proved to be a feasible approach for identifying Enterococcus from environmental matrices increasing the rapidness and accuracy of results. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Fragmentation of organic ions bearing fixed multiple charges observed in MALDI MS.

    Science.gov (United States)

    Lou, Xianwen; Li, Bao; de Waal, Bas F M; Schill, Jurgen; Baker, Matthew B; Bovee, Ralf A A; van Dongen, Joost L J; Milroy, Lech-Gustav; Meijer, E W

    2018-01-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was used to analyze a series of synthetic organic ions bearing fixed multiple charges. Despite the multiple intrinsic charges, only singly charged ions were recorded in each case. In addition to the pseudo-molecular ions formed by counterion adduction, deprotonation and electron capture, a number of fragment ions were also observed. Charge splitting by fragmentation was found to be a viable route for charge reduction leading to the formation of the observed singly charged fragment ions. Unlike multivalent metal ions, organic ions can rearrange and/or fragment during charge reduction. This fragmentation process will evidently complicate the interpretation of the MALDI MS spectrum. Because MALDI MS is usually considered as a soft ionization technique, the fragment ion peaks can easily be erroneously interpreted as impurities. Therefore, the awareness and understanding of the underlying MALDI-induced fragmentation pathways is essential for a proper interpretation of the corresponding mass spectra. Due to the fragment ions generated during charge reduction, special care should be taken in the MALDI MS analysis of multiply charged ions. In this work, the possible mechanisms by which the organic ions bearing fixed multiple charges fragment are investigated. With an improved understanding of the fragmentation mechanisms, MALDI TOF MS should still be a useful technique for the characterization of organic ions with fixed multiple charges. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Differentiation of Clinically Relevant mucorales Rhizopus microsporus and R. arrhizus by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)

    NARCIS (Netherlands)

    Dolatabadi, S.; Kolecka, A.; Versteeg, Matthijs; de Hoog, Sybren G; Boekhout, Teun

    This study addresses the usefulness of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for reliable identification of the two most frequently occuring clinical species of Rhizopus, namely R. arrhizus with its two varieties arrhizus and delemar and R.

  16. Epidemiology of candidemia in Qatar, the Middle East : Performance of MALDI-TOF MS for the identification of Candida species, species distribution, outcome, and susceptibility pattern

    NARCIS (Netherlands)

    Taj-Aldeen, S. J.; Kolecka, A.; Boesten, R.; Alolaqi, A.; Almaslamani, M.; Chandra, P.; Meis, J. F.; Boekhout, T.

    Introduction Bloodstream infections (BSIs) due to Candida spp. constitute the predominant group of hospital-based fungal infections worldwide. A retrospective study evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the

  17. Direct identification of microorganisms from positive blood cultures using the lysis-filtration technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): a multicentre study.

    Science.gov (United States)

    Farina, Claudio; Arena, Fabio; Casprini, Patrizia; Cichero, Paola; Clementi, Massimo; Cosentino, Marina; Degl'Innocenti, Roberto; Giani, Tommaso; Luzzaro, Francesco; Mattei, Romano; Mauri, Carola; Nardone, Maria; Rossolini, Gian Maria; Serna Ortega, Paula Andrea; Vailati, Francesca

    2015-04-01

    Microbial identification from blood cultures is essential to institute optimal antibiotic therapy and improve survival possibilities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify bacteria and yeasts from positive blood cultures broths. The aim of this multicentre study was to evaluate the reliability of the lysis-filtration technique associated with MALDI-TOF MS to directly identify microorganisms from 765 positive blood cultures collected in six Italian hospitals. Overall, 675/765 (78.1%) blood isolates were correctly identified at the species level, with significant differences between Gram-negative and Gram-positive bacteria (92.6%, and 69.8%, respectively). Some difficulties arise in identifying Streptococcus pneumoniae, Staphylococcus aureus, yeasts and anaerobes. The lysis-filtration protocol is a suitable procedure in terms of performance in identifying microorganisms, but it is quite expensive and technically time-consuming since the time of filtration is not regular for all the samples. The application of the MALDI-TOF MS technique to the direct microbial identification from positive blood cultures is a very promising approach, even if more experience must be gained to minimize errors and costs.

  18. Characterisation of the aerobic bacterial flora of boid snakes: application of MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Plenz, Bastian; Schmidt, Volker; Grosse-Herrenthey, Anke; Krüger, Monika; Pees, Michael

    2015-03-14

    The aim of this study was to identify aerobic bacterial isolates from the respiratory tract of boids with matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). From 47 boid snakes, swabs from the oral cavity, tracheal wash samples and, in cases in which postmortem examination was performed, pulmonary tissue samples were taken. Each snake was classified as having inflammation of the respiratory tract and/or oral cavity, or without evidence of inflammation based on combination of clinical, cytological and histopathological findings. Samples collected from the respiratory tract and oral cavity were inoculated onto routine media and bacteria were cultured aerobically. All morphologically distinct individual colonies obtained were analysed using MALDI-TOF MS. Unidentified isolates detected in more than three snakes were selected for further 16S rDNA PCR and sequencing. Among all examined isolates (n=243), 49 per cent (n=119) could be sufficiently speciated using MALDI-TOF MS. Molecular biology revealed several bacterial species that have not been previously described in reptiles. With an average of 6.3 different isolates from the respiratory tract and/or oral cavity, boids with inflammatory disease harboured significantly more bacterial species than boids without inflammatory disease (average 2.8 isolates). British Veterinary Association.

  19. Heterotrophic monitoring at a drinking water treatment plant by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry after different drinking water treatments.

    Science.gov (United States)

    Sala-Comorera, Laura; Blanch, Anicet R; Vilaró, Carles; Galofré, Belén; García-Aljaro, Cristina

    2017-10-01

    The aim of this work was to assess the suitability of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for routine heterotrophic monitoring in a drinking water treatment plant. Water samples were collected from raw surface water and after different treatments during two campaigns over a 1-year period. Heterotrophic bacteria were studied and isolates were identified by MALDI-TOF MS. Moreover, the diversity index and the coefficient of population similarity were also calculated using biochemical fingerprinting of the populations studied. MALDI-TOF MS enabled us to characterize and detect changes in the bacterial community composition throughout the water treatment plant. Raw water showed a large and diverse population which was slightly modified after initial treatment steps (sand filtration and ultrafiltration). Reverse osmosis had a significant impact on the microbial diversity, while the final chlorination step produced a shift in the composition of the bacterial community. Although MALDI-TOF MS could not identify all the isolates since the available MALDI-TOF MS database does not cover all the bacterial diversity in water, this technique could be used to monitor bacterial changes in drinking water treatment plants by creating a specific protein profile database for tracking purposes.

  20. Custom database development and biomarker discovery methods for MALDI-TOF mass spectrometry-based identification of high-consequence bacterial pathogens.

    Science.gov (United States)

    Tracz, Dobryan M; Tyler, Andrea D; Cunningham, Ian; Antonation, Kym S; Corbett, Cindi R

    2017-03-01

    A high-quality custom database of MALDI-TOF mass spectral profiles was developed with the goal of improving clinical diagnostic identification of high-consequence bacterial pathogens. A biomarker discovery method is presented for identifying and evaluating MALDI-TOF MS spectra to potentially differentiate biothreat bacteria from less-pathogenic near-neighbour species. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  1. Independent component analysis for the extraction of reliable protein signal profiles from MALDI-TOF mass spectra.

    Science.gov (United States)

    Mantini, Dante; Petrucci, Francesca; Del Boccio, Piero; Pieragostino, Damiana; Di Nicola, Marta; Lugaresi, Alessandra; Federici, Giorgio; Sacchetta, Paolo; Di Ilio, Carmine; Urbani, Andrea

    2008-01-01

    Independent component analysis (ICA) is a signal processing technique that can be utilized to recover independent signals from a set of their linear mixtures. We propose ICA for the analysis of signals obtained from large proteomics investigations such as clinical multi-subject studies based on MALDI-TOF MS profiling. The method is validated on simulated and experimental data for demonstrating its capability of correctly extracting protein profiles from MALDI-TOF mass spectra. The comparison on peak detection with an open-source and two commercial methods shows its superior reliability in reducing the false discovery rate of protein peak masses. Moreover, the integration of ICA and statistical tests for detecting the differences in peak intensities between experimental groups allows to identify protein peaks that could be indicators of a diseased state. This data-driven approach demonstrates to be a promising tool for biomarker-discovery studies based on MALDI-TOF MS technology. The MATLAB implementation of the method described in the article and both simulated and experimental data are freely available at http://www.unich.it/proteomica/bioinf/.

  2. MALDI-TOF MS contribution to the diagnosis of Campylobacter rectus multiple skull base and brain abscesses

    Directory of Open Access Journals (Sweden)

    D. Martiny

    2017-09-01

    Full Text Available Campylobacter rectus is rarely associated with invasive infection. Both the isolation and the identification requirements of C. rectus are fastidious, probably contributing to an underestimation of its burden. We report the case of a 66-year-old man who developed several skull base and intracerebral abscesses after dental intervention. Campylobacter rectus was isolated from the brain biopsy. Within 45 minutes of reading the bacterial plate, the strain was accurately identified by MALDI-TOF MS. This rapid identification avoided the extra costs and delays present with 16S rRNA gene sequencing and allowed for a rapid confirmation of the adequacy of the empirical antibiotic treatment.

  3. Identification of Candida species isolated from vulvovaginitis in Mashhad, Iran by Use of MALDI-TOF MS

    Directory of Open Access Journals (Sweden)

    Majid Alizadeh

    2017-12-01

     Of the 65 isolates analyzed, 61 (93.8% were recognised by MALDI-TOF mass spectrometry and for four isolates (6.1% only not relabile identifications were achieved. In this study, the most frequently isolated species were Candida albicans (58.5%, followed by Candida tropicalis (16.9%, Candida glabrata (7.7%, Candida parapsilosis (7.7% and Candida guillermondii (3.1%.  Conclusion presented results demonstrate that the MALDI TOF mass spectrometry is a fast and reliable technique, and has the potential to replace conventional phenotypic identification of Candida species and other yeast strains routinely isolated in clinical microbiology laboratories.

  4. Direct identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS from positive blood culture bottles: An opportunity to customize growth conditions for fastidious organisms causing bloodstream infections

    Directory of Open Access Journals (Sweden)

    Megha Sharma

    2017-01-01

    Full Text Available Culture-negative bacteraemia has been an enigmatic entity with respect to its aetiological agents. In an attempt to actively identify those positive blood cultures that escape isolation and detection on routine workflow, an additional step of MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry based detection was carried out directly from the flagged blood culture bottles. Blood samples from 200 blood culture bottles that beeped positive with automated (BACTEC system and showed no growth of organism on routine culture media, were subjected to analysis by MALDI-TOF MS. Forty seven of the 200 (23.5% bacterial aetiology could be established by bottle-based method. Based on these results, growth on culture medium could be achieved for the isolates by providing special growth conditions to the fastidious organisms. Direct identification by MALDI-TOF MS from BACTEC-positive bottles provided an opportunity to isolate those fastidious organisms that failed to grow on routine culture medium by providing them with necessary alterations in growth environment.

  5. Identification of proteins of human colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    Ying-Tao Zhang; Yi-Ping Geng; Le Zhou; Bao-Chang Lai; Lv-Sheng Si; Yi-Li Wang

    2005-01-01

    AIM: To conduct the proteomic analysis of human colorectal carcinoma cell line, SW480 by using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption /ionization-time of flight mass spectrometry (MALDITOFMS).METHODS: The total proteins of human colorectal carcinoma cell line, SW480 were separated with 2-DE by using immobilized pH gradient strips and visualized by staining with silver nitrate. The gel images were acquired by scanner and 2-DE analysis software, Image Master 2D Elite. Nineteen distinct protein spots were excised from gel randomly and digested in gel by TPCK-trypsin. Mass analysis ofthe tryptic digest peptides mixture was performed by using MALDI-TOF MS. Peptide mass fingerprints (PMFs) obtained by the MALDI-TOF analysis were used to search NCBI,SWISS-PROT and MSDB databases by using Mascot software.RESULTS: PMF maps of all spots were obtained by MALDI-TOF MS and thirteen proteins were preliminarily identified.CONCLUSION: The methods of analysis and identification of protein spots of tumor cells in 2-DE gel with silver staining by MALDI-TOF MS derived PMF have been established.Protein expression profile of SW480 has been obtained.It is demonstrated that a combination of proteomics and cell culture is a useful approach to comprehend the process of colon carcinogenesis.

  6. HPLC, NMR and MALDI-TOF MS Analysis of Condensed Tannins from Lithocarpus glaber Leaves with Potent Free Radical Scavenging Activity

    OpenAIRE

    Zhang, Liang Liang; Lin, Yi Ming

    2008-01-01

    Using acid-catalyzed degradation in the presence of cysteamine, the condensed tannins from Lithocarpus glaber leaves were characterized, following thiolysis, by means of reversed-phase HPLC, 13C-NMR and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. The thiolysis reaction products showed the presence of the procyanidin (PC) and prodelphinidin (PD) structures. The 13C-NMR spectrum revealed that the condensed tannins were comprised of PD (7...

  7. Work flow analysis of around-the-clock processing of blood culture samples and integrated MALDI-TOF mass spectrometry analysis for the diagnosis of bloodstream infections.

    Science.gov (United States)

    Schneiderhan, Wilhelm; Grundt, Alexander; Wörner, Stefan; Findeisen, Peter; Neumaier, Michael

    2013-11-01

    Because sepsis has a high mortality rate, rapid microbiological diagnosis is required to enable efficient therapy. The effectiveness of MALDI-TOF mass spectrometry (MALDI-TOF MS) analysis in reducing turnaround times (TATs) for blood culture (BC) pathogen identification when available in a 24-h hospital setting has not been determined. On the basis of data from a total number of 912 positive BCs collected within 140 consecutive days and work flow analyses of laboratory diagnostics, we evaluated different models to assess the TATs for batch-wise and for immediate response (real-time) MALDI-TOF MS pathogen identification of positive BC results during the night shifts. The results were compared to TATs from routine BC processing and biochemical identification performed during regular working hours. Continuous BC incubation together with batch-wise MALDI-TOF MS analysis enabled significant reductions of up to 58.7 h in the mean TATs for the reporting of the bacterial species. The TAT of batch-wise MALDI-TOF MS analysis was inferior by a mean of 4.9 h when compared to the model of the immediate work flow under ideal conditions with no constraints in staff availability. Together with continuous cultivation of BC, the 24-h availability of MALDI-TOF MS can reduce the TAT for microbial pathogen identification within a routine clinical laboratory setting. Batch-wise testing of positive BC loses a few hours compared to real-time identification but is still far superior to classical BC processing. Larger prospective studies are required to evaluate the contribution of rapid around-the-clock pathogen identification to medical decision-making for septicemic patients.

  8. Isolation and Identification of Spoilage Yeasts in Wine Samples by MALDI-TOF MS Biotyper

    Directory of Open Access Journals (Sweden)

    Attila Kántor

    2015-05-01

    Full Text Available Many genera and species of microorganisms can be found in grape musts and wines at various times during the winemaking process. For instance, Saccharomyces, Brettanomyces, and Pediococcus can be found together in wine. There are many species of yeast involved in wine spoilage during storage. Aim of this study was to isolate the spoilage yeasts from wine samples with using special selective agar media and identified on species level by Matrix-Assisted Laser Desorption/Ionization-Time of Fly Mass Spectrometry (MALDI-TOF MS. Six red wines used in this study. We identified 10 yeast species from 152 isolates. The most common species in wine samples was Saccharomyces cerevisiae. We also identified four Candida species, two Zygosaccharomyces species and one species from genus Rhodotorula, Saccharomycodes and Dekkera.

  9. Evaluation of MALDI-TOF mass spectrometry for differentiation of Pichia kluyveri strains isolated from traditional fermentation processes.

    Science.gov (United States)

    De la Torre González, Francisco Javier; Gutiérrez Avendaño, Daniel Oswaldo; Gschaedler Mathis, Anne Christine; Kirchmayr, Manuel Reinhart

    2018-06-06

    Non- Saccharomyces yeasts are widespread microorganisms and some time ago were considered contaminants in the beverage industry. However, nowadays they have gained importance for their ability to produce aromatic compounds, which in alcoholic beverages improves aromatic complexity and therefore the overall quality. Thus, identification and differentiation of the species involved in fermentation processes is vital and can be classified in traditional methods and techniques based on molecular biology. Traditional methods, however, can be expensive, laborious and/or unable to accurately discriminate on strain level. In the present study, a total of 19 strains of Pichia kluyveri isolated from mezcal, tejuino and cacao fermentations were analyzed with rep-PCR fingerprinting and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparative analysis between MS spectra and rep-PCR patterns obtained from these strains showed a high similarity between both methods. However, minimal differences between the obtained rep-PCR and MALDI-TOF MS clusters could be observed. The data shown suggests that MALDI-TOF MS is a promising alternative technique for rapid, reliable and cost-effective differentiation of natives yeast strains isolated from different traditional fermented foods and beverages. This article is protected by copyright. All rights reserved.

  10. Candida "Psilosis" - electromigration techniques and MALDI-TOF mass spectrometry for phenotypical discrimination

    Czech Academy of Sciences Publication Activity Database

    Kubesová, Anna; Šalplachta, Jiří; Horká, Marie; Růžička, F.; Šlais, Karel

    2012-01-01

    Roč. 137, č. 8 (2012), s. 1937-1943 ISSN 0003-2654 R&D Projects: GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : Candida parapsilosis * electromigration techniques * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.969, year: 2012

  11. Weak cation magnetic separation technology and MALDI-TOF-MS in screening serum protein markers in primary type I osteoporosis.

    Science.gov (United States)

    Shi, X L; Li, C W; Liang, B C; He, K H; Li, X Y

    2015-11-30

    We investigated weak cation magnetic separation technology and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) in screening serum protein markers of primary type I osteoporosis. We selected 16 postmenopausal women with osteoporosis and nine postmenopausal women as controls to find a new method for screening biomarkers and establishing a diagnostic model for primary type I osteoporosis. Serum samples were obtained from controls and patients. Serum protein was extracted with the WCX protein chip system; protein fingerprints were examined using MALDI-TOF-MS. The preprocessed and model construction data were handled by the ProteinChip system. The diagnostic models were established using a genetic arithmetic model combined with a support vector machine (SVM). The SVM model with the highest Youden index was selected. Combinations with the highest accuracy in distinguishing different groups of data were selected as potential biomarkers. From the two groups of serum proteins, 123 cumulative MS protein peaks were selected. Significant intensity differences in the protein peaks of 16 postmenopausal women with osteoporosis were screened. The difference in Youden index between the four groups of protein peaks showed that the highest peaks had mass-to-charge ratios of 8909.047, 8690.658, 13745.48, and 15114.52. A diagnosis model was established with these four markers as the candidates, and the model specificity and sensitivity were found to be 100%. Two groups of specimens in the SVM results on the scatterplot were distinguishable. We established a diagnosis model, and provided a new serological method for screening and diagnosis of osteoporosis with high sensitivity and specificity.

  12. DBDA as a Novel Matrix for the Analyses of Small Molecules and Quantification of Fatty Acids by Negative Ion MALDI-TOF MS.

    Science.gov (United States)

    Ling, Ling; Li, Ying; Wang, Sheng; Guo, Liming; Xiao, Chunsheng; Chen, Xuesi; Guo, Xinhua

    2018-04-01

    Matrix interference ions in low mass range has always been a concern when using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze small molecules (matrix, N1,N4-dibenzylidenebenzene-1,4-diamine (DBDA) was synthesized for the analyses of small molecules by negative ion MALDI-TOF MS. Notably, only neat ions ([M-H] - ) of fatty acids without matrix interference appeared in the mass spectra and the limit of detection (LOD) reached 0.3 fmol. DBDA also has great performance towards other small molecules such as amino acids, peptides, and nucleotide. Furthermore, with this novel matrix, the free fatty acids in serum were quantitatively analyzed based on the correlation curves with correlation coefficient of 0.99. In addition, UV-Vis experiments and molecular orbital calculations were performed to explore mechanism about DBDA used as matrix in the negative ion mode. The present work shows that the DBDA matrix is a highly sensitive matrix with few interference ions for analysis of small molecules. Meanwhile, DBDA is able to precisely quantify the fatty acids in real biological samples. Graphical Abstract ᅟ.

  13. Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Julie Gagnaire

    Full Text Available The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF mass spectrometry (MS, correlate delta-toxin expression with accessory gene regulator (agr status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively. In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

  14. Coupling Sodium Dodecyl Sulfate–Capillary Polyacrylamide Gel Electrophoresis with MALDI-TOF-MS via a PTFE Membrane

    Science.gov (United States)

    Lu, Joann J.; Zhu, Zaifang; Wang, Wei; Liu, Shaorong

    2011-01-01

    Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) is a fundamental analytical technique for proteomic research, and SDS–capillary gel electrophoresis (CGE) is its miniaturized version. Compared to conventional slab-gel electrophoresis, SDS-CGE has many advantages such as increased separation efficiency, reduced separation time and automated operation. SDS-CGE is not widely accepted in proteomic research primarily due to the difficulties in identifying the well-resolved proteins. MALDI–TOF–MS is an outstanding platform for protein identifications. Coupling the two would solve the problem but is extremely challenging because the MS detector has no access to the SDS-CGE resolved proteins and the SDS interferes with MS detection. In this work we introduce an approach to address these issues. We discover that poly(tetrafluoroethylene) (PTFE) membranes are excellent materials for collecting SDS-CGE separated proteins. We demonstrate that we can wash off the SDS bound to the collected proteins and identify these proteins on-membrane with MALDI-TOF-MS. We also show that we can immunoblot and Coomassie-stain the proteins collected on these membranes. PMID:21309548

  15. The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus, BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS.

    Directory of Open Access Journals (Sweden)

    Mohammed Almuhayawi

    Full Text Available Detection and identification of anaerobic bacteria in blood cultures (BC is a well-recognized challenge in clinical microbiology. We studied 100 clinical anaerobic BC isolates to evaluate the performance of BacT/ALERT-FN, -FN Plus (BioMérieux, BACTEC-Plus and -Lytic (Becton Dickinson BioSciences BC bottles in detection and time to detection (TTD of anaerobic bacteria. BACTEC Lytic had higher detection rate (94/100, 94% than BacT/ALERT FN Plus (80/100, 80% (p<0.01 in the studied material. There was no significant difference in detection of anaerobic bacteria among the remaining bottle types. The 67 anaerobic bacteria that signalled positive in all four bottle types were analyzed to compare the time to detection (TTD and isolates were directly identified by MALDI-TOF MS. There was a significant difference in TTD among the four bottle types (p<0.0001. The shortest median TTD was 18 h in BACTEC Lytic followed by BacT/ALERT FN (23.5 h, BACTEC Plus (27 h and finally BacT/ALERT FN Plus (38 h bottles. In contrast, MALDI-TOF MS performed similarly in all bottle types with accurate identification in 51/67 (76% BacT/ALERT FN, 51/67 (76% BacT/ALERT FN Plus, 53/67 (79% BACTEC Plus and 50/67 (75% BACTEC Lytic bottles. In conclusion, BACTEC Lytic bottles have significantly better detection rates and shorter TTD compared to the three other bottle types. The anaerobic BC bottles are equally suitable for direct MALDI-TOF MS for rapid and reliable identification of common anaerobic bacteria. Further clinical studies are warranted to investigate the performance of anaerobic BC bottles in detection of anaerobic bacteria and identification by direct MALDI-TOF MS.

  16. Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium.

    Science.gov (United States)

    Idelevich, E A; Schüle, I; Grünastel, B; Wüllenweber, J; Peters, G; Becker, K

    2014-10-01

    Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

  17. Analysis of Phospholipid Mixtures from Biological Tissues by Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS): A Laboratory Experiment

    Science.gov (United States)

    Eibisch, Mandy; Fuchs, Beate; Schiller, Jurgen; Sub, Rosmarie; Teuber, Kristin

    2011-01-01

    Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used to investigate the phospholipid (PL) compositions of tissues and body fluids, often without previous separation of the total mixture into the individual PL classes. Therefore, the questions of whether all PL classes are detectable…

  18. The construction and evaluation of reference spectra for the identification of human pathogenic microorganisms by MALDI-TOF MS.

    Directory of Open Access Journals (Sweden)

    Di Xiao

    Full Text Available Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS is an emerging technique for the rapid and high-throughput identification of microorganisms. There remains a dearth of studies in which a large number of pathogenic microorganisms from a particular country or region are utilized for systematic analyses. In this study, peptide mass reference spectra (PMRS were constructed and evaluated from numerous human pathogens (a total of 1019 strains from 94 species, including enteric (46 species, respiratory (21 species, zoonotic (17 species, and nosocomial pathogens (10 species, using a MALDI-TOF MS Biotyper system (MBS. The PMRS for 380 strains of 52 species were new contributions to the original reference database (ORD. Compared with the ORD, the new reference database (NRD allowed for 28.2% (from 71.5% to 99.7% and 42.3% (from 51.3% to 93.6% improvements in identification at the genus and species levels, respectively. Misidentification rates were 91.7% and 57.1% lower with the NRD than with the ORD for genus and species identification, respectively. Eight genera and 25 species were misidentified. For genera and species that are challenging to accurately identify, identification results must be manually determined and adjusted in accordance with the database parameters. Through augmentation, the MBS demonstrated a high identification accuracy and specificity for human pathogenic microorganisms. This study sought to provide theoretical guidance for using PMRS databases in various fields, such as clinical diagnosis and treatment, disease control, quality assurance, and food safety inspection.

  19. An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS.

    Science.gov (United States)

    Loonen, A J M; Jansz, A R; Stalpers, J; Wolffs, P F G; van den Brule, A J C

    2012-07-01

    Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker's MALDI Sepsityper kit, the commercially available Molzym's MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.

  20. Validation of a for anaerobic bacteria optimized MALDI-TOF MS biotyper database: The ENRIA project.

    Science.gov (United States)

    Veloo, A C M; Jean-Pierre, H; Justesen, U S; Morris, T; Urban, E; Wybo, I; Kostrzewa, M; Friedrich, A W

    2018-03-12

    Within the ENRIA project, several 'expertise laboratories' collaborated in order to optimize the identification of clinical anaerobic isolates by using a widely available platform, the Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Main Spectral Profiles (MSPs) of well characterized anaerobic strains were added to one of the latest updates of the Biotyper database db6903; (V6 database) for common use. MSPs of anaerobic strains nominated for addition to the Biotyper database are included in this validation. In this study, we validated the optimized database (db5989 [V5 database] + ENRIA MSPs) using 6309 anaerobic isolates. Using the V5 database 71.1% of the isolates could be identified with high confidence, 16.9% with low confidence and 12.0% could not be identified. Including the MSPs added to the V6 database and all MSPs created within the ENRIA project, the amount of strains identified with high confidence increased to 74.8% and 79.2%, respectively. Strains that could not be identified using MALDI-TOF MS decreased to 10.4% and 7.3%, respectively. The observed increase in high confidence identifications differed per genus. For Bilophila wadsworthia, Prevotella spp., gram-positive anaerobic cocci and other less commonly encountered species more strains were identified with higher confidence. A subset of the non-identified strains (42.1%) were identified using 16S rDNA gene sequencing. The obtained identities demonstrated that strains could not be identified either due to the generation of spectra of insufficient quality or due to the fact that no MSP of the encountered species was present in the database. Undoubtedly, the ENRIA project has successfully increased the number of anaerobic isolates that can be identified with high confidence. We therefore recommend further expansion of the database to include less frequently isolated species as this would also allow us to gain valuable insight into the clinical

  1. Typing of vancomycin-resistant enterococci with MALDI-TOF mass spectrometry in a nosocomial outbreak setting.

    Science.gov (United States)

    Holzknecht, B J; Dargis, R; Pedersen, M; Pinholt, M; Christensen, J J

    2018-03-23

    To investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm). Fifty-five VREfm isolates, previously characterized by whole-genome sequencing (WGS), were included and analysed by MALDI-TOF MS. To take peak reproducibility into account, ethanol/formic acid extraction and other steps of the protocol were conducted in triplicate. Twenty-seven spectra were generated per isolate, and spectra were visually inspected to determine discriminatory peaks. The presence or absence of these was recorded in a peak scheme. Nine discriminatory peaks were identified. A characteristic pattern of these could distinguish between the three major WGS groups: WGS I, WGS II and WGS III. Only one of 38 isolates belonging to WGS I, WGS II or WGS III was misclassified. However, ten of the 17 isolates not belonging to WGS I, II or III displayed peak patterns indistinguishable from those of the outbreak strain. Using visual inspection of spectra, MALDI-TOF MS typing proved to be useful in differentiating three VREfm outbreak clones from each other. However, as non-outbreak isolates could not be reliably differentiated from outbreak clones, the practical value of this typing method for VREfm outbreak management was limited in our setting. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  2. MALDI-TOF MS is more accurate than VITEK II ANC card and API Rapid ID 32 A system for the identification of Clostridium species.

    Science.gov (United States)

    Kim, Young Jin; Kim, Si Hyun; Park, Hyun-Jung; Park, Hae-Geun; Park, Dongchul; Song, Sae Am; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kook, Joong-Ki; Kim, Hye Ran; Shin, Jeong Hwan

    2016-08-01

    All 50 Clostridium difficile strains were definitely identified by Vitek2 system, Rapid ID 32A system, and MALDI-TOF. For 18 non-difficile Clostridium strains, the identification results were correct in 0, 2, and 17 strains by Vitek2, Rapid ID 32A, and MALDI-TOF, respectively. MALDI-TOF could be used as the primary tool for identification of Clostridium species. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Identification of protein markers for the occurrence of defrosted material in milk through a MALDI-TOF-MS profiling approach.

    Science.gov (United States)

    Arena, Simona; Salzano, Anna Maria; Scaloni, Andrea

    2016-09-16

    Mozzarella di Bufala Campana is a soft, stretched curd Italian cheese made from fresh buffalo milk that obtained the Protected Designation of Origin (PDO) registration in EU legislation. Seasonality of buffalo milk production, rapid cheese decay and transport of its preserving liquid have relevant practical/economic consequences for mozzarella production; consequently, a progressive diffusion of cheese products realized with frozen curd or frozen milk has recently been observed. In order to meet the demand of the dairy producers and consumers for a reduction of starting material adulterations and for the certification of the raw milk used for cheese manufacturing, we have developed a rapid/robust MALDI-TOF-MS polypeptide profiling procedure that assays material quality through the identification of specific markers of its freshness. Massive analysis of fresh and frozen buffalo milks (stored for different times) was realized to this purpose; a tough statistical evaluation of the resulting data ultimately permitted the typing of milk samples. We identified 28 polypeptide markers of the milk freezing storage, among which 13 and 15 showed down- and over-representation, respectively. Quantitative data were confirmed by an independent analytical approach on selected markers. GLYCAM1-derived phosphopeptides (1-53), β-casein-derived phosphopeptides (1-68), β-casein-derived γ2-, γ3- and γ4-fragments, α-lactalbumin and β-lactoglobulin were components showing the highest significance. The occurrence of the first compounds in buffalo milk is here described for the first time; their formation in the frozen material was ascribed to the activity of plasmin or of unknown bacterial proteases/peptidases stable at low temperatures. In conclusion, data reported here suggest the application of this MALDI-TOF-MS polypeptide profiling platform to other high-quality dairy productions, in which milk freshness has important consequences on final product organoleptic properties. In

  4. Typing of vancomycin-resistant enterococci with MALDI-TOF mass spectrometry in a nosocomial outbreak setting

    DEFF Research Database (Denmark)

    Holzknecht, B J; Dargis, R; Pedersen, M

    2018-01-01

    OBJECTIVES: To investigate the usefulness of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) typing as a first-line epidemiological tool in a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VREfm). METHODS: Fifty-five VREfm isolates...

  5. Usefulness of MALDI-TOF mass spectrometry in epidemiological control of etiologic agents of infection

    Directory of Open Access Journals (Sweden)

    Roberto Degl’Innocenti

    2013-08-01

    Full Text Available Introduction: The use of the MALDI-TOF mass spectrometry in the routine of microbiological diagnostics has revolutionized procedures and response times of bacteriology.The use of this technique aims to epidemiological investigations in a hospital environment and represents a further significant opportunity, quickly feasible and extremely economical. Methods: By means of the MALDI-TOF-MS Vitek2 (MS Vitek2 mass spectrometer, accompanied by the AgnosTec-SARAMIS (bioMérieux, France software, were analyzed the spectra of 149 bacterial isolates (139 Staphylococcus aureus and 10 Staphylococcus epidermidis obtained from cultures of 148 patients (141 inpatients and 7 outpatients. Clinical isolates were stored at a temperature of -20°C.The isolates were then thawed and immediately cultured on agar blood medium. The colonies were subjected to analysis by MS Vitek on the day after sowing. The spectra obtained were analyzed and compared using the software AgnosTec-SARAMIS, which allowed the construction of a dendrogram. Results and conclusions: The evaluation of the data collected suggests that mass spectrometry could be an useful tool in epidemiological surveys. Speed of analysis and low costs make the MS Vitek2 an usable tool by many microbiology laboratories.

  6. Use of ribosomal proteins as biomarkers for identification of Flavobacterium psychrophilum by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Fernández-Álvarez, Clara; Torres-Corral, Yolanda; Santos, Ysabel

    2018-01-06

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a rapid methodology for identification of bacteria that is increasingly used in diagnostic laboratories. This work aimed at evaluating the potential of MALDI-TOF-MS for identification of the main serotypes of Flavobacterium psychrophilum isolated from salmonids, and its discrimination from closely related Flavobacterium spp. A mass spectra library was constructed by analysing 70 F. psychrophilum strains representing the serotypes O1, O2a, O2b and O3, including reference and clinical isolates. Peak mass lists were examined using the Mass-Up software for the detection of potential biomarkers, similarity and cluster analysis. Fourteen species-identifying biomarkers were detected in all the F. psychrophilum isolates tested, moreover, sets of serotype-identifying biomarkers ions were selected. F. psychrophilum-specific biomarkers were identified as ribosomal proteins by matching with protein databases. Furthermore, sequence variation corresponding to amino acid exchanges in several biomarker proteins were tentatively assigned. Closely related Flavobacterium species (F. flevense, F. succinicans, F. columnare, F. branchiophilum and F. johnsoniae) could be differentiated from F. psychrophilum by defining species identifying biomarkers and hierarchical cluster analysis. These results demonstrated that MALDI-TOF spectrometry represents a powerful tool for an accurate identification of the fish pathogen F. psychrophilum as well as for epidemiological studies. The results obtained in this study demonstrated that MALDI-TOF mass spectrometry represents a powerful tool that can be used by diagnostic laboratories for rapid identification of the fish pathogen Flavobacterium psychrophilum and its differentiation from other Flavobacterium-related species. Analysis of mass peak lists revealed the potential of the MALDI-TOF technique to identify epidemiologically important serotypes affecting

  7. MALDI-TOF MS Analysis of Condensed Tannins with Potent Antioxidant Activity from the Leaf, Stem Bark and Root Bark of Acacia confusa

    OpenAIRE

    Wei; Zhou; Lin; Liao; Chai

    2010-01-01

    The structures of the condensed tannins from leaf, stem bark and root bark of Acacia confusa were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and their antioxidant activities were measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and ferric reducing/antioxidant power (FRAP) assays. The results showed that the condensed tannins from stem bark and root bark include propelargonidin and procyanidi...

  8. Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit™ for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital

    OpenAIRE

    Tadros, Manal; Petrich, Astrid

    2013-01-01

    Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit – the Sepsityper Kit (Bruker Daltonik, Germany) – and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in com...

  9. MALDI-TOF Mass Spectrometry Enables a Comprehensive and Fast Analysis of Dynamics and Qualities of Stress Responses of Lactobacillus paracasei subsp. paracasei F19

    Science.gov (United States)

    Schott, Ann-Sophie; Behr, Jürgen; Quinn, Jennifer; Vogel, Rudi F.

    2016-01-01

    Lactic acid bacteria (LAB) are widely used as starter cultures in the manufacture of foods. Upon preparation, these cultures undergo various stresses resulting in losses of survival and fitness. In order to find conditions for the subsequent identification of proteomic biomarkers and their exploitation for preconditioning of strains, we subjected Lactobacillus (Lb.) paracasei subsp. paracasei TMW 1.1434 (F19) to different stress qualities (osmotic stress, oxidative stress, temperature stress, pH stress and starvation stress). We analysed the dynamics of its stress responses based on the expression of stress proteins using MALDI-TOF mass spectrometry (MS), which has so far been used for species identification. Exploiting the methodology of accumulating protein expression profiles by MALDI-TOF MS followed by the statistical evaluation with cluster analysis and discriminant analysis of principle components (DAPC), it was possible to monitor the expression of low molecular weight stress proteins, identify a specific time point when the expression of stress proteins reached its maximum, and statistically differentiate types of adaptive responses into groups. Above the specific result for F19 and its stress response, these results demonstrate the discriminatory power of MALDI-TOF MS to characterize even dynamics of stress responses of bacteria and enable a knowledge-based focus on the laborious identification of biomarkers and stress proteins. To our knowledge, the implementation of MALDI-TOF MS protein profiling for the fast and comprehensive analysis of various stress responses is new to the field of bacterial stress responses. Consequently, we generally propose MALDI-TOF MS as an easy and quick method to characterize responses of microbes to different environmental conditions, to focus efforts of more elaborate approaches on time points and dynamics of stress responses. PMID:27783652

  10. MALDI-TOF Mass Spectrometry Enables a Comprehensive and Fast Analysis of Dynamics and Qualities of Stress Responses of Lactobacillus paracasei subsp. paracasei F19.

    Directory of Open Access Journals (Sweden)

    Ann-Sophie Schott

    Full Text Available Lactic acid bacteria (LAB are widely used as starter cultures in the manufacture of foods. Upon preparation, these cultures undergo various stresses resulting in losses of survival and fitness. In order to find conditions for the subsequent identification of proteomic biomarkers and their exploitation for preconditioning of strains, we subjected Lactobacillus (Lb. paracasei subsp. paracasei TMW 1.1434 (F19 to different stress qualities (osmotic stress, oxidative stress, temperature stress, pH stress and starvation stress. We analysed the dynamics of its stress responses based on the expression of stress proteins using MALDI-TOF mass spectrometry (MS, which has so far been used for species identification. Exploiting the methodology of accumulating protein expression profiles by MALDI-TOF MS followed by the statistical evaluation with cluster analysis and discriminant analysis of principle components (DAPC, it was possible to monitor the expression of low molecular weight stress proteins, identify a specific time point when the expression of stress proteins reached its maximum, and statistically differentiate types of adaptive responses into groups. Above the specific result for F19 and its stress response, these results demonstrate the discriminatory power of MALDI-TOF MS to characterize even dynamics of stress responses of bacteria and enable a knowledge-based focus on the laborious identification of biomarkers and stress proteins. To our knowledge, the implementation of MALDI-TOF MS protein profiling for the fast and comprehensive analysis of various stress responses is new to the field of bacterial stress responses. Consequently, we generally propose MALDI-TOF MS as an easy and quick method to characterize responses of microbes to different environmental conditions, to focus efforts of more elaborate approaches on time points and dynamics of stress responses.

  11. Ribosomal subunit protein typing using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification and discrimination of Aspergillus species.

    Science.gov (United States)

    Nakamura, Sayaka; Sato, Hiroaki; Tanaka, Reiko; Kusuya, Yoko; Takahashi, Hiroki; Yaguchi, Takashi

    2017-04-26

    Accurate identification of Aspergillus species is a very important subject. Mass spectral fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is generally employed for the rapid identification of fungal isolates. However, the results are based on simple mass spectral pattern-matching, with no peak assignment and no taxonomic input. We propose here a ribosomal subunit protein (RSP) typing technique using MALDI-TOF MS for the identification and discrimination of Aspergillus species. The results are concluded to be phylogenetic in that they reflect the molecular evolution of housekeeping RSPs. The amino acid sequences of RSPs of genome-sequenced strains of Aspergillus species were first verified and compared to compile a reliable biomarker list for the identification of Aspergillus species. In this process, we revealed that many amino acid sequences of RSPs (about 10-60%, depending on strain) registered in the public protein databases needed to be corrected or newly added. The verified RSPs were allocated to RSP types based on their mass. Peak assignments of RSPs of each sample strain as observed by MALDI-TOF MS were then performed to set RSP type profiles, which were then further processed by means of cluster analysis. The resulting dendrogram based on RSP types showed a relatively good concordance with the tree based on β-tubulin gene sequences. RSP typing was able to further discriminate the strains belonging to Aspergillus section Fumigati. The RSP typing method could be applied to identify Aspergillus species, even for species within section Fumigati. The discrimination power of RSP typing appears to be comparable to conventional β-tubulin gene analysis. This method would therefore be suitable for species identification and discrimination at the strain to species level. Because RSP typing can characterize the strains within section Fumigati, this method has potential as a powerful and reliable tool in

  12. Potential of MALDI-TOF mass spectrometry as a rapid detection technique in plant pathology: identification of plant-associated microorganisms.

    Science.gov (United States)

    Ahmad, Faheem; Babalola, Olubukola O; Tak, Hamid I

    2012-09-01

    Plant diseases caused by plant pathogens substantially reduce crop production every year, resulting in massive economic losses throughout the world. Accurate detection and identification of plant pathogens is fundamental to plant pathogen diagnostics and, thus, plant disease management. Diagnostics and disease-management strategies require techniques to enable simultaneous detection and quantification of a wide range of pathogenic and non-pathogenic microorganisms. Over the past decade, rapid development of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for characterization of microorganisms has enabled substantially improved detection and identification of microorganisms. In the biological sciences, MALDI-TOF MS is used to analyze specific peptides or proteins directly desorbed from intact bacteria, fungal spores, nematodes, and other microorganisms. The ability to record biomarker ions, in a broad m/z range, which are unique to and representative of individual microorganisms, forms the basis of taxonomic identification of microorganisms by MALDI-TOF MS. Recent advances in mass spectrometry have initiated new research, i.e. analysis of more complex microbial communities. Such studies are just beginning but have great potential for elucidation not only of the interactions between microorganisms and their host plants but also those among different microbial taxa living in association with plants. There has been a recent effort by the mass spectrometry community to make data from large scale mass spectrometry experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MALDI-TOF MS as a universal technique for detection of plant pathogens and non-pathogens. The effects of experimental conditions are sufficiently understood, reproducible spectra can be obtained from computational database search, and microorganisms can be rapidly characterized by genus, species

  13. The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond.

    Science.gov (United States)

    Haslam, Carl; Hellicar, John; Dunn, Adrian; Fuetterer, Arne; Hardy, Neil; Marshall, Peter; Paape, Rainer; Pemberton, Michelle; Resemannand, Anja; Leveridge, Melanie

    2016-02-01

    Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays. © 2015 Society for Laboratory Automation and Screening.

  14. Use of MALDI-TOF Mass Spectrometry for the Fast Identification of Gram-Positive Fish Pathogens

    Science.gov (United States)

    Assis, Gabriella B. N.; Pereira, Felipe L.; Zegarra, Alexandra U.; Tavares, Guilherme C.; Leal, Carlos A.; Figueiredo, Henrique C. P.

    2017-01-01

    Gram-positive cocci, such as Streptococcus agalactiae, Lactococcus garvieae, Streptococcus iniae, and Streptococcus dysgalactiae subsp. dysgalactiae, are found throughout the world, particularly in outbreaks in farmed fish, and are thus associated with high economic losses, especially in the cultivation of Nile Tilapia. The aim of this study was to evaluate the efficacy of matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as an alternative for the diagnosis of these pathogens. One hundred and thirty-one isolates from Brazilian outbreaks assisted by the national authority were identified using a MALDI Biotyper from Bruker Daltonics. The results showed an agreement with respect to identification (Kappa = 1) between this technique and 16S ribosomal RNA gene sequencing for S. agalactiae and L. garvieae. However, for S. iniae and S. dysgalactiae subsp. dysgalactiae, perfect agreement was only achieved after the creation of a custom main spectra profile, as well as further comparisons with 16S ribosomal RNA and multilocus sequence analysis. MALDI-TOF MS was shown to be an efficient technology for the identification of these Gram-positive pathogens, yielding a quick and precise diagnosis. PMID:28848512

  15. Evaluation of MALDI-TOF mass spectrometry and Sepsityper Kit™ for the direct identification of organisms from sterile body fluids in a Canadian pediatric hospital.

    Science.gov (United States)

    Tadros, Manal; Petrich, Astrid

    2013-01-01

    Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit - the Sepsityper Kit (Bruker Daltonik, Germany) - and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2%) were blood culture specimens and seven (8.7%) were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8%) and 39 of 50 (78%) blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%. MALDI-TOF MS is a promising tool for the direct identification of organisms cultured from sterile sites.

  16. An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS

    NARCIS (Netherlands)

    M.Sc. A. Jansz; Dr. A.J.C. van den Brule, van den; Dr. P.F.G. Wolffs; Ing J. Stalpers; Drs A.J.M. Loonen

    2011-01-01

    Matrix-assisted laser desorption/ionisation time of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three

  17. MALDI-TOF MS typing enables the classification of brewing yeasts of the genus Saccharomyces to major beer styles.

    Science.gov (United States)

    Lauterbach, Alexander; Usbeck, Julia C; Behr, Jürgen; Vogel, Rudi F

    2017-01-01

    Brewing yeasts of the genus Saccharomyces are either available from yeast distributor centers or from breweries employing their own "in-house strains". During the last years, the classification and characterization of yeasts of the genus Saccharomyces was achieved by using biochemical and DNA-based methods. The current lack of fast, cost-effective and simple methods to classify brewing yeasts to a beer type, may be closed by Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) upon establishment of a database based on sub-proteome spectra from reference strains of brewing yeasts. In this study an extendable "brewing yeast" spectra database was established including 52 brewing yeast strains of the most important types of bottom- and top-fermenting strains as well as beer-spoiling S. cerevisiae var. diastaticus strains. 1560 single spectra, prepared with a standardized sample preparation method, were finally compared against the established database and investigated by bioinformatic analyses for similarities and distinctions. A 100% separation between bottom-, top-fermenting and S. cerevisiae var. diastaticus strains was achieved. Differentiation between Alt and Kölsch strains was not achieved because of the high similarity of their protein patterns. Whereas the Ale strains show a high degree of dissimilarity with regard to their sub-proteome. These results were supported by MDS and DAPC analysis of all recorded spectra. Within five clusters of beer types that were distinguished, and the wheat beer (WB) cluster has a clear separation from other groups. With the establishment of this MALDI-TOF MS spectra database proof of concept is provided of the discriminatory power of this technique to classify brewing yeasts into different major beer types in a rapid, easy way, and focus brewing trails accordingly. It can be extended to yeasts for specialty beer types and other applications including wine making or baking.

  18. MALDI-TOF MS typing enables the classification of brewing yeasts of the genus Saccharomyces to major beer styles

    Science.gov (United States)

    Lauterbach, Alexander; Usbeck, Julia C.; Behr, Jürgen

    2017-01-01

    Brewing yeasts of the genus Saccharomyces are either available from yeast distributor centers or from breweries employing their own “in-house strains”. During the last years, the classification and characterization of yeasts of the genus Saccharomyces was achieved by using biochemical and DNA-based methods. The current lack of fast, cost-effective and simple methods to classify brewing yeasts to a beer type, may be closed by Matrix Assisted Laser Desorption/Ionization–Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) upon establishment of a database based on sub-proteome spectra from reference strains of brewing yeasts. In this study an extendable “brewing yeast” spectra database was established including 52 brewing yeast strains of the most important types of bottom- and top-fermenting strains as well as beer-spoiling S. cerevisiae var. diastaticus strains. 1560 single spectra, prepared with a standardized sample preparation method, were finally compared against the established database and investigated by bioinformatic analyses for similarities and distinctions. A 100% separation between bottom-, top-fermenting and S. cerevisiae var. diastaticus strains was achieved. Differentiation between Alt and Kölsch strains was not achieved because of the high similarity of their protein patterns. Whereas the Ale strains show a high degree of dissimilarity with regard to their sub-proteome. These results were supported by MDS and DAPC analysis of all recorded spectra. Within five clusters of beer types that were distinguished, and the wheat beer (WB) cluster has a clear separation from other groups. With the establishment of this MALDI-TOF MS spectra database proof of concept is provided of the discriminatory power of this technique to classify brewing yeasts into different major beer types in a rapid, easy way, and focus brewing trails accordingly. It can be extended to yeasts for specialty beer types and other applications including wine making or baking. PMID

  19. MALDI-TOF MS typing enables the classification of brewing yeasts of the genus Saccharomyces to major beer styles.

    Directory of Open Access Journals (Sweden)

    Alexander Lauterbach

    Full Text Available Brewing yeasts of the genus Saccharomyces are either available from yeast distributor centers or from breweries employing their own "in-house strains". During the last years, the classification and characterization of yeasts of the genus Saccharomyces was achieved by using biochemical and DNA-based methods. The current lack of fast, cost-effective and simple methods to classify brewing yeasts to a beer type, may be closed by Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS upon establishment of a database based on sub-proteome spectra from reference strains of brewing yeasts. In this study an extendable "brewing yeast" spectra database was established including 52 brewing yeast strains of the most important types of bottom- and top-fermenting strains as well as beer-spoiling S. cerevisiae var. diastaticus strains. 1560 single spectra, prepared with a standardized sample preparation method, were finally compared against the established database and investigated by bioinformatic analyses for similarities and distinctions. A 100% separation between bottom-, top-fermenting and S. cerevisiae var. diastaticus strains was achieved. Differentiation between Alt and Kölsch strains was not achieved because of the high similarity of their protein patterns. Whereas the Ale strains show a high degree of dissimilarity with regard to their sub-proteome. These results were supported by MDS and DAPC analysis of all recorded spectra. Within five clusters of beer types that were distinguished, and the wheat beer (WB cluster has a clear separation from other groups. With the establishment of this MALDI-TOF MS spectra database proof of concept is provided of the discriminatory power of this technique to classify brewing yeasts into different major beer types in a rapid, easy way, and focus brewing trails accordingly. It can be extended to yeasts for specialty beer types and other applications including wine making or baking.

  20. False positives in MALDI-TOF detection of ERβ in mitochondria

    International Nuclear Information System (INIS)

    Schwend, Thomas; Gustafsson, Jan-Ake

    2006-01-01

    Recently, Yang et al. reported that estrogen receptor beta (ERβ) is a mitochondrial protein rather than a nuclear receptor. Because this claim would lead to a significant change in our understanding of estrogen signaling, we have attempted to reproduce the MALDI-TOF data of Yang et al. We separated proteins extracted from mouse liver mitochondria by SDS-PAGE and analysed a gel band covering the molecular weight range of 50-65 kDa by MALDI-TOF/TOF. Analysis of the data with the MASCOT database algorithm provided no evidence for the presence of ERβ in the mitochondria. If we search (as the authors did) with only the peptide masses which match to tryptic fragments of ERβ, ERβ is identified with a significant score of 69. However, fragmentation of these peptides shows that they are not from ERβ. Our conclusion is that ERβ cannot be identified by MALDI-TOF from a mixture of mitochondrial proteins resolved on SDS-PAGE

  1. A Silicon Nanomembrane Detector for Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of Large Proteins

    OpenAIRE

    Park, Jonghoo; Blick, Robert

    2013-01-01

    We describe a MALDI-TOF ion detector based on freestanding silicon nanomembrane technology. The detector is tested in a commercial MALDI-TOF mass spectrometer with equimolar mixtures of proteins. The operating principle of the nanomembrane detector is based on phonon-assisted field emission from these silicon nanomembranes, in which impinging ion packets excite electrons in the nanomembrane to higher energy states. Thereby the electrons can overcome the vacuum barrier and escape from the surf...

  2. Timeframe Dependent Fragment Ions Observed in In-Source Decay Experiments with β-Casein Using MALDI MS.

    Science.gov (United States)

    Sekiya, Sadanori; Nagoshi, Keishiro; Iwamoto, Shinichi; Tanaka, Koichi; Takayama, Mitsuo

    2015-09-01

    The fragment ions observed with time-of-flight (TOF) and quadrupole ion trap (QIT) TOF mass spectrometers (MS) combined with matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiments of phosphorylated analytes β-casein and its model peptide were compared from the standpoint of the residence timeframe of analyte and fragment ions in the MALDI ion source and QIT cell. The QIT-TOF MS gave fragment c-, z'-, z-ANL, y-, and b-ions, and further degraded fragments originating from the loss of neutrals such as H(2)O, NH(3), CH(2)O (from serine), C2H4O (from threonine), and H(3)PO(4), whereas the TOF MS merely showed MALDI source-generated fragment c-, z'-, z-ANL, y-, and w-ions. The fragment ions observed in the QIT-TOF MS could be explained by the injection of the source-generated ions into the QIT cell or a cooperative effect of a little internal energy deposition, a long residence timeframe (140 ms) in the QIT cell, and specific amino acid effects on low-energy CID, whereas the source-generated fragments (c-, z'-, z-ANL, y-, and w-ions) could be a result of prompt radical-initiated fragmentation of hydrogen-abundant radical ions [M + H + H](+) and [M + H - H](-) within the 53 ns timeframe, which corresponds to the delayed extraction time. The further degraded fragment b/y-ions produced in the QIT cell were confirmed by positive- and negative-ion low-energy CID experiments performed on the source-generated ions (c-, z'-, and y-ions). The loss of phosphoric acid (98 u) from analyte and fragment ions can be explained by a slow ergodic fragmentation independent of positive and negative charges.

  3. Timeframe Dependent Fragment Ions Observed in In-Source Decay Experiments with β-Casein Using MALDI MS

    Science.gov (United States)

    Sekiya, Sadanori; Nagoshi, Keishiro; Iwamoto, Shinichi; Tanaka, Koichi; Takayama, Mitsuo

    2015-09-01

    The fragment ions observed with time-of-flight (TOF) and quadrupole ion trap (QIT) TOF mass spectrometers (MS) combined with matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiments of phosphorylated analytes β-casein and its model peptide were compared from the standpoint of the residence timeframe of analyte and fragment ions in the MALDI ion source and QIT cell. The QIT-TOF MS gave fragment c-, z'-, z-ANL, y-, and b-ions, and further degraded fragments originating from the loss of neutrals such as H2O, NH3, CH2O (from serine), C2H4O (from threonine), and H3PO4, whereas the TOF MS merely showed MALDI source-generated fragment c-, z'-, z-ANL, y-, and w-ions. The fragment ions observed in the QIT-TOF MS could be explained by the injection of the source-generated ions into the QIT cell or a cooperative effect of a little internal energy deposition, a long residence timeframe (140 ms) in the QIT cell, and specific amino acid effects on low-energy CID, whereas the source-generated fragments (c-, z'-, z-ANL, y-, and w-ions) could be a result of prompt radical-initiated fragmentation of hydrogen-abundant radical ions [M + H + H]+ and [M + H - H]- within the 53 ns timeframe, which corresponds to the delayed extraction time. The further degraded fragment b/y-ions produced in the QIT cell were confirmed by positive- and negative-ion low-energy CID experiments performed on the source-generated ions (c-, z'-, and y-ions). The loss of phosphoric acid (98 u) from analyte and fragment ions can be explained by a slow ergodic fragmentation independent of positive and negative charges.

  4. Fragmentation of organic ions bearing fixed multiple charges observed in MALDI MS

    NARCIS (Netherlands)

    Lou, X.; Li, B.; de Waal, B.F.M.; Schill, J.; Baker, M.B.; Bovee, R.A.A.; van Dongen, J.L.J.; Milroy, L.G.; Meijer, E.W.

    2018-01-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was used to analyze a series of synthetic organic ions bearing fixed multiple charges. Despite the multiple intrinsic charges, only singly charged ions were recorded in each case. In addition to the

  5. MALDI-TOF Baseline Drift Removal Using Stochastic Bernstein Approximation

    Directory of Open Access Journals (Sweden)

    Howard Daniel

    2006-01-01

    Full Text Available Stochastic Bernstein (SB approximation can tackle the problem of baseline drift correction of instrumentation data. This is demonstrated for spectral data: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF data. Two SB schemes for removing the baseline drift are presented: iterative and direct. Following an explanation of the origin of the MALDI-TOF baseline drift that sheds light on the inherent difficulty of its removal by chemical means, SB baseline drift removal is illustrated for both proteomics and genomics MALDI-TOF data sets. SB is an elegant signal processing method to obtain a numerically straightforward baseline shift removal method as it includes a free parameter that can be optimized for different baseline drift removal applications. Therefore, research that determines putative biomarkers from the spectral data might benefit from a sensitivity analysis to the underlying spectral measurement that is made possible by varying the SB free parameter. This can be manually tuned (for constant or tuned with evolutionary computation (for .

  6. Biomarker- and similarity coefficient-based approaches to bacterial mixture characterization using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Zhang, Lin; Smart, Sonja; Sandrin, Todd R

    2015-11-05

    MALDI-TOF MS profiling has been shown to be a rapid and reliable method to characterize pure cultures of bacteria. Currently, there is keen interest in using this technique to identify bacteria in mixtures. Promising results have been reported with two- or three-isolate model systems using biomarker-based approaches. In this work, we applied MALDI-TOF MS-based methods to a more complex model mixture containing six bacteria. We employed: 1) a biomarker-based approach that has previously been shown to be useful in identification of individual bacteria in pure cultures and simple mixtures and 2) a similarity coefficient-based approach that is routinely and nearly exclusively applied to identification of individual bacteria in pure cultures. Both strategies were developed and evaluated using blind-coded mixtures. With regard to the biomarker-based approach, results showed that most peaks in mixture spectra could be assigned to those found in spectra of each component bacterium; however, peaks shared by two isolates as well as peaks that could not be assigned to any individual component isolate were observed. For two-isolate blind-coded samples, bacteria were correctly identified using both similarity coefficient- and biomarker-based strategies, while for blind-coded samples containing more than two isolates, bacteria were more effectively identified using a biomarker-based strategy.

  7. Geena 2, improved automated analysis of MALDI/TOF mass spectra.

    Science.gov (United States)

    Romano, Paolo; Profumo, Aldo; Rocco, Mattia; Mangerini, Rosa; Ferri, Fabio; Facchiano, Angelo

    2016-03-02

    Mass spectrometry (MS) is producing high volumes of data supporting oncological sciences, especially for translational research. Most of related elaborations can be carried out by combining existing tools at different levels, but little is currently available for the automation of the fundamental steps. For the analysis of MALDI/TOF spectra, a number of pre-processing steps are required, including joining of isotopic abundances for a given molecular species, normalization of signals against an internal standard, background noise removal, averaging multiple spectra from the same sample, and aligning spectra from different samples. In this paper, we present Geena 2, a public software tool for the automated execution of these pre-processing steps for MALDI/TOF spectra. Geena 2 has been developed in a Linux-Apache-MySQL-PHP web development environment, with scripts in PHP and Perl. Input and output are managed as simple formats that can be consumed by any database system and spreadsheet software. Input data may also be stored in a MySQL database. Processing methods are based on original heuristic algorithms which are introduced in the paper. Three simple and intuitive web interfaces are available: the Standard Search Interface, which allows a complete control over all parameters, the Bright Search Interface, which leaves to the user the possibility to tune parameters for alignment of spectra, and the Quick Search Interface, which limits the number of parameters to a minimum by using default values for the majority of parameters. Geena 2 has been utilized, in conjunction with a statistical analysis tool, in three published experimental works: a proteomic study on the effects of long-term cryopreservation on the low molecular weight fraction of serum proteome, and two retrospective serum proteomic studies, one on the risk of developing breat cancer in patients affected by gross cystic disease of the breast (GCDB) and the other for the identification of a predictor of

  8. Characterization of sialylated and fucosylated glycopeptides of beta2-glycoprotein I by a combination of HILIC LC and MALDI MS/MS

    DEFF Research Database (Denmark)

    Kondo, Akira; Thaysen-Andersen, Morten; Hjernø, Karin

    2010-01-01

    were characterized using MALDI quadrupole TOF MS/MS. A total of 23 glycan structures, including sialylated bi- and tri-antennary complex type glycans, were characterized at three N-glycosylation sites, namely Asn-143, Asn-174 and Asn-234, of beta2-GPI. Further exploration of the complementary nature...

  9. Facilitating the Hyphenation of CIEF and MALDI-MS for Two-Dimensional Separation of Proteins

    Science.gov (United States)

    Cheng, Chang; Lu, Joann J.; Wang, Xiayan; Roberts, Jonathan; Liu, Shaorong

    2011-01-01

    Both CIEF and MALDI-MS are frequently used in protein analysis, but hyphenation of the two is not investigated proportionally. One of the major reasons is that the additives (such as carrier ampholytes and detergent) in CIEF severely suppress the MALDI-MS signal, which hampers the hyphenation of the two. In this paper, we develop a simple means to alleviate the above signal-suppressing effect. We first deposit 1 µL of water onto a MALDI-MS target, deliver a fraction of CIEF-separated protein (~0.1 µL) to the water droplet, evaporate the solvent, add 0.5 µL of MALDI matrix to the sample spot, dry the matrix, and move the target plate to a MALDI-TOF-MS for mass spectrum measurement. We optimize the droplet volume and the laser-ablation region. Under the optimized conditions, we improve the signal to noise ratio by 2–10 fold. We also apply this method for two-dimensional separations of standard proteins and Apolipoprotein A-I, a membrane protein expressed in E. Coli cells. PMID:20603827

  10. 2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation

    International Nuclear Information System (INIS)

    Lu, Jian; Zhou, Zhongping; Zheng, Jianzhou; Zhang, Zhuyi; Lu, Rongzhu; Liu, Hanqing; Shi, Haifeng; Tu, Zhigang

    2015-01-01

    Cadmium is a toxic heavy metal present in the environment and in industrial materials. Cadmium has demonstrated carcinogenic activity that induces cell transformation, but how this occurs is unclear. We used 2D-DIGE and MALDI TOF/TOF MS combined with bioinformatics and immunoblotting to investigate the molecular mechanism of cadmium transformation. We found that small GTPases were critical for transformation. Additionally, proteins involved in mitochondrial transcription, DNA repair, and translation also had altered expression patterns in cadmium treated cells. Collectively, our results suggest that activation of small GTPases contributes to cadmium-induced transformation of colon cells. - Highlights: • Colon epithelial cell line is firstly successfully transformed by cadmium. • 2D-DIGE is applied to visualize the differentially expressed proteins. • RhoA plays an important role in cadmium induced malignant transformation. • Bioinformatic and experimental methods are combined to explore new mechanisms.

  11. 2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jian, E-mail: lujian@ujs.edu.cn [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Zhou, Zhongping [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Zheng, Jianzhou [Department of Respiration Medicine, Changzhou No.2 People' s Hospital, Changzhou 213003 (China); Zhang, Zhuyi [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Lu, Rongzhu [School of Medicine, Jiangsu University, Zhenjiang 212013 (China); Liu, Hanqing [School of Pharmacy, Jiangsu University, Zhenjiang 212013 (China); Shi, Haifeng [Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China); Tu, Zhigang, E-mail: tuzg_ujs@ujs.edu.cn [Institute of Life Sciences, Jiangsu University, Zhenjiang 212013 (China)

    2015-10-01

    Cadmium is a toxic heavy metal present in the environment and in industrial materials. Cadmium has demonstrated carcinogenic activity that induces cell transformation, but how this occurs is unclear. We used 2D-DIGE and MALDI TOF/TOF MS combined with bioinformatics and immunoblotting to investigate the molecular mechanism of cadmium transformation. We found that small GTPases were critical for transformation. Additionally, proteins involved in mitochondrial transcription, DNA repair, and translation also had altered expression patterns in cadmium treated cells. Collectively, our results suggest that activation of small GTPases contributes to cadmium-induced transformation of colon cells. - Highlights: • Colon epithelial cell line is firstly successfully transformed by cadmium. • 2D-DIGE is applied to visualize the differentially expressed proteins. • RhoA plays an important role in cadmium induced malignant transformation. • Bioinformatic and experimental methods are combined to explore new mechanisms.

  12. Evaluation of the Biotyper MALDI-TOF MS system for identification of Staphylococcus species.

    Science.gov (United States)

    Zhu, Wenming; Sieradzki, Krzysztof; Albrecht, Valerie; McAllister, Sigrid; Lin, Wen; Stuchlik, Olga; Limbago, Brandi; Pohl, Jan; Kamile Rasheed, J

    2015-10-01

    The Bruker Biotyper MALDI-TOF MS (Biotyper) system, with a modified 30 minute formic acid extraction method, was evaluated by its ability to identify 216 clinical Staphylococcus isolates from the CDC reference collection comprising 23 species previously identified by conventional biochemical tests. 16S rDNA sequence analysis was used to resolve discrepancies. Of these, 209 (96.8%) isolates were correctly identified: 177 (84.7%) isolates had scores ≥2.0, while 32 (15.3%) had scores between 1.70 and 1.99. The Biotyper identification was inconsistent with the biochemical identification for seven (3.2%) isolates, but the Biotyper identifications were confirmed by 16S rDNA analysis. The distribution of low scores was strongly species-dependent, e.g. only 5% of Staphylococcus epidermidis and 4.8% of Staphylococcus aureus isolates scored below 2.0, while 100% of Staphylococcus cohnii, 75% of Staphylococcus sciuri, and 60% of Staphylococcus caprae produced low but accurate Biotyper scores. Our results demonstrate that the Biotyper can reliably identify Staphylococcus species with greater accuracy than conventional biochemicals. Broadening of the reference database by inclusion of additional examples of under-represented species could further optimize Biotyper results. Published by Elsevier B.V.

  13. Detection of Listeria monocytogenes from selective enrichment broth using MALDI-TOF Mass Spectrometry.

    Science.gov (United States)

    Jadhav, Snehal; Sevior, Danielle; Bhave, Mrinal; Palombo, Enzo A

    2014-01-31

    Conventional methods used for primary detection of Listeria monocytogenes from foods and subsequent confirmation of presumptive positive samples involve prolonged incubation and biochemical testing which generally require four to five days to obtain a result. In the current study, a simple and rapid proteomics-based MALDI-TOF MS approach was developed to detect L. monocytogenes directly from selective enrichment broths. Milk samples spiked with single species and multiple species cultures were incubated in a selective enrichment broth for 24h, followed by an additional 6h secondary enrichment. As few as 1 colony-forming unit (cfu) of L. monocytogenes per mL of initial selective broth culture could be detected within 30h. On applying the same approach to solid foods previously implicated in listeriosis, namely chicken pâté, cantaloupe and Camembert cheese, detection was achieved within the same time interval at inoculation levels of 10cfu/mL. Unlike the routine application of MALDI-TOF MS for identification of bacteria from solid media, this study proposes a cost-effective and time-saving detection scheme for direct identification of L. monocytogenes from broth cultures.This article is part of a Special Issue entitled: Trends in Microbial Proteomics. Globally, foodborne diseases are major causes of illness and fatalities in humans. Hence, there is a continual need for reliable and rapid means for pathogen detection from food samples. Recent applications of MALDI-TOF MS for diagnostic microbiology focused on detection of microbes from clinical specimens. However, the current study has emphasized its use as a tool for detecting the major foodborne pathogen, Listeria monocytogenes, directly from selective enrichment broths. This proof-of-concept study proposes a detection scheme that is more rapid and simple compared to conventional methods of Listeria detection. Very low levels of the pathogen could be identified from different food samples post-enrichment in

  14. [EXPRESS IDENTIFICATION OF POSITIVE BLOOD CULTURES USING DIRECT MALDI-TOF MASS SPECTROMETRY].

    Science.gov (United States)

    Popov, D A; Ovseenko, S T; Vostrikova, T Yu

    2015-01-01

    To evaluate the effectiveness of direct identification of pathogens of bacteremia by direct matrix assisted laser desorption ionization time-flight mass spectrometry (mALDI-TOF) compared to routine method. A prospective study included 211 positive blood cultures obtained from 116 patients (106 adults and 10 children, aged from 2 weeks to 77 years old in the ICU after open heart surgery. Incubation was carried out under aerobic vials with a sorbent for antibiotics Analyzer BacT/ALERT 3D 120 (bioMerieux, France) in parallel with the primary sieving blood cultures on solid nutrient media with subsequent identification of pure cultures using MALDI-TOF mass spectrometry analyzer Vitek MS, bioMerieux, France routine method), after appropriate sample preparation we carried out a direct (without screening) MALDI-TOF mass spectrometric study of monocomponental blood cultures (n = 201). using a routine method in 211 positive blood cultures we identified 23 types of microorganisms (Staphylococcus (n = 87), Enterobacteria- ceae (n = 71), Enterococci (n = 20), non-fermentative Gram-negative bacteria (n = 18), others (n = 5). The average time of incubation of samples to obtain a signal of a blood culture growth was 16.2 ± 7.4 h (from 3.75 to 51 hours.) During the first 12 hours of incubation, growth was obtained in 32.4% of the samples, and on the first day in 92.2%. In the direct mass spectrometric analysis mnonocomponental blood cultures (n = 201) is well defined up to 153 species of the sample (76.1%), while the share of successful identification of Gram-negative bacteria was higher than that of Gram-positive (85.4 and 69, 1%, respectively p = 0.01). The high degree of consistency in the results of standard and direct method of identifying blood cultures using MALDI-TOF mass spectrometry (κ = 0.96, p direct mass spectrometric analysis, including sample preparation, was no longer than 1 hour: The method of direct MALDI-TOF mass spectrometry allows to significantly speed up

  15. Proteomic profiling of renal allograft rejection in serum using magnetic bead-based sample fractionation and MALDI-TOF MS.

    Science.gov (United States)

    Sui, Weiguo; Huang, Liling; Dai, Yong; Chen, Jiejing; Yan, Qiang; Huang, He

    2010-12-01

    Proteomics is one of the emerging techniques for biomarker discovery. Biomarkers can be used for early noninvasive diagnosis and prognosis of diseases and treatment efficacy evaluation. In the present study, the well-established research systems of ClinProt Micro solution incorporated unique magnetic bead sample preparation technology, which, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), have become very successful in bioinformatics due to its outstanding performance and reproducibility for discovery disease-related biomarker. We collected fasting blood samples from patients with biopsy-confirmed acute renal allograft rejection (n = 12), chronic rejection (n = 12), stable graft function (n = 12) and also from healthy volunteers (n = 13) to study serum peptidome patterns. Specimens were purified with magnetic bead-based weak cation exchange chromatography and analyzed with a MALDI-TOF mass spectrometer. The results indicated that 18 differential peptide peaks were selected as potential biomarkers of acute renal allograft rejection, and 6 differential peptide peaks were selected as potential biomarkers of chronic rejection. A Quick Classifier Algorithm was used to set up the classification models for acute and chronic renal allograft rejection. The algorithm models recognize 82.64% of acute rejection and 98.96% of chronic rejection episodes, respectively. We were able to identify serum protein fingerprints in small sample sizes of recipients with renal allograft rejection and establish the models for diagnosis of renal allograft rejection. This preliminary study demonstrated that proteomics is an emerging tool for early diagnosis of renal allograft rejection and helps us to better understand the pathogenesis of disease process.

  16. MALDI-TOF mass spectrometry confirms difficulties in separating species of the Avibacterium genus

    DEFF Research Database (Denmark)

    Alispahic, Merima; Christensen, Henrik; Bisgaard, Magne

    2014-01-01

    In the present study a well-characterized strain collection (n = 33) of Avibacterium species was investigated by matrix-assisted laser desorption ionization-time-of flight mass spectrometry (MALDI-TOF MS). The robustness of the currently available reference database (Bruker Biotyper 3.0) was tested...... to determine the degree of identification of these strains. Reproducible signal patterns were obtained from all strains. However, identification of most strains was only possible at genus level. Furthermore, two strains could not be identified by this method. Based on their protein spectra profiles, a MALDI...

  17. Differentiation of Cronobacter spp. by tryptic digestion of the cell suspension followed by MALDI-TOF MS analysis

    Czech Academy of Sciences Publication Activity Database

    Krásný, Lukáš; Rohlová, E.; Růžičková, E.; Šantrůček, J.; Hynek, R.; Hochel, I.

    2014-01-01

    Roč. 98, MAR 2014 (2014), s. 105-113 ISSN 0167-7012 R&D Projects: GA ČR GAP503/10/0664 Institutional support: RVO:61388971 Keywords : Biotyper * Cronobacter * MALDI - TOF Subject RIV: CE - Biochemistry Impact factor: 2.026, year: 2014

  18. MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

    Directory of Open Access Journals (Sweden)

    Marko Jovanović

    2014-04-01

    Full Text Available Human milk oligosaccharides (HMO represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1-3 or 1-4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI quadrupole-time-of-flight (Q-TOF collision-induced dissociation (CID as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.

  19. 2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing

    Czech Academy of Sciences Publication Activity Database

    Petry-Podgorska, Inga; Žídková, Jitka; Flodrová, Dana; Bobálová, Janette

    2010-01-01

    Roč. 878, č. 30 (2010), s. 3143-3148 ISSN 1570-0232 R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : 2D-HPLC * MALDI-TOF/TOF mass spectrometry * barley Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.971, year: 2010

  20. Identification and susceptibility testing of microorganism by direct inoculation from positive blood culture bottles by combining MALDI-TOF and Vitek-2 Compact is rapid and effective.

    Science.gov (United States)

    Romero-Gómez, María-Pilar; Gómez-Gil, Rosa; Paño-Pardo, Jose Ramón; Mingorance, Jesús

    2012-12-01

    The objective of this study was to evaluate the reliability and accuracy of the combined use of MALDI-TOF MS bacterial identification and the Vitek-2 Compact antimicrobial susceptibility testing (AST) directly from positive blood cultures. Direct identification by MALDI-TOF MS and AST were performed in parallel to the standard methods in all positively flagged blood cultures bottles during the study period. Three hundred and twenty four monomicrobial positive blood cultures were included in the present study, with 257 Gram-negative and 67 Gram-positive isolates. MALDI-TOF MS identification directly from blood bottles reported the correct identification for Enterobacteriaceae in 97.7%, non-fermentative Gram-negative bacilli 75.0%, Staphylococcus aureus 75.8%, coagulase negative staphylococci 63.3% and enterococci 63.3%. A total 6156 isolate/antimicrobial agent combinations were tested. Enterobacteriaceae group and non-fermentative Gram-negative Bacilli showed an agreement of 96.67% and 92.30%, respectively, for the Gram-positive cocci the overall agreement found was 97.84%. We conclude that direct identification by MALDI-TOF and inoculation of Vitek-2 Compact AST with positive blood culture bottles yielded very good results and decreased time between initial inoculation of blood culture media and determination of the antibiotic susceptibility for Gram-negative rods and Gram-positive cocci causing bacteremia. Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  1. Lactococcus garvieae endocarditis in a native valve identified by MALDI-TOF MS and PCR-based 16s rRNA in Spain: A case report

    Directory of Open Access Journals (Sweden)

    V. Heras Cañas

    2015-05-01

    Full Text Available Lactococcus garvieae is a Gram-positive, catalase negative coccus arranged in pairs or short chains, well-known as a fish pathogen. We report a case of Infective Endocarditis (IE by L. garvieae in a native valve from a 68-year-old male with unknown history of contact with raw fish and an extensive history of heart disease. This case highlights the reliability of MALDI-TOF MS compared to conventional methods in the identification of rare microorganisms like this.

  2. Development and validation of an extended database for yeast identification by MALDI-TOF MS in Argentina.

    Science.gov (United States)

    Taverna, Constanza Giselle; Mazza, Mariana; Bueno, Nadia Soledad; Alvarez, Christian; Amigot, Susana; Andreani, Mariana; Azula, Natalia; Barrios, Rubén; Fernández, Norma; Fox, Barbara; Guelfand, Liliana; Maldonado, Ivana; Murisengo, Omar Alejandro; Relloso, Silvia; Vivot, Matias; Davel, Graciela

    2018-05-11

    Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microorganisms in clinical laboratories because it is rapid, relatively simple to use, accurate, and can be used for a wide number of microorganisms. Several studies have demonstrated the utility of this technique in the identification of yeasts; however, its performance is usually improved by the extension of the database. Here we developed an in-house database of 143 strains belonging to 42 yeast species in the MALDI Biotyper platform, and we validated the extended database with 388 regional strains and 15 reference strains belonging to 55 yeast species. We also performed an intra- and interlaboratory study to assess reproducibility and analyzed the use of the cutoff values of 1.700 and 2.000 to correctly identify at species level. The creation of an in-house database that extended the manufacturer's database was successful in view of no incorrect identification was introduced. The best performance was observed by using the extended database and a cutoff value of 1.700 with a sensitivity of .94 and specificity of .96. A reproducibility study showed utility to detect deviations and could be used for external quality control. The extended database was able to differentiate closely related species and it has potential in distinguishing the molecular genotypes of Cryptococcus neoformans and Cryptococcus gattii.

  3. Mayfly and fish species identification and sex determination in bleak (Alburnus alburnus) by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Maasz, G; Takács, P; Boda, P; Varbiro, G; Pirger, Z

    2017-12-01

    Besides food quality control of fish or cephalopods, the novel mass spectrometry (MS) approaches could be effective and beneficial methods for the investigation of biodiversity in ecological research. Our aims were to verify the applicability of MALDI-TOF MS in the rapid identification of closely related species, and to further develop it for sex determination in phenotypically similar fish focusing on the low mass range. For MALDI-TOF MS spectra analysis, ClinProTools software was applied, but our observed classification was also confirmed by Self Organizing Map. For verifying the wide applicability of the method, brains from invertebrate and vertebrate species were used in order to detect the species related markers from two mayflies and eight fish as well as sex-related markers within bleak. Seven Ephemera larvae and sixty-one fish species related markers were observed and nineteen sex-related markers were identified in bleak. Similar patterns were observed between the individuals within one species. In contrast, there were markedly diverse patterns between the different species and sexes visualized by SOMs. Two different Ephemera species and male or female fish were identified with 100% accuracy. The various fish species were classified into 8 species with a high level of accuracy (96.2%). Based on MS data, dendrogram was generated from different fish species by using ClinProTools software. This MS-based dendrogram shows relatively high correspondence with the phylogenetic relationships of both the studied species and orders. In summary, MALDI-TOF MS provides a cheap, reliable, sensitive and fast identification tool for researchers in the case of closely related species using mass spectra acquired in a low mass range to define specific molecular profiles. Moreover, we presented evidence for the first time for determination of sex within one fish species by using this method. We conclude that it is a powerful tool that can revolutionize ecological and

  4. Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: role of cysteine alkylation during sample processing.

    Science.gov (United States)

    Franc, Vojtěch; Řehulka, Pavel; Raus, Martin; Stulík, Jiří; Novak, Jan; Renfrow, Matthew B; Šebela, Marek

    2013-10-30

    Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236. In this work, we studied the O-glycosylation in the hinge region of human immunoglobulin A1 (IgA1). Aberrant glycosylation of the protein plays a key role in the pathogenesis of IgA nephropathy. Thus identification of the O-glycan composition of IgA1 is important for a deeper understanding of the disease mechanism, biomarker discovery and validation, and implementation and monitoring of disease-specific therapies. We developed a new procedure for elucidating the heterogeneity of IgA1 O-glycosylation. After running a polyacrylamide gel

  5. Selective extraction of phospholipids from dairy products by micro-solid phase extraction based on titanium dioxide microcolumns followed by MALDI-TOF-MS analysis

    DEFF Research Database (Denmark)

    Calvano, Cosima; Jensen, Ole; Zambonin, Carlo

    2009-01-01

    A new micro-solid phase extraction (micro-SPE) procedure based on titanium dioxide microcolumns was developed for the selective extraction of phospholipids (PLs) from dairy products before matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. All...... the extraction steps (loading, washing, and elution) have been optimized using a synthetic mixture of PLs standard and the procedure was subsequently applied to food samples such as milk, chocolate milk and butter. The whole method demonstrated to be simpler than traditional approaches and it appears very...

  6. A statistical design of experiments for optimizing the MALDI-TOF-MS sample preparation of polymers. An application in the assessment of the thermo-mechanical degradation mechanisms of poly (ethylene terephthalate)

    International Nuclear Information System (INIS)

    Badia, J.D.; Stroemberg, E.; Ribes-Greus, A.; Karlsson, S.

    2011-01-01

    The sample preparation procedure for MALDI-TOF MS of polymers is addressed in this study by the application of a statistical Design of Experiments (DoE). Industrial poly (ethylene terephthalate) (PET) was chosen as model polymer. Different experimental settings (levels) for matrixes, analyte/matrix proportions and concentrations of cationization agent were considered. The quality parameters used for the analysis were signal-to-noise ratio and resolution. A closer inspection of the statistical results provided the study not only with the best combination of factors for the MALDI sample preparation, but also with a better understanding of the influence of the different factors, individually or in combination, to the signal. The application of DoE for the improvement of the MALDI measure of PET stated that the best combination of factors and levels was the following: matrix (dithranol), proportion analyte/matrix/cationization agent (1/15/1, V/V/V), and concentration of cationization agent (2 g L -1 ). In a second part, multiple processing by means of successive injection cycles was used to simulate the thermo-mechanical degradation effects on the oligomeric distribution of PET under mechanical recycling. The application of MALDI-TOF-MS showed that thermo-mechanical degradation primarily affected initially predominant cyclic species. Several degradation mechanisms were proposed, remarking intramolecular transesterification and hydrolysis. The ether links of the glycol unit in PET were shown to act as potential reaction sites, driving the main reactions of degradation.

  7. Comparison of the accuracy of two conventional phenotypic methods and two MALDI-TOF MS systems with that of DNA sequencing analysis for correctly identifying clinically encountered yeasts.

    Science.gov (United States)

    Chao, Qiao-Ting; Lee, Tai-Fen; Teng, Shih-Hua; Peng, Li-Yun; Chen, Ping-Hung; Teng, Lee-Jene; Hsueh, Po-Ren

    2014-01-01

    We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud's dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex

  8. Axial spatial distribution focusing: improving MALDI-TOF/RTOF mass spectrometric performance for high-energy collision-induced dissociation of biomolecules.

    Science.gov (United States)

    Belgacem, O; Pittenauer, E; Openshaw, M E; Hart, P J; Bowdler, A; Allmaier, G

    2016-02-15

    For the last two decades, curved field reflectron technology has been used in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers, assisting in the generation of post-source-decay (PSD) or collision-induced dissociation (CID) without decelerating precursor ions, producing true high-energy CID spectra. The result was the generation of product ion mass spectra with product ions typical of high-energy (10 keV and beyond) collision processes. The disadvantage of this approach was the lack of resolution in CID spectra resulting from the excess laser energy deposition used to generate those MS/MS spectra. The work presented in this study overcomes this limitation and includes comprehensive examples of high-energy and high-resolution CID MALDI-MS/MS spectra of biomolecules. The devices used in this study are TOF/RTOF instruments equipped with a high-vacuum MALDI ion source. High-resolution and high-energy CID spectra result from the use of axial spatial distribution focusing (ASDF) in combination with curved field reflectron technology. A CID spectrum of the P14 R1 peptide exhibits product ion resolution in excess of 10,000 (FWHM) but at the same time yields typical high-energy product ions such as w- and [y-2]-type ion series. High-energy CID spectra of lipids, exemplified by a glycerophospholipid and triglyceride, demonstrate C-C backbone fragmentation elucidating the presence of a hydroxyl group in addition to double-bond positioning. A complex high mannose carbohydrate (Man)8 (GlcNAc)2 was also studied at 20 keV collision energy and revealed further high-energy product ions with very high resolution, allowing unambiguous detection and characterization of cross-ring cleavage-related ions. This is the first comprehensive study using a MALDI-TOF/RTOF instrument equipped with a curved field reflectron and an ASDF device prior to the reflectron. © 2015 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley

  9. Structural Defects in Polyallylcarbosilane Dendrimers and Their PolyolDerivatives Characterized by NMR and MALDI-TOF Mass Spectrometry

    Czech Academy of Sciences Publication Activity Database

    Krupková, Alena; Čermák, Jan; Walterová, Zuzana; Horský, Jiří

    2010-01-01

    Roč. 43, č. 10 (2010), s. 4511-4519 ISSN 0024-9297 R&D Projects: GA MŠk(CZ) LC06070 Institutional research plan: CEZ:AV0Z40720504; CEZ:AV0Z40500505 Keywords : carbosilane dendrimer s * maldi-tof ms * structural defects Subject RIV: CC - Organic Chemistry Impact factor: 4.838, year: 2010

  10. MALDI-TOF typing highlights geographical and fluconazole resistance clusters in Candida glabrata.

    Science.gov (United States)

    Dhieb, C; Normand, A C; Al-Yasiri, M; Chaker, E; El Euch, D; Vranckx, K; Hendrickx, M; Sadfi, N; Piarroux, R; Ranque, S

    2015-06-01

    Utilizing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra for Candida glabrata typing would be a cost-effective and easy-to-use alternative to classical DNA-based typing methods. This study aimed to use MALDI-TOF for the typing of C. glabrata clinical isolates from various geographical origins and test its capacity to differentiate between fluconazole-sensitive and -resistant strains.Both microsatellite length polymorphism (MLP) and MALDI-TOF mass spectra of 58 C. glabrata isolates originating from Marseilles (France) and Tunis (Tunisia) as well as collection strains from diverse geographic origins were analyzed. The same analysis was conducted on a subset of C. glabrata isolates that were either susceptible (MIC ≤ 8 mg/l) or resistant (MIC ≥ 64 mg/l) to fluconazole.According to the seminal results, both MALDI-TOF and MLP classifications could highlight C. glabrata population structures associated with either geographical dispersal barriers (p typing to investigate C. glabrata infection outbreaks and predict the antifungal susceptibility profile of clinical laboratory isolates. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Conventional Morphology Versus PCR Sequencing, rep-PCR, and MALDI-TOF-MS for Identification of Clinical Aspergillus Isolates Collected Over a 2-Year Period in a University Hospital at Kayseri, Turkey.

    Science.gov (United States)

    Atalay, Altay; Koc, Ayse Nedret; Suel, Ahmet; Sav, Hafize; Demir, Gonca; Elmali, Ferhan; Cakir, Nuri; Seyedmousavi, Seyedmojtaba

    2016-09-01

    Aspergillus species cause a wide range of diseases in humans, including allergies, localized infections, or fatal disseminated diseases. Rapid detection and identification of Aspergillus spp. facilitate effective patient management. In the current study we compared conventional morphological methods with PCR sequencing, rep-PCR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the identification of Aspergillus strains. A total of 24 consecutive clinical isolates of Aspergillus were collected during 2012-2014. Conventional morphology and rep-PCR were performed in our Mycology Laboratory. The identification, evaluation, and reporting of strains using MALDI-TOF-MS were performed by BioMérieux Diagnostic, Inc. in Istanbul. DNA sequence analysis of the clinical isolates was performed by the BMLabosis laboratory in Ankara. Samples consisted of 18 (75%) lower respiratory tract specimens, 3 otomycosis (12.5%) ear tissues, 1 sample from keratitis, and 1 sample from a cutaneous wound. According to DNA sequence analysis, 12 (50%) specimens were identified as A. fumigatus, 8 (33.3%) as A. flavus, 3 (12.5%) as A. niger, and 1 (4.2%) as A. terreus. Statistically, there was good agreement between the conventional morphology and rep-PCR and MALDI-TOF methods; kappa values were κ = 0.869, 0.871, and 0.916, respectively (P < 0.001). The good level of agreement between the methods included in the present study and sequence method could be due to the identification of Aspergillus strains that were commonly encountered. Therefore, it was concluded that studies conducted with a higher number of isolates, which include other Aspergillus strains, are required. © 2016 Wiley Periodicals, Inc.

  12. Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Stephan, Roger; Cernela, Nicole; Ziegler, Dominik; Pflüger, Valentin; Tonolla, Mauro; Ravasi, Damiana; Fredriksson-Ahomaa, Maria; Hächler, Herbert

    2011-11-01

    Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Investigating the microstructure of keratin extracted from wool: peptide sequence (MALDI-TOF/TOF) and protein conformation (FTIR)

    Science.gov (United States)

    Keratin was extracted from wool by reduction with 2-mercaptoethanol. It was isolated as intact keratin and characterized by its similar molecular weight, protein composition, and secondary structure to native keratin. Gel electrophoresis patterns and MALDI-TOF/TOF peptide sequences provided the ide...

  14. Rapid identification of moulds and arthroconidial yeasts from positive blood cultures by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    de Almeida, João N; Sztajnbok, Jaques; da Silva, Afonso Rafael; Vieira, Vinicius Adriano; Galastri, Anne Layze; Bissoli, Leandro; Litvinov, Nadia; Del Negro, Gilda Maria Barbaro; Motta, Adriana Lopes; Rossi, Flávia; Benard, Gil

    2016-11-01

    Moulds and arthroconidial yeasts are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens hastens initiation of targeted antifungal therapy, thereby improving the patients' prognosis. We describe a new strategy that enabled the identification of moulds and arthroconidial yeasts directly from positive blood cultures by MALDI-TOF mass spectrometry (MS). Positive blood cultures (BCs) with Gram staining showing hyphae and/or arthroconidia were prospectively selected and submitted to an in-house protein extraction protocol. Mass spectra were obtained by Vitek MS™ system, and identifications were carried out with in the research use only (RUO) mode with an extended database (SARAMIS™ [v.4.12] plus in-house database). Fusarium solani, Fusarium verticillioides, Exophiala dermatitidis, Saprochaete clavata, and Trichosporon asahii had correct species ID by MALDI-TOF MS analysis of positive BCs. All cases were related to critically ill patients with high mortality fungemia and direct ID from positive BCs was helpful for rapid administration of targeted antifungal therapy. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. New methods of microbiological identification using MALDI-TOF

    Directory of Open Access Journals (Sweden)

    Jacyr Pasternak

    2012-03-01

    Full Text Available Rapid diagnosis of pathogens is decisive to guarantee adequatetherapy in infections: culture methods are precise and sensitive, butrather slow. New resources are available to enable faster diagnosis,and the most promising is MALDI-TOF technology: mass spectrometryapplied to microbiological diagnosis. Times as fast as 10 to 15 minutes to etiological diagnosis are possible after a positive blood culture result. We hope to have this technology in our laboratory, ANVISA permitting and improving their very slow rate of doing things… MALDI-TOF is basically putting a sample of culture or an enriched suspension of the probable pathogen over a small spot with a matrix and vaporizing it with a laser pulse: the products are aspired into a chamber, ionized and reach detectors at variable times: the detectors show time of arrival and quantity of the product, and each pathogen has its characteristic spectrum analyzed by a software.

  16. Defining Diagnostic Biomarkers Using Shotgun Proteomics and MALDI-TOF Mass Spectrometry.

    Science.gov (United States)

    Armengaud, Jean

    2017-01-01

    Whole-cell MALDI-TOF has become a robust and widely used tool to quickly identify any pathogen. In addition to being routinely used in hospitals, it is also useful for low cost dereplication in large scale screening procedures of new environmental isolates for environmental biotechnology or taxonomical applications. Here, I describe how specific biomarkers can be defined using shotgun proteomics and whole-cell MALDI-TOF mass spectrometry. Based on MALDI-TOF spectra recorded on a given set of pathogens with internal calibrants, m/z values of interest are extracted. The proteins which contribute to these peaks are deduced from label-free shotgun proteomics measurements carried out on the same sample. Quantitative information based on the spectral count approach allows ranking the most probable candidates. Proteogenomic approaches help to define whether these proteins give the same m/z values along the whole taxon under consideration or result in heterogeneous lists. These specific biomarkers nicely complement conventional profiling approaches and may help to better define groups of organisms, for example at the subspecies level.

  17. Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis.

    Directory of Open Access Journals (Sweden)

    Aneta Nowakiewicz

    Full Text Available The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS. From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson's index of diversity, Rand and Wallace coefficients. Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the

  18. MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex.

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    Carolina Firacative

    Full Text Available BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this

  19. Improvement of MALDI-TOF MS profiling for the differentiation of species within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

    Science.gov (United States)

    Šedo, Ondrej; Nemec, Alexandr; Křížová, Lenka; Kačalová, Magdaléna; Zdráhal, Zbyněk

    2013-12-01

    MALDI-TOF MS is currently becoming the method of choice for rapid identification of bacterial species in routine diagnostics. Yet, this method suffers from the inability to differentiate reliably between some closely related bacterial species including those of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, namely A. baumannii and Acinetobacter nosocomialis. In the present study, we evaluated a protocol which was different from that used in the Bruker Daltonics identification system (MALDI BioTyper) to improve species identification using a taxonomically precisely defined set of 105 strains representing the four validly named species of the ACB complex. The novel protocol is based on the change in matrix composition from alpha-cyano-4-hydroxycinnamic acid (saturated solution in water:acetonitrile:trifluoroacetic acid, 47.5:50:2.5, v/v) to ferulic acid (12.5mgml(-1) solution in water:acetonitrile:formic acid 50:33:17, v/v), while the other steps of sample processing remain unchanged. Compared to the standard protocol, the novel one extended the range of detected compounds towards higher molecular weight, produced signals with better mass resolution, and allowed the detection of species-specific signals. As a result, differentiation of A. nosocomialis and A. baumannii strains by cluster analysis was improved and 13 A. nosocomialis strains, assigned erroneously or ambiguously by using the standard protocol, were correctly identified. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Comparison of the accuracy of two conventional phenotypic methods and two MALDI-TOF MS systems with that of DNA sequencing analysis for correctly identifying clinically encountered yeasts.

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    Qiao-Ting Chao

    Full Text Available We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS systems (Bruker Biotyper and Vitek MS and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database, Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud's dextrose agar systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD system, 39 (43.8%, including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5% by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii

  1. Investigations on aberrant glycosylation of glycosphingolipids in colorectal cancer tissues using liquid chromatography and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS).

    Science.gov (United States)

    Holst, Stephanie; Stavenhagen, Kathrin; Balog, Crina I A; Koeleman, Carolien A M; McDonnell, Liam M; Mayboroda, Oleg A; Verhoeven, Aswin; Mesker, Wilma E; Tollenaar, Rob A E M; Deelder, André M; Wuhrer, Manfred

    2013-11-01

    Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.

  2. Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)*

    Science.gov (United States)

    Holst, Stephanie; Stavenhagen, Kathrin; Balog, Crina I. A.; Koeleman, Carolien A. M.; McDonnell, Liam M.; Mayboroda, Oleg A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

    2013-01-01

    Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment. PMID:23878401

  3. An innovative strategy for sulfopeptides analysis using MALDI-TOF MS reflectron positive ion mode.

    Science.gov (United States)

    Cantel, Sonia; Brunel, Luc; Ohara, Keiichiro; Enjalbal, Christine; Martinez, Jean; Vasseur, Jean-Jacques; Smietana, Michael

    2012-08-01

    Sulfation of tyrosine residues is a key posttranslational modification in the regulation of various cellular processes. As such, the detection and localization of tyrosine sulfation is an essential step toward the elucidation of the physiological and pathological roles of this process. Despite substantial advances, intact sulfated peptides are still difficult to detect by MALDI-MS due to the extreme lability of the sulfo-moiety. The present report demonstrates for the first time how intact sulfated peptides can be directly and specifically detected by MALDI-MS in positive reflectron mode by using pyrenemethylguanidine (pmg) as a noncovalent derivatizing agent and an ionization enhancer. This new method allows the determination of the degree of sulfation of sulfopeptides pure or in mixtures. Moreover, the observation of specific peaks in the mass spectra enables a rapid and unambiguous discrimination between phospho- and sulfopeptides. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A silicon nanomembrane detector for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of large proteins.

    Science.gov (United States)

    Park, Jonghoo; Blick, Robert H

    2013-10-11

    We describe a MALDI-TOF ion detector based on freestanding silicon nanomembrane technology. The detector is tested in a commercial MALDI-TOF mass spectrometer with equimolar mixtures of proteins. The operating principle of the nanomembrane detector is based on phonon-assisted field emission from these silicon nanomembranes, in which impinging ion packets excite electrons in the nanomembrane to higher energy states. Thereby the electrons can overcome the vacuum barrier and escape from the surface of the nanomembrane via field emission. Ion detection is demonstrated of apomyoglobin (16,952 Da), aldolase (39,212 Da), bovine serum albumin (66,430 Da), and their equimolar mixtures. In addition to the three intact ions, a large number of fragment ions are also revealed by the silicon nanomembrane detector, which are not observable with conventional detectors.

  5. A Silicon Nanomembrane Detector for Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS of Large Proteins

    Directory of Open Access Journals (Sweden)

    Jonghoo Park

    2013-10-01

    Full Text Available We describe a MALDI-TOF ion detector based on freestanding silicon nanomembrane technology. The detector is tested in a commercial MALDI-TOF mass spectrometer with equimolar mixtures of proteins. The operating principle of the nanomembrane detector is based on phonon-assisted field emission from these silicon nanomembranes, in which impinging ion packets excite electrons in the nanomembrane to higher energy states. Thereby the electrons can overcome the vacuum barrier and escape from the surface of the nanomembrane via field emission. Ion detection is demonstrated of apomyoglobin (16,952 Da, aldolase (39,212 Da, bovine serum albumin (66,430 Da, and their equimolar mixtures. In addition to the three intact ions, a large number of fragment ions are also revealed by the silicon nanomembrane detector, which are not observable with conventional detectors.

  6. Ga+ TOF-SIMS lineshape analysis for resolution enhancement of MALDI MS spectra of a peptide mixture

    International Nuclear Information System (INIS)

    Malyarenko, D.I.; Chen, H.; Wilkerson, A.L.; Tracy, E.R.; Cooke, W.E.; Manos, D.M.; Sasinowski, M.; Semmes, O.J.

    2004-01-01

    The use of mass spectrometry to obtain molecular profiles indicative of alteration of concentrations of peptides in body fluids is currently the subject of intense investigation. For surface-based time-of-flight mass spectrometry the reliability and specificity of such profiling methods depend both on the resolution of the measuring instrument and on the preparation of samples. The present work is a part of a program to use Ga + beam TOF-SIMS alone, and as an adjunct to MALDI, in the development of reliable protein and peptide markers for diseases. Here, we describe techniques to prepare samples of relatively high-mass peptides, which serve as calibration standards and proxies for biomarkers. These are: Arg8-vasopressin, human angiotensin II, and somatostatin. Their TOF-SIMS spectra show repeatable characteristic features, with mass resolution exceeding 2000, including parent peaks and chemical adducts. The lineshape analysis for high-resolution parent peaks is shown to be useful for filter construction and deconvolution of inferior resolution SELDI-TOF spectra of calibration peptide mixture

  7. An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS.

    Science.gov (United States)

    Tsuchida, Sachio; Murata, Syota; Miyabe, Akiko; Satoh, Mamoru; Takiwaki, Masaki; Matsushita, Kazuyuki; Nomura, Fumio

    2018-05-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-μm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min. Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. The influence of incubation time, sample preparation and exposure to oxygen on the quality of the MALDI-TOF MS spectrum of anaerobic bacteria.

    Science.gov (United States)

    Veloo, A C M; Elgersma, P E; Friedrich, A W; Nagy, E; van Winkelhoff, A J

    2014-12-01

    With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), bacteria can be identified quickly and reliably. This accounts especially for anaerobic bacteria. Because growth rate and oxygen sensitivity differ among anaerobic bacteria, we aimed to study the influence of incubation time, exposure to oxygen and sample preparation on the quality of the spectrum using the Bruker system. Also, reproducibility and inter-examiner variability were determined. Twenty-six anaerobic species, representing 17 genera, were selected based on gram-stain characteristics, growth rate and colony morphology. Inter-examiner variation showed that experience in the preparation of the targets can be a significant variable. The influence of incubation time was determined between 24 and 96 h of incubation. Reliable species identification was obtained after 48 h of incubation for gram-negative anaerobes and after 72 h for gram-positive anaerobes. Exposure of the cultures to oxygen did not influence the results of the MALDI-TOF MS identifications of all tested gram-positive species. Fusobacterium necrophorum and Prevotella intermedia could not be identified after >24 h and 48 h of exposure to oxygen, respectively. Other tested gram-negative bacteria could be identified after 48 h of exposure to oxygen. Most of the tested species could be identified using the direct spotting method. Bifidobacterium longum and Finegoldia magna needed on-target extraction with 70% formic acid in order to obtain reliable species identification and Peptoniphilus ivorii a full extraction. Spectrum quality was influenced by the amount of bacteria spotted on the target, the homogeneity of the smear and the experience of the examiner. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

  9. Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

    Science.gov (United States)

    Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

    2014-01-01

    Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

  10. Quantitative lipidomic analysis of plasma and plasma lipoproteins using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Serna, Jorge; García-Seisdedos, David; Alcázar, Alberto; Lasunción, Miguel Ángel; Busto, Rebeca; Pastor, Óscar

    2015-07-01

    Knowledge of the plasma lipid composition is essential to clarify the specific roles of different lipid species in various pathophysiological processes. In this study, we developed an analytical strategy combining high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and off-line coupling with matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF/MS) to determine the composition of plasma and major lipoproteins at two levels, lipid classes and lipid species. We confirmed the suitability of MALDI-TOF/MS as a quantitative measurement tool studying the linearity and repeatability for triglycerides (TG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, data obtained with this method were correlated with other lipid classes and species measurements using currently available technologies. To establish the potential utility of our approach, human plasma very low density- (VLDL), low density- (LDL) and high density- (HDL) lipoproteins from 10 healthy donors were separated using ultracentrifugation, and compositions of nine lipid classes, cholesteryl esters (CE), TG, free cholesterol (FC), PE, phosphatidylinositol (PI), sulfatides (S), PC, lysophosphatidylcholine (LPC) and sphingomyelin (SM), analyzed. In total, 157 lipid species in plasma, 182 in LDL, 171 in HDL, and 148 in VLDL were quantified. The lipidomic profile was consistent with known differences in lipid classes, but also revealed unexpected differences in lipid species distribution of lipoproteins, particularly for LPC and SM. In summary, the methodology developed in this study constitutes a valid approach to determine the lipidomic composition of plasma and lipoproteins. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. LIMPIC: a computational method for the separation of protein MALDI-TOF-MS signals from noise

    Directory of Open Access Journals (Sweden)

    Di Nicola Marta

    2007-03-01

    Full Text Available Abstract Background Mass spectrometry protein profiling is a promising tool for biomarker discovery in clinical proteomics. However, the development of a reliable approach for the separation of protein signals from noise is required. In this paper, LIMPIC, a computational method for the detection of protein peaks from linear-mode MALDI-TOF data is proposed. LIMPIC is based on novel techniques for background noise reduction and baseline removal. Peak detection is performed considering the presence of a non-homogeneous noise level in the mass spectrum. A comparison of the peaks collected from multiple spectra is used to classify them on the basis of a detection rate parameter, and hence to separate the protein signals from other disturbances. Results LIMPIC preprocessing proves to be superior than other classical preprocessing techniques, allowing for a reliable decomposition of the background noise and the baseline drift from the MALDI-TOF mass spectra. It provides lower coefficient of variation associated with the peak intensity, improving the reliability of the information that can be extracted from single spectra. Our results show that LIMPIC peak-picking is effective even in low protein concentration regimes. The analytical comparison with commercial and freeware peak-picking algorithms demonstrates its superior performances in terms of sensitivity and specificity, both on in-vitro purified protein samples and human plasma samples. Conclusion The quantitative information on the peak intensity extracted with LIMPIC could be used for the recognition of significant protein profiles by means of advanced statistic tools: LIMPIC might be valuable in the perspective of biomarker discovery.

  12. Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis.

    Science.gov (United States)

    Spraggins, Jeffrey M; Rizzo, David G; Moore, Jessica L; Noto, Michael J; Skaar, Eric P; Caprioli, Richard M

    2016-06-01

    MALDI imaging mass spectrometry is a powerful analytical tool enabling the visualization of biomolecules in tissue. However, there are unique challenges associated with protein imaging experiments including the need for higher spatial resolution capabilities, improved image acquisition rates, and better molecular specificity. Here we demonstrate the capabilities of ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR IMS platforms as they relate to these challenges. High spatial resolution MALDI-TOF protein images of rat brain tissue and cystic fibrosis lung tissue were acquired at image acquisition rates >25 pixels/s. Structures as small as 50 μm were spatially resolved and proteins associated with host immune response were observed in cystic fibrosis lung tissue. Ultra-high speed MALDI-TOF enables unique applications including megapixel molecular imaging as demonstrated for lipid analysis of cystic fibrosis lung tissue. Additionally, imaging experiments using MALDI FTICR IMS were shown to produce data with high mass accuracy (z 5000) for proteins up to ∼20 kDa. Analysis of clear cell renal cell carcinoma using MALDI FTICR IMS identified specific proteins localized to healthy tissue regions, within the tumor, and also in areas of increased vascularization around the tumor. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Source-identifying biomarker ions between environmental and clinical Burkholderia pseudomallei using whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Niyompanich, Suthamat; Jaresitthikunchai, Janthima; Srisanga, Kitima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

    2014-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, which is an endemic disease in Northeast Thailand and Northern Australia. Environmental reservoirs, including wet soils and muddy water, serve as the major sources for contributing bacterial infection to both humans and animals. The whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has recently been applied as a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In this present study, we employed a whole-cell MALDI-TOF MS approach for assessing its potency in clustering a total of 11 different B. pseudomallei isolates (consisting of 5 environmental and 6 clinical isolates) with respect to their origins and to further investigate the source-identifying biomarker ions belonging to each bacterial group. The cluster analysis demonstrated that six out of eleven isolates were grouped correctly to their sources. Our results revealed a total of ten source-identifying biomarker ions, which exhibited statistically significant differences in peak intensity between average environmental and clinical mass spectra using ClinProTools software. Six out of ten mass ions were assigned as environmental-identifying biomarker ions (EIBIs), including, m/z 4,056, 4,214, 5,814, 7,545, 7,895, and 8,112, whereas the remaining four mass ions were defined as clinical-identifying biomarker ions (CIBIs) consisting of m/z 3,658, 6,322, 7,035, and 7,984. Hence, our findings represented, for the first time, the source-specific biomarkers of environmental and clinical B. pseudomallei.

  14. Evaluation of MALDI-TOF mass spectrometry for the competitiveness analysis of selected indigenous cowpea (Vigna unguiculata L. Walp.) Bradyrhizobium strains from Kenya.

    Science.gov (United States)

    Ndungu, Samuel Mathu; Messmer, Monika M; Ziegler, Dominik; Thuita, Moses; Vanlauwe, Bernard; Frossard, Emmanuel; Thonar, Cécile

    2018-06-01

    Cowpea N 2 fixation and yield can be enhanced by selecting competitive and efficient indigenous rhizobia. Strains from contrasting agro-ecologies of Kilifi and Mbeere (Kenya) were screened. Two pot experiments were established consisting of 13 Bradyrhizobium strains; experiment 1 (11 Mbeere + CBA + BK1 from Burkina Faso), experiment 2 (12 Kilifi + CBA). Symbiotic effectiveness was assessed (shoot biomass, SPAD index and N uptake). Nodule occupancy of 13 simultaneously co-inoculated strains in each experiment was analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to assess competitiveness. Strains varied in effectiveness and competitiveness. The four most efficient strains were further evaluated in a field trial in Mbeere during the 2014 short rains. Strains from bacteroids of cowpea nodules from pot and field experiments were accurately identified as Bradyrhizobium by MALDI-TOF based on the SARAMIS™ database. In the field, abundant indigenous populations 7.10 × 10 3 rhizobia g -1 soil, outcompeted introduced strains. As revealed by MALDI-TOF, indigenous strains clustered into six distinct groups (I, II, III, IV, V and VI), group III were most abundant occupying 80% of nodules analyzed. MALDI-TOF was rapid, affordable and reliable to identify Bradyrhizobium strains directly from nodule suspensions in competition pot assays and in the field with abundant indigenous strains thus, its suitability for future competition assays. Evaluating strain competitiveness and then symbiotic efficacy is proposed in bioprospecting for potential cowpea inoculant strains.

  15. Characterization of Novel Fusaricidins Produced by Paenibacillus polymyxa-M1 Using MALDI-TOF Mass Spectrometry

    Science.gov (United States)

    Vater, Joachim; Niu, Ben; Dietel, Kristin; Borriss, Rainer

    2015-09-01

    Paenibacillus polymyxa-M1 is a potent producer of bioactive compounds, such as lipopeptides, polyketides, and lantibiotics of biotechnological and medical interest. Genome sequencing revealed nine gene clusters for nonribosomal biosynthesis of such agents. Here we report on the investigation of the fusaricidins, a complex of cyclic lipopeptides containing 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid component by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). More than 20 variants of these compounds were detected and characterized in detail. Mass spectrometric sequence analysis was performed by MALDI-LIFT-TOF/TOF fragment analysis. The obtained product ion spectra show a specific processing in the fatty acid part. GHPD is cleaved between the α- and ß-position yielding two fragments a and b, one bearing the end-standing guanidine group and another one comprising the residual two C-atoms of GHPD with the attached peptide moiety. The complete sequence of all fusaricidins was derived from sets of bn- and yn-ions. The fusaricidin complex can be divided into four lipopeptide families, three of them showing variations of the amino acid in position 3, Val or Ile for the first and Tyr or Phe for families 2 and 3, respectively. A collection of novel fusaricidins was detected differing from those of families 1-3 by an additional residue of 71 Da (family 4). LIFT-TOF/TOF fragment spectra of these species imply that in their peptide moiety, an Ala-residue is attached by an ester bond to the free hydroxyl group of Thr4. More than 10 novel fusaricidins were characterized mass spectrometrically.

  16. Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF

    OpenAIRE

    Tanner, Hannah; Evans, Jason T.; Gossain, Savita; Hussain, Abid

    2017-01-01

    Background Patient mortality is significantly reduced by rapid identification of bacteria from sterile sites. MALDI-TOF can identify bacteria directly from positive blood cultures and multiple sample preparation methods are available. We evaluated three sample preparation methods and two MALDI-TOF score cut-off values. Positive blood culture bottles with organisms present in Gram stains were prospectively analysed by MALDI-TOF. Three lysis reagents (Saponin, SDS, and SepsiTyper lysis bufer) w...

  17. MALDI-TOF and cluster-TOF-SIMS imaging of Fabry disease biomarkers

    Science.gov (United States)

    Touboul, David; Roy, Sandrine; Germain, Dominique P.; Chaminade, Pierre; Brunelle, Alain; Laprevote, Olivier

    2007-02-01

    Fabry disease is an X-linked disorder of glycosphingolipid metabolism, in which a partial or total deficiency of [alpha]-galactosidase A, a lysosomal enzyme, results in the progressive accumulation of neutral glycosphingolipids (globotriaosylceramide and digalactosylceramide) in most fluids and tissues of the body. Few information is available about the composition and distribution in tissues of the accumulated glycosphingolipids species. Mass spectrometry imaging is an innovative technique, which can provide pieces of information about the distribution of numerous biological compounds, such as lipids, directly on the tissue sections. MALDI-TOF and cluster-TOF-SIMS imaging approaches were used to study the localization of lipids (cholesterol, cholesterol sulfate, vitamin E, glycosphingolipids ...) on skin and kidney sections of patients affected by the Fabry disease. Numerous information on pathophysiology were enlightened by both techniques.

  18. Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF.

    Science.gov (United States)

    Tanner, Hannah; Evans, Jason T; Gossain, Savita; Hussain, Abid

    2017-01-18

    Patient mortality is significantly reduced by rapid identification of bacteria from sterile sites. MALDI-TOF can identify bacteria directly from positive blood cultures and multiple sample preparation methods are available. We evaluated three sample preparation methods and two MALDI-TOF score cut-off values. Positive blood culture bottles with organisms present in Gram stains were prospectively analysed by MALDI-TOF. Three lysis reagents (Saponin, SDS, and SepsiTyper lysis bufer) were applied to each positive culture followed by centrifugation, washing and protein extraction steps. Methods were compared using the McNemar test and 16S rDNA sequencing was used to assess discordant results. In 144 monomicrobial cultures, using ≥2.000 as the cut-off value, species level identifications were obtained from 69/144 (48%) samples using Saponin, 86/144 (60%) using SDS, and 91/144 (63%) using SepsiTyper. The difference between SDS and SepsiTyper was not statistically significant (P = 0.228). Differences between Saponin and the other two reagents were significant (P direct MALDI-TOF identification were observed in monomicrobial cultures. In 32 polymicrobial cultures, MALDI-TOF identified one organism in 34-75% of samples depending on the method. This study demonstrates two inexpensive in-house detergent lysis methods are non-inferior to a commercial kit for analysis of positive blood cultures by direct MALDI-TOF in a clinical diagnostic microbiology laboratory.

  19. Evaluation of MALDI-TOF Mass Spectrometry and Sepsityper Kit™ for the Direct Identification of Organisms from Sterile Body Fluids in a Canadian Pediatric Hospital

    Directory of Open Access Journals (Sweden)

    Manal Tadros

    2013-01-01

    Full Text Available Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS can be used to identify bacteria directly from positive blood and sterile fluid cultures. The authors evaluated a commercially available kit – the Sepsityper Kit (Bruker Daltonik, Germany – and MALDI-TOF MS for the rapid identification of organisms from 80 flagged positive blood culture broths, of which 73 (91.2% were blood culture specimens and seven (8.7% were cerebrospinal fluid specimens, in comparison with conventional identification methods. Correct identification to the genus and species levels was obtained in 75 of 80 (93.8% and 39 of 50 (78% blood culture broths, respectively. Applying the blood culture analysis module, a newly developed software tool, improved the species identification of Gram-negative organisms from 94.7% to 100% and of Gram-positive organisms from 66.7% to 70%.

  20. A Novel Rapid MALDI-TOF-MS-Based Method for Measuring Urinary Globotriaosylceramide in Fabry Patients

    Science.gov (United States)

    Alharbi, Fahad J.; Geberhiwot, Tarekegn; Hughes, Derralynn A.; Ward, Douglas G.

    2016-04-01

    Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of α-galactosidase A, resulting in the accumulation of glycosphingolipids in various organs. Globotriaosylceramide (Gb3) and its isoforms and analogues have been identified and quantified as biomarkers of disease severity and treatment efficacy. The current study aimed to establish rapid methods for urinary Gb3 extraction and quantitation. Urine samples from 15 Fabry patients and 21 healthy control subjects were processed to extract Gb3 by mixing equal volumes of urine, methanol containing an internal standard, and chloroform followed by sonication and centrifugation. Thereafter, the lower phase was analyzed by MALDI-TOF MS and the relative peak areas of the internal standard and four major species of Gb3 determined. The results showed high reproducibility with intra- and inter-assay coefficients variation of 9.9% and 13.7%, respectively. The limit of detection was 0.15 ng/μL and the limit of quantitation was 0.30 ng/μL. Total urinary Gb3 levels in both genders of classic Fabry patients were significantly higher than in healthy controls (p < 0.0001). Gb3 levels in Fabry males were higher than in Fabry females (p = 0.08). We have established a novel assay for urinary total Gb3 that takes less than 15 min from start to finish.

  1. Semi Quantitative MALDI TOF for Antimicrobial Susceptibility Testing in Staphylococcus aureus

    Science.gov (United States)

    2017-08-31

    Semi- quantitative MALDI-TOF for antimicrobial susceptibility testing in Staphylococcus 1 aureus 2 3 4 Tucker Maxson,a Cheryl L. Taylor-Howell,a...Timothy D. Minoguea# 5 6 Diagnostic Systems Division, United States Army Medical Research Institute of Infectious 7 Disease, Fort Detrick, MD...USAa 8 9 Running Title: Quantitative MALDI for AST in S. aureus 10 #Address correspondence to Timothy D. Minogue, timothy.d.minogue.civ@mail.mil

  2. Using Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Complemented with Selected 16S rRNA and gyrB Genes Sequencing to Practically Identify Clinical Important Viridans Group Streptococci (VGS).

    Science.gov (United States)

    Zhou, Menglan; Yang, Qiwen; Kudinha, Timothy; Zhang, Li; Xiao, Meng; Kong, Fanrong; Zhao, Yupei; Xu, Ying-Chun

    2016-01-01

    There are challenges in viridans group streptococci (VGS) identification especially for the mitis group. Few studies have investigated the performance of MALDI-TOF MS system in VGS identification. Using 16S rRNA gene and gyrB gene sequencing as a gold standard, the performance of two MALDI-TOF MS instruments in the identification of 181 VGS clinical isolates was studied. The Bruker Biotyper and Vitek MS IVD systems correctly identified 88.4% and 98.9% of the 181 isolates, respectively. The Vitek MS RUO system was the least reliable, only correctly identifying 38.7% of the isolates to species level with several misidentifications and invalid results. The Bruker Biotyper system was very unreliable in the identification of species within the mitis group. Among 22 non-pneumococci isolates (S. mitis/S. oralis/S. pseudopneumoniae), Biotyper misidentified 21 of them as S. pneumoniae leading to a low sensitivity and low positive predictive value in these species. In contrast, the Vitek MS IVD demonstrated a better resolution for pneumococci and non-pneumococci despite the inability to distinguish between S. mitis/S. oralis. For more accurate species-level identification, further improvements in the VGS spectra databases are needed. Based on MALDI-TOF analysis and selected 16S rRNA gene plus gyrB genes sequencing, we designed a practical VGS identification algorithm.

  3. MALDI MS-based Composition Analysis of the Polymerization Reaction of Toluene Diisocyanate (TDI) and Ethylene Glycol (EG).

    Science.gov (United States)

    Ahn, Yeong Hee; Lee, Yeon Jung; Kim, Sung Ho

    2015-01-01

    This study describes an MS-based analysis method for monitoring changes in polymer composition during the polyaddition polymerization reaction of toluene diisocyanate (TDI) and ethylene glycol (EG). The polymerization was monitored as a function of reaction time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The resulting series of polymer adducts terminated with various end-functional groups were precisely identified and the relative compositions of those series were estimated. A new MALDI MS data interpretation method was developed, consisting of a peak-resolving algorithm for overlapping peaks in MALDI MS spectra, a retrosynthetic analysis for the generation of reduced unit mass peaks, and a Gaussian fit-based selection of the most prominent polymer series among the reconstructed unit mass peaks. This method of data interpretation avoids errors originating from side reactions due to the presence of trace water in the reaction mixture or MALDI analysis. Quantitative changes in the relative compositions of the resulting polymer products were monitored as a function of reaction time. These results demonstrate that the mass data interpretation method described herein can be a powerful tool for estimating quantitative changes in the compositions of polymer products arising during a polymerization reaction.

  4. MALDI-TOF mass spectrometry and high-consequence bacteria: safety and stability of biothreat bacterial sample testing in clinical diagnostic laboratories.

    Science.gov (United States)

    Tracz, Dobryan M; Tober, Ashley D; Antonation, Kym S; Corbett, Cindi R

    2018-03-01

    We considered the application of MALDI-TOF mass spectrometry for BSL-3 bacterial diagnostics, with a focus on the biosafety of live-culture direct-colony testing and the stability of stored extracts. Biosafety level 2 (BSL-2) bacterial species were used as surrogates for BSL-3 high-consequence pathogens in all live-culture MALDI-TOF experiments. Viable BSL-2 bacteria were isolated from MALDI-TOF mass spectrometry target plates after 'direct-colony' and 'on-plate' extraction testing, suggesting that the matrix chemicals alone cannot be considered sufficient to inactivate bacterial culture and spores in all samples. Sampling of the instrument interior after direct-colony analysis did not recover viable organisms, suggesting that any potential risks to the laboratory technician are associated with preparation of the MALDI-TOF target plate before or after testing. Secondly, a long-term stability study (3 years) of stored MALDI-TOF extracts showed that match scores can decrease below the threshold for reliable species identification (<1.7), which has implications for proficiency test panel item storage and distribution.

  5. Microbial identification and automated antibiotic susceptibility testing directly from positive blood cultures using MALDI-TOF MS and VITEK 2.

    Science.gov (United States)

    Wattal, C; Oberoi, J K

    2016-01-01

    The study addresses the utility of Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight mass spectrometry (MALDI-TOF MS) using VITEK MS and the VITEK 2 antimicrobial susceptibility testing (AST) system for direct identification (ID) and timely AST from positive blood culture bottles using a lysis-filtration method (LFM). Between July and December 2014, a total of 140 non-duplicate mono-microbial blood cultures were processed. An aliquot of positive blood culture broth was incubated with lysis buffer before the bacteria were filtered and washed. Micro-organisms recovered from the filter were first identified using VITEK MS and its suspension was used for direct AST by VITEK 2 once the ID was known. Direct ID and AST results were compared with classical methods using solid growth. Out of the 140 bottles tested, VITEK MS resulted in 70.7 % correct identification to the genus and/ or species level. For the 103 bottles where identification was possible, there was agreement in 97 samples (94.17 %) with classical culture. Compared to the routine method, the direct AST resulted in category agreement in 860 (96.5 %) of 891 bacteria-antimicrobial agent combinations tested. The results of direct ID and AST were available 16.1 hours before those of the standard approach on average. The combined use of VITEK MS and VITEK 2 directly on samples from positive blood culture bottles using a LFM technique can result in rapid and reliable ID and AST results in blood stream infections to result in early institution of targeted treatment. The combination of LFM and AST using VITEK 2 was found to expedite AST more reliably.

  6. Identification of Low Molecular Weight Glutenin Alleles by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) in Common Wheat (Triticum aestivum L.)

    Science.gov (United States)

    Islam, Shahidul; Applebee, Marie; Appels, Rudi; Yan, Yueming; Ma, Wujun

    2015-01-01

    Low molecular weight glutenin subunits (LMW-GS) play an important role in determining dough properties and breadmaking quality. However, resolution of the currently used methodologies for analyzing LMW-GS is rather low which prevents an efficient use of genetic variations associated with these alleles in wheat breeding. The aim of the current study is to evaluate and develop a rapid, simple, and accurate method to differentiate LMW-GS alleles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A set of standard single LMW-GS allele lines as well as a suite of well documented wheat cultivars were collected from France, CIMMYT, and Canada. Method development and optimization were focused on protein extraction procedures and MALDI-TOF instrument settings to generate reproducible diagnostic spectrum peak profiles for each of the known wheat LMW-GS allele. Results revealed a total of 48 unique allele combinations among the studied genotypes. Characteristic MALDI-TOF peak patterns were obtained for 17 common LMW-GS alleles, including 5 (b, a or c, d, e, f), 7 (a, b, c, d or i, f, g, h) and 5 (a, b, c, d, f) patterns or alleles for the Glu-A3, Glu-B3, and Glu-D3 loci, respectively. In addition, some reproducible MALDI-TOF peak patterns were also obtained that did not match with any known alleles. The results demonstrated a high resolution and throughput nature of MALDI-TOF technology in analyzing LMW-GS alleles, which is suitable for application in wheat breeding programs in processing a large number of wheat lines with high accuracy in limited time. It also suggested that the variation of LMW-GS alleles is more abundant than what has been defined by the current nomenclature system that is mainly based on SDS-PAGE system. The MALDI-TOF technology is useful to differentiate these variations. An international joint effort may be needed to assign allele symbols to these newly identified alleles and determine their effects on end

  7. MALDI-TOF MS analysis of condensed tannins with potent antioxidant activity from the leaf, stem bark and root bark of Acacia confusa.

    Science.gov (United States)

    Wei, Shu-Dong; Zhou, Hai-Chao; Lin, Yi-Ming; Liao, Meng-Meng; Chai, Wei-Ming

    2010-06-15

    The structures of the condensed tannins from leaf, stem bark and root bark of Acacia confusa were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and their antioxidant activities were measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and ferric reducing/antioxidant power (FRAP) assays. The results showed that the condensed tannins from stem bark and root bark include propelargonidin and procyanidin, and the leaf condensed tannins include propelargonidin, procyanidin and prodelphinidin, all with the procyanidin dominating. The condensed tannins had different polymer chain lengths, varying from trimers to undecamers for leaf and root bark and to dodecamers for stem bark. The condensed tannins extracted from the leaf, stem bark and root bark all showed a very good DPPH radical scavenging activity and ferric reducing power.

  8. Dansyl-peptides matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI-MS/MS analysis of the proteome.

    Science.gov (United States)

    Chiappetta, Giovanni; Ndiaye, Sega; Demey, Emmanuelle; Haddad, Iman; Marino, Gennaro; Amoresano, Angela; Vinh, Joëlle

    2010-10-30

    Peptide tagging is a useful tool to improve matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS-Cl). DNS-Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI-MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N-terminal DNS-Cl sulfonation improves the peptide fragmentation and promotes the generation of b-fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI-MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage. Copyright © 2010 John Wiley & Sons, Ltd.

  9. Identification of phlebotomine sand flies using one MALDI-TOF MS reference database and two mass spectrometer systems.

    Science.gov (United States)

    Mathis, Alexander; Depaquit, Jérôme; Dvořák, Vit; Tuten, Holly; Bañuls, Anne-Laure; Halada, Petr; Zapata, Sonia; Lehrter, Véronique; Hlavačková, Kristýna; Prudhomme, Jorian; Volf, Petr; Sereno, Denis; Kaufmann, Christian; Pflüger, Valentin; Schaffner, Francis

    2015-05-10

    Rapid, accurate and high-throughput identification of vector arthropods is of paramount importance in surveillance programmes that are becoming more common due to the changing geographic occurrence and extent of many arthropod-borne diseases. Protein profiling by MALDI-TOF mass spectrometry fulfils these requirements for identification, and reference databases have recently been established for several vector taxa, mostly with specimens from laboratory colonies. We established and validated a reference database containing 20 phlebotomine sand fly (Diptera: Psychodidae, Phlebotominae) species by using specimens from colonies or field-collections that had been stored for various periods of time. Identical biomarker mass patterns ('superspectra') were obtained with colony- or field-derived specimens of the same species. In the validation study, high quality spectra (i.e. more than 30 evaluable masses) were obtained with all fresh insects from colonies, and with 55/59 insects deep-frozen (liquid nitrogen/-80 °C) for up to 25 years. In contrast, only 36/52 specimens stored in ethanol could be identified. This resulted in an overall sensitivity of 87 % (140/161); specificity was 100 %. Duration of storage impaired data counts in the high mass range, and thus cluster analyses of closely related specimens might reflect their storage conditions rather than phenotypic distinctness. A major drawback of MALDI-TOF MS is the restricted availability of in-house databases and the fact that mass spectrometers from 2 companies (Bruker, Shimadzu) are widely being used. We have analysed fingerprints of phlebotomine sand flies obtained by automatic routine procedure on a Bruker instrument by using our database and the software established on a Shimadzu system. The sensitivity with 312 specimens from 8 sand fly species from laboratory colonies when evaluating only high quality spectra was 98.3 %; the specificity was 100 %. The corresponding diagnostic values with 55 field

  10. Comparison of different tandem mass spectrometric techniques (ESI-IT, ESI- and IP-MALDI-QRTOF and vMALDI-TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L.

    Science.gov (United States)

    Koulakiotis, Nikolaos Stavros; Pittenauer, Ernst; Halabalaki, Maria; Tsarbopoulos, Anthony; Allmaier, Günter

    2012-03-30

    The expensive spice saffron originating from the stigmas of Crocus sativus L. and also applied in traditional Chinese medicine (TCM) constitutes a complex mixture of glycoconjugates varying not only in the aglycon structure, but also in glycosylation pattern. Therefore, various tandem mass spectrometric techniques were evaluated for their usefulness in structural elucidation. Three selected constituents of the stigmas of Crocus sativus L., trans- and cis-crocin-4 as well as picrocrocin, were isolated and purified by HPLC and finally analyzed by ESI-MS (ion trap, QqRTOF), IP-MALDI-MS (QqRTOF) and vMALDI-MS (TOF/RTOF) in combination with tandem mass spectrometry in collision energy regimes ranging from a few eV (LE) to 20 keV (HE) collisions for the first time. These data aid in structurally elucidating minor, unknown glycoconjugates originating from this plant-derived spice. LE-CID of isomeric crocins on either an ion trap with ESI or a QqRTOF-instrument with ESI or IP-MALDI as desorption/ionization technique only yielded a limited number of structurally diagnostic sodiated product ions related to the carbohydrate moiety as well as to the intact aglycon in contrast to true HE-CID. The low MW constituent picrocrocin did not yield useful LE-CID spectra, but showed a high number of structurally diagnostic product ions by HE-CID utilizing a vMALDI TOF/RTOF-instrument. The highest number of structurally diagnostic product ions allowing also determination of the carbohydrate linkage of the gentiobiose-moiety of isomeric crocins ((0,4)A(2), (3,5)A(2) product ions indicating a 1→6 carbohydrate linkage) was only achievable by HE-CID. Fragmentation of the aglycon was not observed by any collision energy regime applied. Copyright © 2012 John Wiley & Sons, Ltd.

  11. NMR and MALDI-TOF MS based characterization of exopolysaccharides in anaerobic microbial aggregates from full-scale reactors

    KAUST Repository

    Gonzalez-Gil, Graciela

    2015-09-22

    Anaerobic granular sludge is composed of multispecies microbial aggregates embedded in a matrix of extracellular polymeric substances (EPS). Here we characterized the chemical fingerprint of the polysaccharide fraction of EPS in anaerobic granules obtained from full-scale reactors treating different types of wastewater. Nuclear magnetic resonance (NMR) signals of the polysaccharide region from the granules were very complex, likely as a result of the diverse microbial population in the granules. Using nonmetric multidimensional scaling (NMDS), the 1H NMR signals of reference polysaccharides (gellan, xanthan, alginate) and those of the anaerobic granules revealed that there were similarities between the polysaccharides extracted from granules and the reference polysaccharide alginate. Further analysis of the exopolysaccharides from anaerobic granules, and reference polysaccharides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that exopolysaccharides from two of the anaerobic granular sludges studied exhibited spectra similar to that of alginate. The presence of sequences related to the synthesis of alginate was confirmed in the metagenomes of the granules. Collectively these results suggest that alginate-like exopolysaccharides are constituents of the EPS matrix in anaerobic granular sludge treating different industrial wastewater. This finding expands the engineered environments where alginate has been found as EPS constituent of microbial aggregates.

  12. NMR and MALDI-TOF MS based characterization of exopolysaccharides in anaerobic microbial aggregates from full-scale reactors

    KAUST Repository

    Gonzalez-Gil, Graciela; Thomas, Ludivine; Emwas, Abdul-Hamid M.; Lens, Piet N. L.; Saikaly, Pascal

    2015-01-01

    Anaerobic granular sludge is composed of multispecies microbial aggregates embedded in a matrix of extracellular polymeric substances (EPS). Here we characterized the chemical fingerprint of the polysaccharide fraction of EPS in anaerobic granules obtained from full-scale reactors treating different types of wastewater. Nuclear magnetic resonance (NMR) signals of the polysaccharide region from the granules were very complex, likely as a result of the diverse microbial population in the granules. Using nonmetric multidimensional scaling (NMDS), the 1H NMR signals of reference polysaccharides (gellan, xanthan, alginate) and those of the anaerobic granules revealed that there were similarities between the polysaccharides extracted from granules and the reference polysaccharide alginate. Further analysis of the exopolysaccharides from anaerobic granules, and reference polysaccharides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that exopolysaccharides from two of the anaerobic granular sludges studied exhibited spectra similar to that of alginate. The presence of sequences related to the synthesis of alginate was confirmed in the metagenomes of the granules. Collectively these results suggest that alginate-like exopolysaccharides are constituents of the EPS matrix in anaerobic granular sludge treating different industrial wastewater. This finding expands the engineered environments where alginate has been found as EPS constituent of microbial aggregates.

  13. Direct identification from Bact/Alert™ blood culture bottles by MALDI-TOF

    Directory of Open Access Journals (Sweden)

    Vesselina Kroumova

    2011-12-01

    Full Text Available Bacterial identification from blood culture using traditional methods needs about 48 hours, since positivization, to be performed. Rapid bacterial identification can result in clinical and economic benefits. To provide rapid pathogen identification for targeted antibiotic treatment, in this study we tested an our previously described homemade method for bacterial identification using MALDI-TOF directly from positive BACTEC blood culture, on positive BacT/ALERT blood culture. A total of 108 bacteria were identified by MALDI-TOF with a positive identification obtained for 98% of Gram negative and 84,3% of Gram positive bacteria.The average of identification score obtained using the protocol described in this study was 2,047 for Gram positive and 2,204 for Gram negative microorganisms. Data here described show that this method is also useful when BacT/ALERT bottles are used and even if these bottles have activated charcoal as inhibitor of antibiotics.

  14. Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation.

    Science.gov (United States)

    Labas, Valérie; Teixeira-Gomes, Ana-Paula; Bouguereau, Laura; Gargaros, Audrey; Spina, Lucie; Marestaing, Aurélie; Uzbekova, Svetlana

    2018-03-20

    Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

    Science.gov (United States)

    Heap, Rachel E; Hope, Anthony G; Pearson, Lesley-Anne; Reyskens, Kathleen M S E; McElroy, Stuart P; Hastie, C James; Porter, David W; Arthur, J Simon C; Gray, David W; Trost, Matthias

    2017-12-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC 50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

  16. Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet.

    Science.gov (United States)

    Musyimi, Harrison K; Guy, Jason; Narcisse, Damien A; Soper, Steven A; Murray, Kermit K

    2005-12-01

    We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.

  17. Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

    DEFF Research Database (Denmark)

    Guillaumie, Fanny; Sterling, J.D.; Jensen, K.J.

    2003-01-01

    Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through...... into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (alpha-D-GalA)(14) and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)(15)). When UDP-GalA was added...

  18. DBDA as a Novel Matrix for the Analyses of Small Molecules and Quantification of Fatty Acids by Negative Ion MALDI-TOF MS

    Science.gov (United States)

    Ling, Ling; Li, Ying; Wang, Sheng; Guo, Liming; Xiao, Chunsheng; Chen, Xuesi; Guo, Xinhua

    2018-01-01

    Matrix interference ions in low mass range has always been a concern when using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze small molecules (fatty acids without matrix interference appeared in the mass spectra and the limit of detection (LOD) reached 0.3 fmol. DBDA also has great performance towards other small molecules such as amino acids, peptides, and nucleotide. Furthermore, with this novel matrix, the free fatty acids in serum were quantitatively analyzed based on the correlation curves with correlation coefficient of 0.99. In addition, UV-Vis experiments and molecular orbital calculations were performed to explore mechanism about DBDA used as matrix in the negative ion mode. The present work shows that the DBDA matrix is a highly sensitive matrix with few interference ions for analysis of small molecules. Meanwhile, DBDA is able to precisely quantify the fatty acids in real biological samples. [Figure not available: see fulltext.

  19. Qualitative and quantitative analysis of pharmaceutical compounds by MALDI-TOF mass spectrometry.

    NARCIS (Netherlands)

    Kampen, J.J. van; Burgers, P.C.; Groot, R. de; Luider, T.M.

    2006-01-01

    In this report, we discuss key issues for the successful application of MALDI-TOF mass spectrometry to quantify drugs. These include choice and preparation of matrix, nature of cationization agent, automation, and data analysis procedures. The high molecular weight matrix

  20. MALDI-TOF mass spectrometry for rapid diagnosis of postoperative endophthalmitis.

    Science.gov (United States)

    Mailhac, Adriane; Durand, Harmonie; Boisset, Sandrine; Maubon, Danièle; Berger, Francois; Maurin, Max; Chiquet, Christophe; Bidart, Marie

    2017-01-30

    This study describes an innovative strategy for rapid detection and identification of bacteria causing endophthalmitis, combining the use of an automated blood culture system with MALDI-TOF mass spectrometry methodology. Using this protocol, we could identify 96% of 45 bacterial strains isolated from vitreous samples collected in acute post-operative endophthalmitis patients. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. The Technical and Biological Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Based Typing: Employment of Bioinformatics in a Multicenter Study.

    Science.gov (United States)

    Oberle, Michael; Wohlwend, Nadia; Jonas, Daniel; Maurer, Florian P; Jost, Geraldine; Tschudin-Sutter, Sarah; Vranckx, Katleen; Egli, Adrian

    2016-01-01

    The technical, biological, and inter-center reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) typing data has not yet been explored. The aim of this study is to compare typing data from multiple centers employing bioinformatics using bacterial strains from two past outbreaks and non-related strains. Participants received twelve extended spectrum betalactamase-producing E. coli isolates and followed the same standard operating procedure (SOP) including a full-protein extraction protocol. All laboratories provided visually read spectra via flexAnalysis (Bruker, Germany). Raw data from each laboratory allowed calculating the technical and biological reproducibility between centers using BioNumerics (Applied Maths NV, Belgium). Technical and biological reproducibility ranged between 96.8-99.4% and 47.6-94.4%, respectively. The inter-center reproducibility showed a comparable clustering among identical isolates. Principal component analysis indicated a higher tendency to cluster within the same center. Therefore, we used a discriminant analysis, which completely separated the clusters. Next, we defined a reference center and performed a statistical analysis to identify specific peaks to identify the outbreak clusters. Finally, we used a classifier algorithm and a linear support vector machine on the determined peaks as classifier. A validation showed that within the set of the reference center, the identification of the cluster was 100% correct with a large contrast between the score with the correct cluster and the next best scoring cluster. Based on the sufficient technical and biological reproducibility of MALDI-TOF MS based spectra, detection of specific clusters is possible from spectra obtained from different centers. However, we believe that a shared SOP and a bioinformatics approach are required to make the analysis robust and reliable.

  2. The Technical and Biological Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS Based Typing: Employment of Bioinformatics in a Multicenter Study.

    Directory of Open Access Journals (Sweden)

    Michael Oberle

    Full Text Available The technical, biological, and inter-center reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS typing data has not yet been explored. The aim of this study is to compare typing data from multiple centers employing bioinformatics using bacterial strains from two past outbreaks and non-related strains.Participants received twelve extended spectrum betalactamase-producing E. coli isolates and followed the same standard operating procedure (SOP including a full-protein extraction protocol. All laboratories provided visually read spectra via flexAnalysis (Bruker, Germany. Raw data from each laboratory allowed calculating the technical and biological reproducibility between centers using BioNumerics (Applied Maths NV, Belgium.Technical and biological reproducibility ranged between 96.8-99.4% and 47.6-94.4%, respectively. The inter-center reproducibility showed a comparable clustering among identical isolates. Principal component analysis indicated a higher tendency to cluster within the same center. Therefore, we used a discriminant analysis, which completely separated the clusters. Next, we defined a reference center and performed a statistical analysis to identify specific peaks to identify the outbreak clusters. Finally, we used a classifier algorithm and a linear support vector machine on the determined peaks as classifier. A validation showed that within the set of the reference center, the identification of the cluster was 100% correct with a large contrast between the score with the correct cluster and the next best scoring cluster.Based on the sufficient technical and biological reproducibility of MALDI-TOF MS based spectra, detection of specific clusters is possible from spectra obtained from different centers. However, we believe that a shared SOP and a bioinformatics approach are required to make the analysis robust and reliable.

  3. An impulse-driven liquid-droplet deposition interface for combining LC with MALDI MS and MS/MS.

    Science.gov (United States)

    Young, J Bryce; Li, Liang

    2006-03-01

    A simple and robust impulse-driven droplet deposition system was developed for off-line liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS). The system uses a solenoid operated with a pulsed voltage power supply to generate impulses that dislodge the hanging droplets from the LC outlet directly to a MALDI plate via a momentum transfer process. There is no contact between the LC outlet and the collection surface. The system is compatible with solvents of varying polarity and viscosity, and accommodates the use of hydrophobic and hydrophilic MALDI matrices. MALDI spots are produced on-line with the separation, and do not require further processing before MS analysis. It is shown that high quality MALDI spectra from 5 fmol of pyro-Glu-fibrinopeptide deposition after LC separation could be obtained using the device, indicating that there was no sample loss in the interface. To demonstrate the analytical performance of the system as a proteome analysis tool, a range of BSA digest concentrations covering about 3 orders of magnitude, from 5 fmol to 1 pmol, were analyzed by LC-MALDI quadrupole time-of-flight MS, yielding 6 and 57% amino acid sequence coverage, respectively. In addition, a complex protein mixture of an E. coli cell extract was tryptically digested and analyzed by LC-MALDI MS, resulting in the detection of a total of 409 unique peptides from 100 fractions of 15-s intervals.

  4. MALDI-TOF mass spectrometry and microsatellite markers to evaluate Candida parapsilosis transmission in neonatal intensive care units.

    Science.gov (United States)

    Pulcrano, G; Roscetto, E; Iula, V D; Panellis, D; Rossano, F; Catania, M R

    2012-11-01

    Recent studies on outbreaks of Candida showed an increased incidence of bloodstream infections in neonatal intensive care units (NICUs) caused by C. parapsilosis species, highlighting the need for the proper identification and epidemiology of these species. Several systems are available for molecular epidemiological and taxonomic studies of fungal infections: pulsed-field gel electrophoresis (PFGE) represents the gold standard for typing, but is also one of the most lengthy and expensive, while simple sequence repeats (SSRs) is based on polymerase chain reaction (PCR) amplification and is, therefore, faster. Only recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to identify and type microorganisms involved in nosocomial outbreaks. In our study, 19 strains of C. parapsilosis isolated from the blood cultures of neonates admitted to the University Hospital Federico II were genotyped by the amplification of eight SSR markers and by MALDI-TOF MS. Electrophoretic and spectrometric profile results were compared in order to identify similarities among the isolates and to study microevolutionary changes in the C. parapsilosis population. The discriminatory power and the unweighted pair group method with arithmetic mean (UPGMA) dendrograms generated were compared in order to evaluate the correlation of the groups established by the analysis of the clusters by both methods. Both methods were rapid and effective in highlighting identical strains and studying microevolutionary changes in the population. Our study evidenced that mass spectroscopy is a useful technique not only for the identification but also for monitoring the spread of strains, which is critical to control nosocomial infections.

  5. Rapid identification and susceptibility testing of Candida spp. from positive blood cultures by combination of direct MALDI-TOF mass spectrometry and direct inoculation of Vitek 2.

    Science.gov (United States)

    Idelevich, Evgeny A; Grunewald, Camilla M; Wüllenweber, Jörg; Becker, Karsten

    2014-01-01

    Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥ 1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤ 1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.

  6. Rapid identification and susceptibility testing of Candida spp. from positive blood cultures by combination of direct MALDI-TOF mass spectrometry and direct inoculation of Vitek 2.

    Directory of Open Access Journals (Sweden)

    Evgeny A Idelevich

    Full Text Available Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥ 1.5 with two identical consecutive propositions, 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤ 1 double dilution step was 88.6%. Very major errors (VMEs (false-susceptibility, major errors (false-resistance and minor errors (false categorization involving intermediate result amounted to 33.3% (of resistant isolates, 1.9% (of susceptible isolates and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.

  7. Quantitative Interpretation of MALDI-TOF Mass Spectra of Imperfect Carbosilane Dendrimers.

    Czech Academy of Sciences Publication Activity Database

    Krupková, Alena; Čermák, Jan; Walterová, Zuzana; Horský, Jiří

    2007-01-01

    Roč. 79, 4 (2007) , s. 1639-1645 ISSN 0003-2700 R&D Projects: GA MŠk(CZ) LC06070 Institutional research plan: CEZ:AV0Z40720504; CEZ:AV0Z40500505 Keywords : carbosilane dendrimer s * MALDI-TOF * statistics of defects Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 5.287, year: 2007

  8. Time is of essence; rapid identification of veterinary pathogens using MALDI TOF

    DEFF Research Database (Denmark)

    Nonnemann, Bettina; Dalsgaard, Inger; Pedersen, Karl

    Rapid and accurate identification of microbial pathogens is a cornerstone for timely and correct treatment of diseases of livestock and fish. The utility of the MALDI-TOF technique in the diagnostic laboratory is directly related to the quality of mass spectra and quantity of different microbial...

  9. MALDI-TOF MS for the Identification of Cultivable Organic-Degrading Bacteria in Contaminated Groundwater near Unconventional Natural Gas Extraction Sites

    Directory of Open Access Journals (Sweden)

    Inês C. Santos

    2017-08-01

    Full Text Available Groundwater quality and quantity is of extreme importance as it is a source of drinking water in the United States. One major concern has emerged due to the possible contamination of groundwater from unconventional oil and natural gas extraction activities. Recent studies have been performed to understand if these activities are causing groundwater contamination, particularly with respect to exogenous hydrocarbons and volatile organic compounds. The impact of contaminants on microbial ecology is an area to be explored as alternatives for water treatment are necessary. In this work, we identified cultivable organic-degrading bacteria in groundwater in close proximity to unconventional natural gas extraction. Pseudomonas stutzeri and Acinetobacter haemolyticus were identified using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS, which proved to be a simple, fast, and reliable method. Additionally, the potential use of the identified bacteria in water and/or wastewater bioremediation was studied by determining the ability of these microorganisms to degrade toluene and chloroform. In fact, these bacteria can be potentially applied for in situ bioremediation of contaminated water and wastewater treatment, as they were able to degrade both compounds.

  10. MALDI-TOF MS for the Identification of Cultivable Organic-Degrading Bacteria in Contaminated Groundwater near Unconventional Natural Gas Extraction Sites.

    Science.gov (United States)

    Santos, Inês C; Martin, Misty S; Carlton, Doug D; Amorim, Catarina L; Castro, Paula M L; Hildenbrand, Zacariah L; Schug, Kevin A

    2017-08-10

    Groundwater quality and quantity is of extreme importance as it is a source of drinking water in the United States. One major concern has emerged due to the possible contamination of groundwater from unconventional oil and natural gas extraction activities. Recent studies have been performed to understand if these activities are causing groundwater contamination, particularly with respect to exogenous hydrocarbons and volatile organic compounds. The impact of contaminants on microbial ecology is an area to be explored as alternatives for water treatment are necessary. In this work, we identified cultivable organic-degrading bacteria in groundwater in close proximity to unconventional natural gas extraction. Pseudomonas stutzeri and Acinetobacter haemolyticus were identified using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS), which proved to be a simple, fast, and reliable method. Additionally, the potential use of the identified bacteria in water and/or wastewater bioremediation was studied by determining the ability of these microorganisms to degrade toluene and chloroform. In fact, these bacteria can be potentially applied for in situ bioremediation of contaminated water and wastewater treatment, as they were able to degrade both compounds.

  11. [MALDI-TOF MASS-SPECTROMETRIC ANAIYSIS OF LEPTOSPIRA SPP. USED IN SERODIAGNOSTICS OF LEPTOSPIROSIS].

    Science.gov (United States)

    Zyeva, E V; Stoyanova, N A; Tokarevich, N K; Totolyan, Areg A

    2015-01-01

    Creation of a classification model of Leptospira spp. serovar model using ClinProTools 3.0 software and evaluation of use of MALDI-TOF MS as a method of quality control of reference strains of leptospira. 10 reference strains of Leptospira spp. were used in the study according to microscopic agglutination reaction from the collection of Pasteur RIEM. All the strains were cultivated for 10 days in Terskikh medium at 28 degrees C. Cell extracts were obtained by ethanol/formic acid method. α-cyano-4-hydroxycinnamic acid solution was used as a matrix. Mass-spectra were obtained in Microflex mass-spectrometer (Bruker Daltonics, Germany). External validation of the test-model was carried out using novel spectra of every reference strain during their repeated reseeding. Values of cross-validation and confirmatory ability of the optimal model, built on a genetic algorithm, was 99.14 and 100%, respectively. This model contained 11 biomarker peaks (m/z 2959, 3447, 3548, 3764, 3895, 5221, 5917, 6173, 6701, 7013, 8364) for serovar classification. Results of the external validation have shown a 100% correct classification in serovar classesin Sejroe, Ballum, Tarassovi; Copenhageni, Mozdoc, Grippotyphosa and Patoc, that indicates a high prognostic ability of the model in these classes. However, data from verification matrix have shown, that 50%.of the spectra from Canicola and Pomona serovars were classified as Patoc class, that could be associated with cross serological activity of Patoc serovar L. biflexa with pathogenic leptospirae. MALDI-TOF mass-spectrometry method combined with building and using the classification model could be a useful instrument for intra-laboratory control of leptospira reseeding.

  12. Peptide Peak Detection for Low Resolution MALDI-TOF Mass Spectrometry.

    Science.gov (United States)

    Yao, Jingwen; Utsunomiya, Shin-Ichi; Kajihara, Shigeki; Tabata, Tsuyoshi; Aoshima, Ken; Oda, Yoshiya; Tanaka, Koichi

    2014-01-01

    A new peak detection method has been developed for rapid selection of peptide and its fragment ion peaks for protein identification using tandem mass spectrometry. The algorithm applies classification of peak intensities present in the defined mass range to determine the noise level. A threshold is then given to select ion peaks according to the determined noise level in each mass range. This algorithm was initially designed for the peak detection of low resolution peptide mass spectra, such as matrix-assisted laser desorption/ionization Time-of-Flight (MALDI-TOF) mass spectra. But it can also be applied to other type of mass spectra. This method has demonstrated obtaining a good rate of number of real ions to noises for even poorly fragmented peptide spectra. The effect of using peak lists generated from this method produces improved protein scores in database search results. The reliability of the protein identifications is increased by finding more peptide identifications. This software tool is freely available at the Mass++ home page (http://www.first-ms3d.jp/english/achievement/software/).

  13. Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS

    Science.gov (United States)

    Sides, Cynthia R.; Liyanage, Rohana; Lay, Jackson O.; Philominathan, Sagaya Theresa Leena; Matsushita, Osamu; Sakon, Joshua

    2012-03-01

    Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.

  14. Two Classifiers Based on Serum Peptide Pattern for Prediction of HBV-Induced Liver Cirrhosis Using MALDI-TOF MS

    Directory of Open Access Journals (Sweden)

    Yuan Cao

    2013-01-01

    Full Text Available Chronic infection with hepatitis B virus (HBV is associated with the majority of cases of liver cirrhosis (LC in China. Although liver biopsy is the reference method for evaluation of cirrhosis, it is an invasive procedure with inherent risk. The aim of this study is to discover novel noninvasive specific serum biomarkers for the diagnosis of HBV-induced LC. We performed bead fractionation/MALDI-TOF MS analysis on sera from patients with LC. Thirteen feature peaks which had optimal discriminatory performance were obtained by using support-vector-machine-(SVM- based strategy. Based on the previous results, five supervised machine learning methods were employed to construct classifiers that discriminated proteomic spectra of patients with HBV-induced LC from those of controls. Here, we describe two novel methods for prediction of HBV-induced LC, termed LC-NB and LC-MLP, respectively. We obtained a sensitivity of 90.9%, a specificity of 94.9%, and overall accuracy of 93.8% on an independent test set. Comparisons with the existing methods showed that LC-NB and LC-MLP held better accuracy. Our study suggests that potential serum biomarkers can be determined for discriminating LC and non-LC cohorts by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These two classifiers could be used for clinical practice in HBV-induced LC assessment.

  15. Identification of strains with phenotypes similar to those of Staphylococcus aureus isolated from table chicken eggs using MALDI-TOF MS and genotyping methods

    Directory of Open Access Journals (Sweden)

    Marek Agnieszka

    2015-06-01

    Full Text Available The aim of the study was to identify the affinity of 10 Staphylococcus strains isolated from table chicken eggs to specific species. Preliminary analysis performed by API ID32 Staph test identified these strains as S. aureus, but they exhibited a negative reaction in the tube coagulase test. Thus, the analysed strains were initially characterised as Staphylococcus aureus-like (SAL. Further characterisation was performed by genotypic methods, using restriction fragment length polymorphism (RFLP of the coagulase gene (coa and sequencing of the gene rpoB. An attempt was also made to identify the isolated Staphylococcus strains by MALDI-TOF mass spectrometry. The results indicated that none of the strains tested belonged to the species S. aureus. The rpoB sequences of five isolates showed the highest sequence similarity to S. haemolyticus, three isolates to S. chromogenes, and one isolate to S. epidermidis. One strain (SAL4 remained unidentified in this analysis. The results obtained using mass spectrometry were comparable to those based on gene sequence analysis. Strain SAL4, which could not be identified by sequencing, was identified by MALDI-TOF as Staphylococcus chromogenes.

  16. Characterising phase variations in MALDI-TOF data and correcting

    Directory of Open Access Journals (Sweden)

    Michael C Fitzgerald

    2005-01-01

    Full Text Available Abstract: The use of MALDI-TOF mass spectrometry as a means of analyzing the proteome has been evaluated extensively in recent years. One of the limitations of this technique that has impeded the development of robust data analysis algorithms is the variability in the location of protein ion signals along the x-axis. We studied technical variations of MALDI-TOF measurements in the context of proteomics profiling. By acquiring a benchmark data set with five replicates, we estimated 76% to 85% of the total variance is due to phase variation. We devised a lobster plot, so named because of the resemblance to a lobster claw, to help detect the phase variation in replicates. We also investigated a peak alignment algorithm to remove the phase variation. This operation is analogous to the normalization step in microarray data analysis. Only after this critical step can features of biological interest be clearly revealed. With the help of principal component analysis, we demonstrated that after peak alignment, the differences among replicates are reduced. We compared this approach to peak alignment with a model-based calibration approach in which there was known information about peaks in common among all spectra. Finally, we examined the potential value at each point in an analysis pipeline of having a set of methods available that includes parametric, semiparametric and nonparametric methods; among such methods are those that benefit from the use of prior information.

  17. Proteomic analysis of plasma proteins in diabetic retinopathy patients by two dimensional electrophoresis and MALDI-Tof-MS.

    Science.gov (United States)

    Gopalakrishnan, Vidhya; Purushothaman, Parthiban; Bhaskar, Anusha

    2015-01-01

    Diabetic retinopathy is a highly specific vascular complication of diabetes mellitus and progresses from mild non-proliferative abnormalities characterized by increased vascular permeability to moderate and severe proliferative diabetic retinopathy characterized by the growth of blood vessels on the retina. The aim of the study was to identify the differentially expressed proteins in diabetic retinopathy using two-dimensional electrophoresis. Blood sample was drawn from subjects with diabetes mellitus (without retinopathy) who served as controls and patients with diabetic retinopathy in tubes containing EDTA as anticoagulant. Albumin and immunoglobulin IgG collectively removed to enrich proteins of lower abundance. 2de was carried out to see if there are any differentially expressed proteins. Approximately 48 and 61 spots were identified in control and diabetic retinopathy respectively, of which three protein spots RBP1 (retinol-binding protein 1), NUD10 (Diphosphoinositol polyphosphohydrolase 3 alpha), NGB (neuroglobin) were down regulated and HBG2 (hemoglobin) and BY55 (CD 160 antigen) were upregulated in diabetic retinopathy. These five protein spots were excised and were subjected to in-gel tryptic digestion, and their identities were determined by ultraflex MALDI-TOF-MS. We report a comprehensive patient-based plasma proteomic approach to the identification of potential biomarkers for diabetic retinopathy screening and detection. We identified 5 different proteins that were differentially expressed in the plasma of control diabetic patients (without retinopathy). Among these five proteins the expression of neuroglobin (NGB) protein varied significantly and may be a potential biomarker in diabetic retinopathy. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

    Science.gov (United States)

    Barnini, Simona; Ghelardi, Emilia; Brucculeri, Veronica; Morici, Paola; Lupetti, Antonella

    2015-06-18

    Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

  19. MALDI-TOF and SELDI-TOF analysis: “tandem” techniques to identify potential biomarker in fibromyalgia

    Directory of Open Access Journals (Sweden)

    A. Lucacchini

    2011-11-01

    Full Text Available Fibromyalgia (FM is characterized by the presence of chronic widespread pain throughout the musculoskeletal system and diffuse tenderness. Unfortunately, no laboratory tests have been appropriately validated for FM and correlated with the subsets and activity. The aim of this study was to apply a proteomic technique in saliva of FM patients: the Surface Enhance Laser Desorption/Ionization Time-of-Flight (SELDI-TOF. For this study, 57 FM patients and 35 HC patients were enrolled. The proteomic analysis of saliva was carried out using SELDI-TOF. The analysis was performed using different chip arrays with different characteristics of binding. The statistical analysis was performed using cluster analysis and the difference between two groups was underlined using Student’s t-test. Spectra analysis highlighted the presence of several peaks differently expressed in FM patients compared with controls. The preliminary results obtained by SELDI-TOF analysis were compared with those obtained in our previous study performed on whole saliva of FM patients by using electrophoresis. The m/z of two peaks, increased in FM patients, seem to overlap well with the molecular weight of calgranulin A and C and Rho GDP-dissociation inhibitor 2, which we had found up-regulated in our previous study. These preliminary results showed the possibility of identifying potential salivary biomarker through salivary proteomic analysis with MALDI-TOF and SELDI-TOF in FM patients. The peaks observed allow us to focus on some of the particular pathogenic aspects of FM, the oxidative stress which contradistinguishes this condition, the involvement of proteins related to the cytoskeletal arrangements, and central sensibilization.

  20. [MALDI-TOF mass spectrometry: Evaluation of the preanalytical phase for identification of molds].

    Science.gov (United States)

    Maldonado, Ivana; García Ramírez, Dolores; Striebeck, Pablo; Lafage, Marcelo; Fernández Canigia, Liliana

    In order to optimize the identification of molds with MALDI-TOF MS, three protein extraction-methodologies were evaluated against 44 isolates: water extraction (WE), zirconium extraction (ZE) and the provider's recommended method (PRM). Two data bases were compared, Bruker (BK) and Bruker+National Institutes of Health. Considering both databases, results were respectively as follows: correct identification (CI) at gender level, 10 and 16 by WE; 27 and 32 by ZE and 18 and 23 by PRM; CI at species level, 5 and 7 by WE; 17 and 20 by ZE and 11 and 14 by PRM; non-reliable identification, 18 and 12 by WE; 9 and 4 by ZE and by PRM. No peaks were observed in 16 by WE, 8 by ZE and 17 by PRM. ZE showed the best perfomance (p<0.05). Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  1. Využití metod HPLC a MALDI-TOF MS pro stanovení glutenu v bezlepkových surovinách a potravinách

    Czech Academy of Sciences Publication Activity Database

    Gabrovská, D.; Rysová, J.; Šalplachta, Jiří; Řehulka, Pavel; Chmelík, Josef

    2002-01-01

    Roč. 96, č. 6 (2002), s. 486-487 ISSN 0009-2770. [Sjezd chemických společností /54./. Brno, 30.06.2002-04.07.2002] R&D Projects: GA MZe QD1023 Institutional research plan: CEZ:AV0Z4031919 Keywords : gluten * gluten -free * MALDI-TOF Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.336, year: 2002

  2. Identification of differentially expressed proteins between human esophageal immortalized and carcinomatous cell lines by two-dimensional electrophoresis and MALDI-TOF-mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    Xing-Dong Xiong; Li-Yan Xu; Zhong-Ying Shen; Wei-Jia Cai; Jian-Min Luo; Ya-Li Han; En-Min Li

    2002-01-01

    AIM: To identify the differentially expressed proteins between the human immortalized esophageal epithelial cell line (SHEE) and the malignant transformed esophageal carcinoma cell line (SHEEC), and to explore new ways for studying esophageal carcinoma associated genes. METHODS: SHEE and SHEEC cell lines were used to separate differentially expressed proteins by two-dimensional electrophoresis/The silver-stained 2-D gels was scanned with EDAS290 digital camera system and analyzed with the PDQuest 6.2 Software. Six spots in which the differentially expressed protein was more obvious were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).RESULTS: There were 107±4.58 and 115±9.91 protein spots observed in SHEE and SHEEC respectively, and the majority of these spots between the two cell lines matched each other (r=-0.772), only a few were expressed differentially. After analyzed by MALDI-TOF-MS and database search for the six differentially expressed proteins, One new protein as well as other five sequence-known proteins including RNPEP-like protein, human rRNA gene upstream sequence binding transcription factor, uracil DNA glycosylase,Annexin A2 and p300/CBP-associated factor were preliminarily identified.CONCLUSION: These differentially expressed proteins might play an importance role during malignant transformation of SHEEC from SHEE. The identification of these proteins may serve as a new way for studying esophageal carcinoma associated genes.

  3. MALDI-TOF identification of the human Gut microbiome in people with and without diarrhea in Senegal.

    Directory of Open Access Journals (Sweden)

    Bissoume Samb-Ba

    Full Text Available BACKGROUND: In Africa, there are several problems with the specific identification of bacteria. Recently, MALDI-TOF mass spectrometry has become a powerful tool for the routine microbial identification in many clinical laboratories. METHODOLOGY/PRINCIPAL FINDINGS: This study was conducted using feces from 347 individuals (162 with diarrhea and 185 without diarrhea sampled in health centers in Dakar, Senegal. Feces were transported from Dakar to Marseille, France, where they were cultured using different culture conditions. The isolated colonies were identified using MALDI-TOF. If a colony was unidentified, 16S rRNA sequencing was performed. Overall, 2,753 isolates were tested, allowing for the identification of 189 bacteria from 5 phyla, including 2 previously unknown species, 11 species not previously reported in the human gut, 10 species not previously reported in humans, and 3 fungi. 2,718 bacterial isolates (98.8% out of 2,750 yielded an accurate identification using mass spectrometry, as did the 3 Candida albicans isolates. Thirty-two bacterial isolates not identified by MALDI-TOF (1.2% were identified by sequencing, allowing for the identification of 2 new species. The number of bacterial species per fecal sample was significantly higher among patients without diarrhea (8.6±3 than in those with diarrhea (7.3±3.4; P = 0.0003. A modification of the gut microbiota was observed between the two groups. In individuals with diarrhea, major commensal bacterial species such as E. coli were significantly decreased (85% versus 64%, as were several Enterococcus spp. (E. faecium and E. casseliflavus and anaerobes, such as Bacteroides spp. (B. uniformis and B. vulgatus and Clostridium spp. (C. bifermentans, C. orbiscindens, C. perfringens, and C. symbosium. Conversely, several Bacillus spp. (B. licheniformis, B. mojavensis, and B. pumilus were significantly more frequent among patients with diarrhea. CONCLUSIONS/SIGNIFICANCE: MALDI-TOF is a

  4. Copolymer fingerprints of polystyrene-block-polyisoprene by MALDI-ToF-MS

    NARCIS (Netherlands)

    Willemse, R.X.E.; Staal, B.B.P.; Donkers, E.H.D.; Herk, van A.M.

    2004-01-01

    MALDI-ToF-MS mass spectra of copolymers contain a lot of information on both chain length distribution (CLD) and chemical composition distribution (CCD). In this paper an approach for extracting detailed information from a MALDI-ToF-MS mass spectrum is presented that enables the study of

  5. Comparison of multilocus sequence typing, RAPD, and MALDI-TOF mass spectrometry for typing of β-lactam-resistant Klebsiella pneumoniae strains.

    Science.gov (United States)

    Sachse, Svea; Bresan, Stephanie; Erhard, Marcel; Edel, Birgit; Pfister, Wolfgang; Saupe, Angela; Rödel, Jürgen

    2014-12-01

    Extended spectrum of β-lactam (ESBL) resistance of Klebsiella pneumoniae has become an increasing problem in hospital infections. Typing of isolates is important to establish the intrahospital surveillance of resistant clones. In this study, the discriminatory potential of randomly amplified polymorphic DNA and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were compared with multilocus sequence typing (MLST) by using 17 β-lactam-resistant K. pneumoniae isolates of different genotypes. MLST alleles were distributed in 8 sequence types (STs). Among ESBL strains of the same ST, the presence of different β-lactamase genes was common. RAPD band patterns also revealed 8 types that corresponded to MLST-defined genotypes in 15 out of 17 cases. MALDI-TOF analysis could differentiate 5 clusters of strains. The results of this work show that RAPD may be usable as a rapid screening method for the intrahospital surveillance of K. pneumoniae, allowing a discrimination of clonally related strains. MALDI-TOF-based typing was not strongly corresponding to genotyping and warrants further investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Short communication: Evaluation of MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci.

    Science.gov (United States)

    Cameron, M; Perry, J; Middleton, J R; Chaffer, M; Lewis, J; Keefe, G P

    2018-01-01

    This study evaluated MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci (CNS). A total of 861 CNS isolates were used in the study, covering 21 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 804) and the remainder were identified by sequencing of hsp60 (n = 56) and tuf (n = 1). The genotypic identification was considered the gold standard identification. Using a direct transfer protocol and the existing commercial database, MALDI-TOF mass spectrometry showed a typeability of 96.5% (831/861) and an accuracy of 99.2% (824/831). Using a custom reference spectra expanded database, which included an additional 13 in-house created reference spectra, isolates were identified by MALDI-TOF mass spectrometry with 99.2% (854/861) typeability and 99.4% (849/854) accuracy. Overall, MALDI-TOF mass spectrometry using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. LC coupled to ESI, MALDI and ICP MS - A multiple hyphenation for metalloproteomic studies.

    Science.gov (United States)

    Coufalíková, Kateřina; Benešová, Iva; Vaculovič, Tomáš; Kanický, Viktor; Preisler, Jan

    2017-05-22

    A new multiple detection arrangement for liquid chromatography (LC) that supplements conventional electrospray ionization (ESI) mass spectrometry (MS) detection with two complementary detection techniques, matrix-assisted laser desorption/ionization (MALDI) MS and substrate-assisted laser desorption inductively coupled plasma (SALD ICP) MS has been developed. The combination of the molecular and elemental detectors in a single separation run is accomplished by utilizing a commercial MALDI target made of conductive plastic. The proposed platform provides a number of benefits in today's metalloproteomic applications, which are demonstrated by analysis of a metallothionein mixture. To maintain metallothionein complexes, separation is carried out at a neutral pH. The effluent is split; a major portion is directed to ESI MS while the remaining 1.8% fraction is deposited onto a plastic MALDI target. Dried droplets are overlaid with MALDI matrix and analysed consecutively by MALDI MS and SALD ICP MS. In the ESI MS spectra, the MT isoform complexes with metals and their stoichiometry are determined; the apoforms are revealed in the MALDI MS spectra. Quantitative determination of metallothionein isoforms is performed via determination of metals in the complexes of the individual protein isoforms using SALD ICP MS. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS

    Directory of Open Access Journals (Sweden)

    Patrick Broyer

    2018-05-01

    Full Text Available Sepsis is the leading cause of death among patients in intensive care units (ICUs requiring an early diagnosis to introduce efficient therapeutic intervention. Rapid identification (ID of a causative pathogen is key to guide directed antimicrobial selection and was recently shown to reduce hospitalization length in ICUs. Direct processing of positive blood cultures by MALDI-TOF MS technology is one of the several currently available tools used to generate rapid microbial ID. However, all recently published protocols are still manual and time consuming, requiring dedicated technician availability and specific strategies for batch processing. We present here a new prototype instrument for automated preparation of Vitek®MS slides directly from positive blood culture broth based on an “all-in-one” extraction strip. This bench top instrument was evaluated on 111 and 22 organisms processed using artificially inoculated blood culture bottles in the BacT/ALERT® 3D (SA/SN blood culture bottles or the BacT/ALERT VirtuoTM system (FA/FN Plus bottles, respectively. Overall, this new preparation station provided reliable and accurate Vitek MS species-level identification of 87% (Gram-negative bacteria = 85%, Gram-positive bacteria = 88%, and yeast = 100% when used with BacT/ALERT® 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75% with Virtuo® instruments, respectively. The prototype was then evaluated in a clinical microbiology laboratory on 102 clinical blood culture bottles and compared to routine laboratory ID procedures. Overall, the correlation of ID on monomicrobial bottles was 83% (Gram-negative bacteria = 89%, Gram-positive bacteria = 79%, and yeast = 78%, demonstrating roughly equivalent performance between manual and automatized extraction methods. This prototype instrument exhibited a high level of performance regardless of bottle type or BacT/ALERT system. Furthermore, blood culture workflow could

  9. On-target digestion of collected bacteria for MALDI mass spectrometry.

    Science.gov (United States)

    Dugas, Alton J; Murray, Kermit K

    2008-10-03

    An on-target protein digestion system was developed for the identification of microorganisms in collected bioaerosols using off-line matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Bacteria analysis techniques based on MALDI-MS were adapted for use with an orthogonal MALDI quadrupole-time-of-flight mass spectrometer. Bioaerosols were generated using a pneumatic nebulizer and infused into a chamber for sampling. An Andersen N6 single-stage impactor was used to collect the bioaerosols on a MALDI target. On-target digestion was carried out inside temporary mini-wells placed over the impacted samples. The wells served as miniature reactors for proteolysis. Collected test aerosol particles containing the protein cytochrome c and E. coli bacteria were proteolyzed in situ using trypsin or cyanogen bromide. A total of 19 unique proteins were identified for E. coli. Using the TOF-MS spectra of the digested samples, peptide mass mapping was performed using the MASCOT search engine and an iterative search technique.

  10. Direct bacterial identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry: A systematic review and meta-analysis.

    Science.gov (United States)

    Ruiz-Aragón, Jesús; Ballestero-Téllez, Mónica; Gutiérrez-Gutiérrez, Belén; de Cueto, Marina; Rodríguez-Baño, Jesús; Pascual, Álvaro

    2017-10-27

    The rapid identification of bacteraemia-causing pathogens could assist clinicians in the timely prescription of targeted therapy, thereby reducing the morbidity and mortality of this infection. In recent years, numerous techniques that rapidly and directly identify positive blood cultures have been marketed, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) being one of the most commonly used. The aim of this systematic review and meta-analysis was to evaluate the accuracy of MALDI-TOF (Bruker ® ) for the direct identification of positive blood culture bottles. A meta-analysis was performed to summarize the results of the 32 studies evaluated. The overall quality of the studies was moderate. For Gram-positive bacteria, overall rates of correct identification of the species ranged from 0.17 to 0.98, with a cumulative rate (random-effects model) of 0.72 (95% CI: 0.64-0.80). For Gram-negative bacteria, correct identification rates ranged from 0.66 to 1.00, with a cumulative effect of 0.92 (95% CI: 0.88-0.95). For Enterobacteriaceae, the rate was 0.96 (95% CI: 0.94-0.97). MALDI-TOF mass spectrometry shows high accuracy for the correct identification of Gram-negative bacteria, particularly Enterobacteriaceae, directly from positive blood culture bottles, and moderate accuracy for the identification of Gram-positive bacteria (low for some species). Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  11. MALDI-TOF identification of Gram-negative bacteria directly from blood culture bottles containing charcoal: Sepsityper® kits versus centrifugation-filtration method.

    Science.gov (United States)

    Riederer, Kathleen; Cruz, Kristian; Shemes, Stephen; Szpunar, Susan; Fishbain, Joel T

    2015-06-01

    Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has dramatically altered the way microbiology laboratories identify clinical isolates. Direct blood culture (BC) detection may be hampered, however, by the presence of charcoal in BC bottles currently in clinical use. This study evaluates an in-house process for extraction and MALDI-TOF identification of Gram-negative bacteria directly from BC bottles containing charcoal. Three hundred BC aliquots were extracted by a centrifugation-filtration method developed in our research laboratory with the first 96 samples processed in parallel using Sepsityper® kits. Controls were colonies from solid media with standard phenotypic and MALDI-TOF identification. The identification of Gram-negative bacteria was successful more often via the in-house method compared to Sepsityper® kits (94.7% versus 78.1%, P≤0.0001). Our in-house centrifugation-filtration method was further validated for isolation and identification of Gram-negative bacteria (95%; n=300) directly from BC bottles containing charcoal. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins

    DEFF Research Database (Denmark)

    Kjellström, Sven; Jensen, Ole Nørregaard

    2004-01-01

    Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-di...... acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix....

  13. Matrix normalized MALDI-TOF quantification of a fluorotelomer-based acrylate polymer.

    Science.gov (United States)

    Rankin, Keegan; Mabury, Scott A

    2015-05-19

    The degradation of fluorotelomer-based acrylate polymers (FTACPs) has been hypothesized to serve as a source of the environmental contaminants, perfluoroalkyl carboxylates (PFCAs). Studies have relied on indirect measurement of presumed degradation products to evaluate the environmental fate of FTACPs; however, this approach leaves a degree of uncertainty. The present study describes the development of a quantitative matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry method as the first direct analysis method for FTACPs. The model FTACP used in this study was poly(8:2 FTAC-co-HDA), a copolymer of 8:2 fluorotelomer acrylate (8:2 FTAC) and hexadecyl acrylate (HDA). Instead of relying on an internal standard polymer, the intensities of 40 poly(8:2 FTAC-co-HDA) signals (911-4612 Da) were normalized to the signal intensity of a matrix-sodium cluster (659 Da). We termed this value the normalized polymer response (P(N)). By using the same dithranol solution for the sample preparation of poly(8:2 FTAC-co-HDA) standards, calibration curves with coefficient of determinations (R(2)) typically >0.98 were produced. When poly(8:2 FTAC-co-HDA) samples were prepared with the same dithranol solution as the poly(8:2 FTAC-co-HDA) standards, quantification to within 25% of the theoretical concentration was achieved. This approach minimized the sample-to-sample variability that typically plagues MALDI-TOF, and is the first method developed to directly quantify FTACPs.

  14. Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein

    NARCIS (Netherlands)

    Severinovskaya, O. V.; Kovalska, V B; Losytskyy, M Yu; Cherepanov, V. V.; Subramaniam, V.; Yarmoluk, S M

    2014-01-01

    Aim. To study the α-synuclein (ASN) aggregates of different structural origin, namely amyloid fibrils and spherical oligomers, in comparison with a native protein. Methods. MALDI TOF mass spectrometry and atomic force microscopy (AFM). Results. The mass spectra of native and fibrillar ASN have

  15. Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil

    Czech Academy of Sciences Publication Activity Database

    Uhlík, Ondřej; Strejček, M.; Junková, P.; Šanda, Miloslav; Hroudová, Miluše; Vlček, Čestmír; Macková, Martina; Macek, Tomáš

    2011-01-01

    Roč. 77, č. 19 (2011), s. 6858-6866 ISSN 0099-2240 Grant - others:GA MŠk(CZ) ME09024; GA ČR(CZ) GA525/09/1058; GA MŠk(CZ) 2B06156 Program:GA; 2B Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : MALDI-TOF MS * bioremediation * MALDI Biotyper * bacterial identification Subject RIV: CC - Organic Chemistry Impact factor: 3.829, year: 2011

  16. Atypical yeasts identified as Saccharomyces cerevisiae by MALDI-TOF MS and gene sequencing are the main responsible of fermentation of chicha, a traditional beverage from Peru.

    Science.gov (United States)

    Vallejo, Juan Andrés; Miranda, Patricia; Flores-Félix, José David; Sánchez-Juanes, Fernando; Ageitos, José M; González-Buitrago, José Manuel; Velázquez, Encarna; Villa, Tomás G

    2013-12-01

    Chicha is a drink prepared in several Andean countries from Inca's times by maize fermentation. Currently this fermentation is carried out in familiar artesanal "chicherías" that make one of the most known types of chicha, the "chicha de jora". In this study we isolate and identify the yeasts mainly responsible of the fermentation process in this type of chicha in 10 traditional "chicherías" in Cusco region in Peru. We applied by first time MALDI-TOF MS analysis for the identification of yeast of non-clinic origin and the results showed that all of yeast strains isolated belong to the species Saccharomyces cerevisiae. These results agree with those obtained after the analysis of the D1/D2 and 5.8S-ITS regions. However the chicha strains have a phenotypic profile that differed in more than 40% as compared to that of current S. cerevisiae strains. To the best of our knowledge this is the first report concerning the yeasts involved in chicha fermentation. Copyright © 2013 Elsevier GmbH. All rights reserved.

  17. MALDI-TOF mass spectrometry for early identification of bacteria grown in blood culture bottles.

    Science.gov (United States)

    Zabbe, Jean-Benoît; Zanardo, Laura; Mégraud, Francis; Bessède, Emilie

    2015-08-01

    This note reports an interesting way to rapidly identify bacteria grown from blood culture bottles. Chocolate agar plates were inoculated with 1 drop of the positive blood bottle medium. After a 3-hour incubation, the growth veil was submitted to MALDI-TOF mass spectrometry: 77% of the bacteria present have been correctly identified. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. State-of-the-art nanoplatform-integrated MALDI-MS impacting resolutions in urinary proteomics.

    Science.gov (United States)

    Gopal, Judy; Muthu, Manikandan; Chun, Se-Chul; Wu, Hui-Fen

    2015-06-01

    Urine proteomics has become a subject of interest, since it has led to a number of breakthroughs in disease diagnostics. Urine contains information not only from the kidney and the urinary tract but also from other organs, thus urinary proteome analysis allows for identification of biomarkers for both urogenital and systemic diseases. The following review gives a brief overview of the analytical techniques that have been in practice for urinary proteomics. MALDI-MS technique and its current application status in this area of clinical research have been discussed. The review comments on the challenges facing the conventional MALDI-MS technique and the upgradation of this technique with the introduction of nanotechnology. This review projects nano-based techniques such as nano-MALDI-MS, surface-assisted laser desorption/ionization, and nanostructure-initiator MS as the platforms that have the potential in trafficking MALDI-MS from the lab to the bedside. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives.

    Science.gov (United States)

    Brun, Sophie; Madrid, Hugo; Gerrits Van Den Ende, Bert; Andersen, Birgitte; Marinach-Patrice, Carine; Mazier, Dominique; De Hoog, G Sybren

    2013-01-01

    The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species-complexes of morphologically similar taxa. This study aimed to assess if strains of four closely-related plant pathogens, i.e., accurately Alternaria dauci (ten strains), Alternaria porri (six), Alternaria solani (ten), and Alternaria tomatophila (ten) could be identified using multilocus phylogenetic analysis and Matrix-Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) profiling of proteins. Phylogenetic analyses were performed on three loci, i.e., the internal transcribed spacer (ITS) region of rRNA, and the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major antigen (Alt a 1) genes. Phylogenetic trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades. However, none provided significant bootstrap support for all four species, which could only be achieved when results for the three loci were combined. MALDI-TOF-based dendrograms showed three major clusters. The first comprised all A. dauci strains, the second included five strains of A. porri and one of A. solani, and the third included all strains of A. tomatophila, as well as all but one strain of A. solani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely-related plant pathogens. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  20. Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

    Science.gov (United States)

    Ayyadurai, Saravanan; Flaudrops, Christophe; Raoult, Didier; Drancourt, Michel

    2010-11-12

    Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates.

  1. Matrix Optical Absorption in UV-MALDI MS.

    Science.gov (United States)

    Robinson, Kenneth N; Steven, Rory T; Bunch, Josephine

    2018-03-01

    In ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI MS) matrix compound optical absorption governs the uptake of laser energy, which in turn has a strong influence on experimental results. Despite this, quantitative absorption measurements are lacking for most matrix compounds. Furthermore, despite the use of UV-MALDI MS to detect a vast range of compounds, investigations into the effects of laser energy have been primarily restricted to single classes of analytes. We report the absolute solid state absorption spectra of the matrix compounds α-cyano-4-hydroxycinnamic acid (CHCA), para-nitroaniline (PNA), 2-mercaptobenzothiazole (MBT), 2,5-dihydroxybenzoic acid (2,5-DHB), and 2,4,6-trihydroxyacetophenone (THAP). The desorption/ionization characteristics of these matrix compounds with respect to laser fluence was investigated using mixed systems of matrix with either angiotensin II, PC(34:1) lipid standard, or haloperidol, acting as representatives for typical classes of analyte encountered in UV-MALDI MS. The first absolute solid phase spectra for PNA, MBT, and THAP are reported; additionally, inconsistencies between previously published spectra for CHCA are resolved. In light of these findings, suggestions are made for experimental optimization with regards to matrix and laser wavelength selection. The relationship between matrix optical cross-section and wavelength-dependant threshold fluence, fluence of maximum ion yield, and R, a new descriptor for the change in ion intensity with fluence, are described. A matrix cross-section of 1.3 × 10 -17 cm -2 was identified as a potential minimum for desorption/ionization of analytes. Graphical Abstract ᅟ.

  2. Comprehensive MALDI-TOF biotyping of the non-redundant Harvard Pseudomonas aeruginosa PA14 transposon insertion mutant library.

    Science.gov (United States)

    Oumeraci, Tonio; Jensen, Vanessa; Talbot, Steven R; Hofmann, Winfried; Kostrzewa, Markus; Schlegelberger, Brigitte; von Neuhoff, Nils; Häussler, Susanne

    2015-01-01

    Pseudomonas aeruginosa is a gram-negative bacterium that is ubiquitously present in the aerobic biosphere. As an antibiotic-resistant facultative pathogen, it is a major cause of hospital-acquired infections. Its rapid and accurate identification is crucial in clinical and therapeutic environments. In a large-scale MALDI-TOF mass spectrometry-based screen of the Harvard transposon insertion mutant library of P. aeruginosa strain PA14, intact-cell proteome profile spectra of 5547 PA14 transposon mutants exhibiting a plethora of different phenotypes were acquired and analyzed. Of all P. aeruginosa PA14 mutant profiles 99.7% were correctly identified as P. aeruginosa with the Biotyper software on the species level. On the strain level, 99.99% of the profiles were mapped to five different individual P. aeruginosa Biotyper database entries. A principal component analysis-based approach was used to determine the most important discriminatory mass features between these Biotyper groups. Although technical replicas were consistently categorized to specific Biotyper groups in 94.2% of the mutant profiles, biological replicas were not, indicating that the distinct proteotypes are affected by growth conditions. The PA14 mutant profile collection presented here constitutes the largest coherent P. aeruginosa MALDI-TOF spectral dataset publicly available today. Transposon insertions in thousands of different P. aeruginosa genes did not affect species identification from MALDI-TOF mass spectra, clearly demonstrating the robustness of the approach. However, the assignment of the individual spectra to sub-groups proved to be non-consistent in biological replicas, indicating that the differentiation between biotyper groups in this nosocomial pathogen is unassured.

  3. Detection of Serum Peptides in Patients with Lung Squamous Cell Carcinoma by MALDI-TOF-MS and Analysis of Their Correlation with Chemotherapy Efficacy

    Directory of Open Access Journals (Sweden)

    Guanhua ZHAO

    2017-05-01

    Full Text Available Background and objective Treatment options for patients with squamous cell carcinoma of the lung (SCC are limited in chemotherapy. However, not all patients could benefit form standard platinum regimen. Considering the dismal prognosis of patients with advanced SCC, a greater focus on selecting sensitive chemotherapy regimens remains of upmost importance to improve outcomes in this disease. In this study, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect pre-chemotherapy serum peptides in advanced lung squamous cell carcinoma patients accepting paclitaxel combined with platinum chemotherapy and to analyze the correlation between serum peptides and chemotherapy efficacy. Methods Patients with advanced lung squamous cell carcinoma received paclitaxel combining with platinum chemotherapy and evaluated the efficacy every two cycles. Evaluation of complete response (CR or partial response (PR patients defined as sensitive group, progressive disease (PD patients defined as resistant group. Serum samples were collected from patients with lung squamous cell carcinoma. Eighty-one patients were randomly divided into training group (sensitive group I and resistant group I and validation group (sensitive group II and resistant group II according to the ratio of 3:1. Serum samples were pretreated and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS was used to detect serum peptide fingerprints. ClinProTools software was used to analyze the differences between the sensitive group I and the resistant group I. Three kinds of biological algorithms (SNN, GA, QC built in CPT software were used to establish the curative effect prediction model respectively and the optimal algorithm was selected. The validation group was used for blind verification. Results Thirty sensitive patients and 31 resistant patients were enrolled in the training group. Ten sensitive patients and 10

  4. Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

    Science.gov (United States)

    Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

    2008-02-01

    Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

  5. Analysis of antioxidants in insulation cladding of copper wire: a comparison of different mass spectrometric techniques (ESI-IT, MALDI-RTOF and RTOF-SIMS).

    Science.gov (United States)

    Schnöller, Johannes; Pittenauer, Ernst; Hutter, Herbert; Allmaier, Günter

    2009-12-01

    Commercial copper wire and its polymer insulation cladding was investigated for the presence of three synthetic antioxidants (ADK STAB AO412S, Irganox 1010 and Irganox MD 1024) by three different mass spectrometric techniques including electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS), matrix-assisted laser desorption/ionization reflectron time-of-flight (TOF) mass spectrometry (MALDI-RTOF-MS) and reflectron TOF secondary ion mass spectrometry (RTOF-SIMS). The samples were analyzed either directly without any treatment (RTOF-SIMS) or after a simple liquid/liquid extraction step (ESI-IT-MS, MALDI-RTOF-MS and RTOF-SIMS). Direct analysis of the copper wire itself or of the insulation cladding by RTOF-SIMS allowed the detection of at least two of the three antioxidants but at rather low sensitivity as molecular radical cations and with fairly strong fragmentation (due to the highly energetic ion beam of the primary ion gun). ESI-IT- and MALDI-RTOF-MS-generated abundant protonated and/or cationized molecules (ammoniated or sodiated) from the liquid/liquid extract. Only ESI-IT-MS allowed simultaneous detection of all three analytes in the extract of insulation claddings. The latter two so-called 'soft' desorption/ionization techniques exhibited intense fragmentation only by applying low-energy collision-induced dissociation (CID) tandem MS on a multistage ion trap-instrument and high-energy CID on a tandem TOF-instrument (TOF/RTOF), respectively. Strong differences in the fragmentation behavior of the three analytes could be observed between the different CID spectra obtained from either the IT-instrument (collision energy in the very low eV range) or the TOF/RTOF-instrument (collision energy 20 keV), but both delivered important structural information. Copyright 2009 John Wiley & Sons, Ltd.

  6. A differential centrifugation protocol and validation criterion for enhancing mass spectrometry (MALDI-TOF) results in microbial identification using blood culture growth bottles.

    Science.gov (United States)

    March-Rosselló, G A; Muñoz-Moreno, M F; García-Loygorri-Jordán de Urriés, M C; Bratos-Pérez, M A

    2013-05-01

    Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) is a widely used tool in clinical microbiology for rapidly identifying microorganisms. This technique can be applied directly on positive blood cultures without the need for its culturing, thereby, reducing the time required for microbiological diagnosis. The present study proposes an innovative identification protocol applied to positive blood culture bottles using MALDI-TOF. We have processed 100 positive blood culture bottles, of which 36 of 37 Gram-negative bacteria (97.3 %) were correctly identified directly with 100 % of Enterobacteriaceae and other Gram-negative rods and 87.5 % of non-fermenting Gram-negative rods. We also correctly identified directly 62 of 63 of Gram-positive bacteria (98.4 %) with 100 % of Streptococcus, Enterococcus, and Gram-positive bacilli and 98 % of Staphylococcus. Applying the differential centrifugation protocol at the moment the automatic blood culture incubation system gives a positive reading together with the proposed validation criterion offers 98 % sensitivity (95 % confidence interval: 95.2-100 %). The MALDI-TOF system, thus, provides a rapid and reliable system for identifying microorganisms from blood culture growth bottles.

  7. Determination of post-culture processing with carbohydrates by MALDI-MS and TMS derivatization GC-MS.

    Science.gov (United States)

    Wunschel, David S; Wahl, Karen L; Melville, Angela M; Sorensen, Christina M; Colburn, Heather A; Valentine, Nancy B; Stamper, Casey L

    2011-10-15

    Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC-MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC-MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone - PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC-MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC-MS was

  8. Direct identification of bacteria from BacT/ALERT anaerobic positive blood cultures by MALDI-TOF MS: MALDI Sepsityper kit versus an in-house saponin method for bacterial extraction.

    Science.gov (United States)

    Meex, Cécile; Neuville, Florence; Descy, Julie; Huynen, Pascale; Hayette, Marie-Pierre; De Mol, Patrick; Melin, Pierrette

    2012-11-01

    In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66 % of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52 % of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90 % of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction

  9. Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS

    Energy Technology Data Exchange (ETDEWEB)

    Garabedian, Alyssa; Benigni, Paolo; Ramirez, Cesar; Baker, Erin M.; Liu, Tao; Smith, Richard D.; Fernandez-Lima, Francisco

    2018-05-01

    Abstract. In the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150–250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMSCID- TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptide biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10–200 nM range, while simultaneously achieving discovery measurements

  10. Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries.

    Science.gov (United States)

    Chin, Jefferson; Wood, Elizabeth; Peters, Grace S; Drexler, Dieter M

    2016-02-01

    In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample. © 2015 Society for Laboratory Automation and Screening.

  11. Using MALDI-TOF mass spectrometry as a rapid and accurate diagnostic tool in infective endocarditis: a case report of a patient with mitral valve infective endocarditis caused by Abiotrophia defectiva

    DEFF Research Database (Denmark)

    Holler, Jon Gitz; Pedersen, Line; Calum, Henrik

    2011-01-01

    A case of infective endocarditis caused by Abiotrophia defectiva is presented. The use of MALDI-TOF mass spectrometry as a rapid and accurate diagnostic tool in infective endocarditis is discussed.......A case of infective endocarditis caused by Abiotrophia defectiva is presented. The use of MALDI-TOF mass spectrometry as a rapid and accurate diagnostic tool in infective endocarditis is discussed....

  12. Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET.

    Science.gov (United States)

    Beeman, Katrin; Baumgärtner, Jens; Laubenheimer, Manuel; Hergesell, Karlheinz; Hoffmann, Martin; Pehl, Ulrich; Fischer, Frank; Pieck, Jan-Carsten

    2017-12-01

    Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated "in-line reader" for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

  13. Structural characterization of native high-methoxylated pectin using nuclear magnetic resonance spectroscopy and ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comparative use of 2,5-dihydroxybenzoic acid and nor-harmane as UV-MALDI matrices.

    Science.gov (United States)

    Monge, María Eugenia; Negri, R Martín; Kolender, Adriana A; Erra-Balsells, Rosa

    2007-01-01

    The successful analysis by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS) of native and hydrolyzed high-methoxylated pectin samples is described. In order to find the optimal conditions for UV-MALDI-TOF MS analysis several experimental variables were studied such as: different UV-MALDI matrices (nor-harmane, 2,5-dihydroxybenzoic acid), sample preparation methods (mixture, sandwich), inorganic salt addition (doping salts, NaCl, KCl, NH(4)Cl), ion mode (positive, negative), linear and reflectron mode, etc. nor-Harmane has never been used as a UV-MALDI matrix for the analysis of pectins but its use avoids pre-treatment of the sample, such as an enzymatic digestion or an acid hydrolysis, and there is no need to add salts, making the analysis easier and faster. This study suggested an alternative way of analyzing native high-methoxylated pectins, with UV-MALDI-TOF MS, by using nor-harmane as the matrix in negative ion mode. The analysis by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy of the native and hydrolyzed pectin is also briefly described. Copyright (c) 2007 John Wiley & Sons, Ltd.

  14. MALDI-MS drug analysis in biological samples: opportunities and challenges.

    Science.gov (United States)

    Steuer, Andrea E; Poetzsch, Michael; Kraemer, Thomas

    2016-09-01

    Drug analysis represents a large field in different disciplines. Plasma is commonly considered to be the biosample of choice for that purpose. However, concentrations often do not represent the levels present within deeper compartments and therefore cannot sufficiently explain efficacy or toxicology of drugs. MALDI-MS in drug analysis is of great interest for high-throughput quantification and particularly spatially resolved tissue imaging. The current perspective article will deal with challenges and opportunities of MALDI-MS drug analysis in different biological samples. A particular focus will be on hair samples. Recent applications were included, reviewed for their instrumental setup and sample preparation and pros and cons as well as future perspectives are critically discussed.

  15. Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS

    Science.gov (United States)

    Garabedian, Alyssa; Benigni, Paolo; Ramirez, Cesar E.; Baker, Erin S.; Liu, Tao; Smith, Richard D.; Fernandez-Lima, Francisco

    2017-09-01

    In the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150-250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMS-CID-TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptide biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10-200 nM range, while simultaneously achieving discovery measurements of not initially targeted peptides as markers from the same proteins and, eventually, other proteins. [Figure not available: see fulltext.

  16. An On-Target Desalting and Concentration Sample Preparation Protocol for MALDI-MS and MS/MS Analysis

    DEFF Research Database (Denmark)

    Zhang, Xumin; Wang, Quanhui; Lou, Xiaomin

    2012-01-01

    2DE coupled with MALDI-MS is one of the most widely used and powerful analytic technologies in proteomics study. The MALDI sample preparation method has been developed and optimized towards the combination of simplicity, sample-cleaning, and sample concentration since its introduction. Here we...... present a protocol of the so-called Sample loading, Matrix loading, and on-target Wash (SMW) method which fulfills the three criteria by taking advantage of the AnchorChip™ targets. Our method is extremely simple and no pre-desalting or concentration is needed when dealing with samples prepared from 2DE...

  17. Investigating quantitation of phosphorylation using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Parker, Laurie; Engel-Hall, Aaron; Drew, Kevin; Steinhardt, George; Helseth, Donald L; Jabon, David; McMurry, Timothy; Angulo, David S; Kron, Stephen J

    2008-04-01

    Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate because of physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5-10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear that this behavior is highly complex and needs to be further explored. John Wiley & Sons, Ltd

  18. CE-TOF/MS: fundamental concepts, instrumental considerations and applications.

    Science.gov (United States)

    Staub, Aline; Schappler, Julie; Rudaz, Serge; Veuthey, Jean-Luc

    2009-05-01

    This review discusses the fundamental principles of TOF analyzers and covers the great progress that has been made in this area in recent years (i.e. orthogonal acceleration, reflectron). This paper also gives an overview of applications performed by CE coupled to TOF/MS detection. The main domains of interest include the analysis of biomolecules and natural compounds.

  19. The effect of temperature on the stability of compounds used as UV-MALDI-MS matrix: 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, alpha-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, nor-harmane and harmane.

    Science.gov (United States)

    Tarzi, Olga I; Nonami, Hiroshi; Erra-Balsells, Rosa

    2009-02-01

    The thermal stability of several commonly used crystalline matrix-assisted ultraviolet laser desorption/ionization mass spectrometry (UV-MALDI-MS) matrices, 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THA), alpha-cyano-4-hydroxycinnamic acid (CHC), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid; SA), 9H-pirido[3,4-b]indole (nor-harmane; nor-Ho), 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho), perchlorate of nor-harmanonium ([nor-Ho+H]+) and perchlorate of harmanonium ([Ho+H]+) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI-MS), ultraviolet laserdesorption/ionization-time-of-flight-mass spectrometry (UV-LDI-TOF-MS) and electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV-absorption, fluorescence emission and excitation spectroscopy) and 1H nuclear magnetic resonance spectroscopy (1H-NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO2, yielding the trans-/cis-4-hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and 1H-NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well-known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV-MALDI-MS. Commercial SA (SA 98%; trans-SA/cis-SA 5:1) showed mainly cis- to-trans thermal isomerization and, with very poor yield, loss of CO2, yielding (3',5'-dimethoxy-4'-hydroxyphenyl)-1-ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV-MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted. Copyright (c) 2008 John Wiley & Sons, Ltd.

  20. The MR-TOF-MS isobar separator for the TITAN facility at TRIUMF

    Energy Technology Data Exchange (ETDEWEB)

    Jesch, Christian; Dickel, Timo, E-mail: t.dickel@gsi.de; Plaß, Wolfgang R. [Justus-Liebig-University (Germany); Short, Devin [Simon Fraser University (Canada); Ayet San Andres, Samuel [Justus-Liebig-University (Germany); Dilling, Jens [TRIUMF (Canada); Geissel, Hans; Greiner, Florian; Lang, Johannes [Justus-Liebig-University (Germany); Leach, Kyle G. [Simon Fraser University (Canada); Lippert, Wayne; Scheidenberger, Christoph [Justus-Liebig-University (Germany); Yavor, Mikhail I. [Institute for Analytical Instrumentation, Russian Academy of Science (Russian Federation)

    2015-11-15

    At TRIUMF’s Ion Trap for Atomic and Nuclear Science (TITAN) a multiple-reflection time-of-flight mass spectrometer (MR-TOF-MS) will extend TITAN’s capabilities and facilitate mass measurements and in-trap decay spectroscopy of exotic nuclei that so far have not been possible due to strong isobaric contamination. This MR-TOF-MS will also enable mass measurements of very short-lived nuclides (half-life > 1 ms) that are produced in very low quantities (a few detected ions overall). In order to allow the installation of an MR-TOF-MS in the restricted space on the platform, on which the TITAN facility is located, novel mass spectrometric methods have been developed. Transport, cooling and distribution of the ions inside the device is done using a buffer gas-filled RFQ-based ion beam switchyard. Mass selection is achieved using a dynamic retrapping technique after time-of-flight analysis in an electrostatic isochronous reflector system. Only due to the combination of these novel methods the realization of an MR-TOF-MS based isobar separator at TITAN has become possible. The device has been built, commissioned off-line and is currently under installation at TITAN.

  1. The MR-TOF-MS isobar separator for the TITAN facility at TRIUMF

    Science.gov (United States)

    Jesch, Christian; Dickel, Timo; Plaß, Wolfgang R.; Short, Devin; Ayet San Andres, Samuel; Dilling, Jens; Geissel, Hans; Greiner, Florian; Lang, Johannes; Leach, Kyle G.; Lippert, Wayne; Scheidenberger, Christoph; Yavor, Mikhail I.

    2015-11-01

    At TRIUMF's Ion Trap for Atomic and Nuclear Science (TITAN) a multiple-reflection time-of-flight mass spectrometer (MR-TOF-MS) will extend TITAN's capabilities and facilitate mass measurements and in-trap decay spectroscopy of exotic nuclei that so far have not been possible due to strong isobaric contamination. This MR-TOF-MS will also enable mass measurements of very short-lived nuclides (half-life > 1 ms) that are produced in very low quantities (a few detected ions overall). In order to allow the installation of an MR-TOF-MS in the restricted space on the platform, on which the TITAN facility is located, novel mass spectrometric methods have been developed. Transport, cooling and distribution of the ions inside the device is done using a buffer gas-filled RFQ-based ion beam switchyard. Mass selection is achieved using a dynamic retrapping technique after time-of-flight analysis in an electrostatic isochronous reflector system. Only due to the combination of these novel methods the realization of an MR-TOF-MS based isobar separator at TITAN has become possible. The device has been built, commissioned off-line and is currently under installation at TITAN.

  2. An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project

    DEFF Research Database (Denmark)

    Zhang, Xumin; Shi, Liang; Shu, Shaokung

    2007-01-01

    An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The ...

  3. Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Fujita, Yukiko; Naka, Takashi; Doi, Takeshi; Yano, Ikuya

    2005-05-01

    Direct estimation of the molecular mass of single molecular species of trehalose 6-monomycolate (TMM), a ubiquitous cell-wall component of mycobacteria, was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. When less than 1 microg TMM was analysed by MALDI-TOF mass spectrometry, quasimolecular ions [M+Na]+ of each molecular species were demonstrated and the numbers of carbons and double bonds (or cyclopropane rings) were determined. Since the introduction of oxygen atoms such as carbonyl, methoxy and ester groups yielded the appropriate shift of mass ions, the major subclasses of mycolic acid (alpha, methoxy, keto and wax ester) were identified without resorting to hydrolytic procedures. The results showed a marked difference in the molecular species composition of TMM among mycobacterial species. Unexpectedly, differing from other mycoloyl glycolipids, TMM from Mycobacterium tuberculosis showed a distinctive mass pattern, with abundant odd-carbon-numbered monocyclopropanoic (or monoenoic) alpha-mycolates besides dicyclopropanoic mycolate, ranging from C75 to C85, odd- and even-carbon-numbered methoxymycolates ranging from C83 to C94 and even- and odd-carbon-numbered ketomycolates ranging from C83 to C90. In contrast, TMM from Mycobacterium bovis (wild strain and BCG substrains) possessed even-carbon-numbered dicyclopropanoic alpha-mycolates. BCG Connaught strain lacked methoxymycolates almost completely. These results were confirmed by MALDI-TOF mass analysis of mycolic acid methyl esters liberated by alkaline hydrolysis and methylation of the original TMM. Wax ester-mycoloyl TMM molecular species were demonstrated for the first time as an intact form in the Mycobacterium avium-intracellulare group, M. phlei and M. flavescens. The M. avium-intracellulare group possessed predominantly C85 and C87 wax ester-mycoloyl TMM, while M. phlei and the rapid growers tested contained C80, C81, C82 and C83 wax ester

  4. Lipidomic fingerprint of almonds (Prunus dulcis L. cv Nonpareil) using TiO₂ nanoparticle based matrix solid-phase dispersion and MALDI-TOF/MS and its potential in geographical origin verification.

    Science.gov (United States)

    Shen, Qing; Dong, Wei; Yang, Mei; Li, Linqiu; Cheung, Hon-Yeung; Zhang, Zhifeng

    2013-08-14

    A matrix solid-phase dispersion (MSPD) procedure with titanium dioxide (TiO2) nanoparticles (NP) as sorbent was developed for the selective extraction of phospholipids from almond samples, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) was employed for analysis. A remarkable increase in the signals of phospholipid accompanied by a decrease in those of triacylglycerols and diacylglycerols was observed in the relevant mass spectra. The proposed method was applied to five batches of almonds originating from four geographical areas, whereas principal component analysis (PCA) was utilized to normalize the relative amounts of the identified phospholipid species. The results indicated that the lipidomic fingerprint of almonds was successfully established by the negative ion mode spectrum, and the ratio of m/z 833.6 to 835.6 as well as m/z 821.6 could be introduced as potential markers for the differentiation of the tested almonds with different geographical origins. The whole method is of great promise for selective separation of phospholipids from nonphospholipids, especially the glycerides, and superior in fast screening and characterization of phospholipids in almond samples.

  5. Aplicación de la Espectrometría de Masas Maldi tof/tof a la Endocrinología, Inmunología y Patología del paiche Arapaima gigas

    OpenAIRE

    Cueva Távara, Mario David

    2016-01-01

    RESUMEN El pez amazónico gigante “paiche” Arapaima gigas es una especie prometedora para la piscicultura tropical debido a sus características anatómicas, sin embargo, su completa domesticación es problemática debido a la falta de información biológica. Los progresos recientes en el campo de la biotecnología molecular, llamados “omicas”, están revolucionando la ictiología básica y aplicada; en particular la proteómica por espectrometría doble masa MALDI TOF/TOF, que permite la detección...

  6. Combined use of ESI-QqTOF-MS and ESI-QqTOF-MS/MS with mass-spectral library search for qualitative analysis of drugs.

    Science.gov (United States)

    Pavlic, Marion; Libiseller, Kathrin; Oberacher, Herbert

    2006-09-01

    The potential of the combined use of ESI-QqTOF-MS and ESI-QqTOF-MS/MS with mass-spectral library search for the identification of therapeutic and illicit drugs has been evaluated. Reserpine was used for standardizing experimental conditions and for characterization of the performance of the applied mass spectrometric system. Experiments revealed that because of the mass accuracy, the stability of calibration, and the reproducibility of fragmentation, the QqTOF mass spectrometer is an appropriate platform for establishment of a tandem-mass-spectral library. Three-hundred and nineteen substances were used as reference samples to build the spectral library. For each reference compound, product-ion spectra were acquired at ten different collision-energy values between 5 eV and 50 eV. For identification of unknown compounds, a library search algorithm was developed. The closeness of matching between a measured product-ion spectrum and a spectrum stored in the library was characterized by a value called "match probability", which took into account the number of matched fragment ions, the number of fragment ions observed in the two spectra, and the sum of the intensity differences calculated for matching fragments. A large value for the match probability indicated a close match between the measured and the reference spectrum. A unique feature of the library search algorithm-an implemented spectral purification option-enables characterization of multi-contributor fragment-ion spectra. With the aid of this software feature, substances comprising only 1.0% of the total amount of binary mixtures were unequivocally assigned, in addition to the isobaric main contributors. The spectral library was successfully applied to the characterization of 39 forensic casework samples.

  7. Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

    Science.gov (United States)

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

  8. Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation.

    Science.gov (United States)

    Goodwin, Richard J A; Nilsson, Anna; Borg, Daniel; Langridge-Smith, Pat R R; Harrison, David J; Mackay, C Logan; Iverson, Suzanne L; Andrén, Per E

    2012-08-30

    Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Clinical significance of coryneform Gram-positive rods from blood identified by MALDI-TOF mass spectrometry and their susceptibility profiles - a retrospective chart review.

    Science.gov (United States)

    Mushtaq, Ammara; Chen, Derrick J; Strand, Gregory J; Dylla, Brenda L; Cole, Nicolynn C; Mandrekar, Jayawant; Patel, Robin

    2016-07-01

    With the advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), most Gram-positive rods (GPRs) are readily identified; however, their clinical relevance in blood cultures remains unclear. Herein, we assessed the clinical significance of GPRs isolated from blood and identified in the era of MALDI-TOF MS. A retrospective chart review of patients presenting to the Mayo Clinic, Rochester, MN, from January 1, 2013, to October 13, 2015, was performed. Any episode of a positive blood culture for a GPR was included. We assessed the number of bottles positive for a given isolate, time to positivity of blood cultures, patient age, medical history, interpretation of culture results by the healthcare team and whether infectious diseases consultation was obtained. We also evaluated the susceptibility profiles of a larger collection of GPRs tested in the clinical microbiology laboratory of the Mayo Clinic, Rochester, MN from January 1, 2013, to October 31, 2015. There were a total of 246 GPRs isolated from the blood of 181 patients during the study period. 56% (n = 101) were deemed contaminants by the healthcare team and were not treated; 33% (n = 59) were clinically determined to represent true bacteremia and were treated; and 8% (n = 14) were considered of uncertain significance, with patients prescribed treatment regardless. Patient characteristics associated with an isolate being treated on univariate analysis included younger age (P = 0.02), identification to the species level (P = 0.02), higher number of positive blood culture sets (P < 0.0001), lower time to positivity (P < 0.0001), immunosuppression (P = 0.03), and recommendation made by an infectious disease consultant (P = 0.0005). On multivariable analysis, infectious diseases consultation (P = 0.03), higher number of positive blood culture sets (P = 0.0005) and lower time to positivity (P = 0.03) were associated with an isolate being treated. 100, 83, 48 and 34% of GPRs

  10. Novel Accurate Bacterial Discrimination by MALDI-Time-of-Flight MS Based on Ribosomal Proteins Coding in S10-spc-alpha Operon at Strain Level S10-GERMS

    Science.gov (United States)

    Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

    2013-08-01

    Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S 10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

  11. Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

    Science.gov (United States)

    Christner, Martin; Trusch, Maria; Rohde, Holger; Kwiatkowski, Marcel; Schlüter, Hartmut; Wolters, Manuel; Aepfelbacher, Martin; Hentschke, Moritz

    2014-01-01

    Background In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification. Methods Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak. Results Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates. Conclusions MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow. PMID:25003758

  12. Fundamentals of MALDI-ToF-MS analysis applications in bio-diagnosis, tissue engineering and drug delivery

    CERN Document Server

    Hosseini, Samira

    2017-01-01

    This book presents the fundamentals and applications of Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF-MS) technique. It highlights the basic principles, the history of invention as well as the mechanism of ionization and mass determination using this technique. It describes the fundamental principles and methods for MALDI spectra interpretation and determination of exact chemical structures from experimental data. This book guides the reader through the interpretation of MALDI data where complex macromolecular spectra are simplified in order to present the major principles behind data interpretation. In addition, each chapter describes how MALDI-ToF-MS analysis provides necessary understanding of the copolymer systems that have been designed for specialized biomedical applications.

  13. Detection of mono- and di-hexoses as metabolites of 4-bromoaniline using HPLC-TOF-MS/MS.

    Science.gov (United States)

    Major, H; Castro-Perez, J; Nicholson, J K; Wilson, I D

    2003-08-01

    1. The metabolic fate of 4-bromoaniline (4-BrA) was investigated in rat following intraperitoneal administration at 50 mg kg(-1) using HPLC-TOF-MS/MS. 2. The sensitivity provided by the use of TOF-MS/MS, aided by the distinctive isotope pattern resulting from the presence of the bromine substituent in the molecule, enabled the detection of many previously uncharacterized metabolites in the samples. 3. Several groups of minor metabolites were detected in the urine that corresponded to a number of isomeric hexose and di-hexose-containing conjugates (possibly glucosides and diglucosides) of 4-BrA. 4. As well as hexose and di-hexose conjugates of 4-BrA, several further groups of metabolites that also contained either a sulphamate or sulphate group in addition to the sugar moieties were also detected.

  14. Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality : Effect of sample preparation on MALDI-MS of synthetic polymers

    NARCIS (Netherlands)

    Kooijman, Pieter C.; Kok, Sander; Honing, Maarten

    2017-01-01

    Rationale: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides detailed and in-depth information about the molecular characteristics of synthetic polymers. To obtain the most accurate results the sample preparation parameters should be chosen to suit the sample and the

  15. The combination of simple MALDI matrices for the improvement of intact glycoproteins and glycans analysis

    Czech Academy of Sciences Publication Activity Database

    Laštovičková, Markéta; Chmelík, Josef; Bobálová, Janette

    2009-01-01

    Roč. 281, 1-2 (2009), s. 82-88 ISSN 1387-3806 R&D Projects: GA AV ČR IAA600040701; GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : glycoproteins * binary matrices * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.117, year: 2009

  16. MALDI MS

    Indian Academy of Sciences (India)

    Unknown

    revealed that there was an upper limit on the size of the molecules that can be .... 5. Sample preparation. The sample preparation for polymer MALDI must ac- ..... copy are in qualitative agreement with those obtained from other conventional ...

  17. Characterization of Dickeya and Pectobacterium species by capillary electrophoretic techniques and MALDI-TOF MS

    Czech Academy of Sciences Publication Activity Database

    Šalplachta, Jiří; Kubesová, Anna; Horký, J.; Matoušková, H.; Tesařová, Marie; Horká, Marie

    2015-01-01

    Roč. 407, č. 25 (2015), s. 7625-7635 ISSN 1618-2642 R&D Projects: GA MV VG20112015021 Institutional support: RVO:68081715 Keywords : bacteria * electrophoretic techniques * MALDI Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.125, year: 2015 http://hdl.handle.net/11104/0250090

  18. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories.

    Science.gov (United States)

    van Veen, S Q; Claas, E C J; Kuijper, Ed J

    2010-03-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.

  19. Differentiation of Vitis vinifera varieties by MALDI-MS analysis of the grape seed proteins.

    Science.gov (United States)

    Pesavento, Ivana Chiara; Bertazzo, Antonella; Flamini, Riccardo; Vedova, Antonio Dalla; De Rosso, Mirko; Seraglia, Roberta; Traldi, Pietro

    2008-02-01

    Until now the study of pathogenic related proteins in grape juice and wine, performed by ESI-MS, LC/ESI-MS, and MALDI/MS, has been proposed for differentiation of varieties. In fact, chitinases and thaumatin-like proteins persist through the vinification process and cause hazes and sediments in bottled wines. An additional instrument, potentially suitable for the grape varieties differentiation, has been developed by MALDI/MS for the grape seed protein analysis. The hydrosoluble protein profiles of seeds extract from three different Vitis vinifera grape (red and white) varieties were analyzed and compared. In order to evaluate the environmental conditions and harvest effects, the seed protein profiles of one grape variety from different locations and harvests were studied. (c) 2008 John Wiley & Sons, Ltd.

  20. Comparison of feature selection and classification for MALDI-MS data

    Directory of Open Access Journals (Sweden)

    Yang Mary

    2009-07-01

    Full Text Available Abstract Introduction In the classification of Mass Spectrometry (MS proteomics data, peak detection, feature selection, and learning classifiers are critical to classification accuracy. To better understand which methods are more accurate when classifying data, some publicly available peak detection algorithms for Matrix assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS data were recently compared; however, the issue of different feature selection methods and different classification models as they relate to classification performance has not been addressed. With the application of intelligent computing, much progress has been made in the development of feature selection methods and learning classifiers for the analysis of high-throughput biological data. The main objective of this paper is to compare the methods of feature selection and different learning classifiers when applied to MALDI-MS data and to provide a subsequent reference for the analysis of MS proteomics data. Results We compared a well-known method of feature selection, Support Vector Machine Recursive Feature Elimination (SVMRFE, and a recently developed method, Gradient based Leave-one-out Gene Selection (GLGS that effectively performs microarray data analysis. We also compared several learning classifiers including K-Nearest Neighbor Classifier (KNNC, Naïve Bayes Classifier (NBC, Nearest Mean Scaled Classifier (NMSC, uncorrelated normal based quadratic Bayes Classifier recorded as UDC, Support Vector Machines, and a distance metric learning for Large Margin Nearest Neighbor classifier (LMNN based on Mahanalobis distance. To compare, we conducted a comprehensive experimental study using three types of MALDI-MS data. Conclusion Regarding feature selection, SVMRFE outperformed GLGS in classification. As for the learning classifiers, when classification models derived from the best training were compared, SVMs performed the best with respect to the expected testing

  1. BioSunMS: a plug-in-based software for the management of patients information and the analysis of peptide profiles from mass spectrometry

    Directory of Open Access Journals (Sweden)

    Zhang Xuemin

    2009-02-01

    Full Text Available Abstract Background With wide applications of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS, statistical comparison of serum peptide profiles and management of patients information play an important role in clinical studies, such as early diagnosis, personalized medicine and biomarker discovery. However, current available software tools mainly focused on data analysis rather than providing a flexible platform for both the management of patients information and mass spectrometry (MS data analysis. Results Here we presented a plug-in-based software, BioSunMS, for both the management of patients information and serum peptide profiles-based statistical analysis. By integrating all functions into a user-friendly desktop application, BioSunMS provided a comprehensive solution for clinical researchers without any knowledge in programming, as well as a plug-in architecture platform with the possibility for developers to add or modify functions without need to recompile the entire application. Conclusion BioSunMS provides a plug-in-based solution for managing, analyzing, and sharing high volumes of MALDI-TOF or SELDI-TOF MS data. The software is freely distributed under GNU General Public License (GPL and can be downloaded from http://sourceforge.net/projects/biosunms/.

  2. Secondary metabolite profiling of Curcuma species grown at different locations using GC/TOF and UPLC/Q-TOF MS.

    Science.gov (United States)

    Lee, Jueun; Jung, Youngae; Shin, Jeoung-Hwa; Kim, Ho Kyoung; Moon, Byeong Cheol; Ryu, Do Hyun; Hwang, Geum-Sook

    2014-07-04

    Curcuma, a genus of rhizomatous herbaceous species, has been used as a spice, traditional medicine, and natural dye. In this study, the metabolite profile of Curcuma extracts was determined using gas chromatography-time of flight mass spectrometry (GC/TOF MS) and ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) to characterize differences between Curcuma aromatica and Curcuma longa grown on the Jeju-do or Jin-do islands, South Korea. Previous studies have performed primary metabolite profiling of Curcuma species grown in different regions using NMR-based metabolomics. This study focused on profiling of secondary metabolites from the hexane extract of Curcuma species. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) plots showed significant differences between the C. aromatica and C. longa metabolite profiles, whereas geographical location had little effect. A t-test was performed to identify statistically significant metabolites, such as terpenoids. Additionally, targeted profiling using UPLC/Q-TOF MS showed that the concentration of curcuminoids differed depending on the plant origin. Based on these results, a combination of GC- and LC-MS allowed us to analyze curcuminoids and terpenoids, the typical bioactive compounds of Curcuma, which can be used to discriminate Curcuma samples according to species or geographical origin.

  3. Secondary Metabolite Profiling of Curcuma Species Grown at Different Locations Using GC/TOF and UPLC/Q-TOF MS

    Directory of Open Access Journals (Sweden)

    Jueun Lee

    2014-07-01

    Full Text Available Curcuma, a genus of rhizomatous herbaceous species, has been used as a spice, traditional medicine, and natural dye. In this study, the metabolite profile of Curcuma extracts was determined using gas chromatography-time of flight mass spectrometry (GC/TOF MS and ultrahigh-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS to characterize differences between Curcuma aromatica and Curcuma longa grown on the Jeju-do or Jin-do islands, South Korea. Previous studies have performed primary metabolite profiling of Curcuma species grown in different regions using NMR-based metabolomics. This study focused on profiling of secondary metabolites from the hexane extract of Curcuma species. Principal component analysis (PCA and partial least-squares discriminant analysis (PLS-DA plots showed significant differences between the C. aromatica and C. longa metabolite profiles, whereas geographical location had little effect. A t-test was performed to identify statistically significant metabolites, such as terpenoids. Additionally, targeted profiling using UPLC/Q-TOF MS showed that the concentration of curcuminoids differed depending on the plant origin. Based on these results, a combination of GC- and LC-MS allowed us to analyze curcuminoids and terpenoids, the typical bioactive compounds of Curcuma, which can be used to discriminate Curcuma samples according to species or geographical origin.

  4. Application of the MALDI Biotyper to clinical microbiology: progress and potential.

    Science.gov (United States)

    Kostrzewa, Markus

    2018-03-01

    The introduction of the MALDI Biotyper in laboratories substantially changed microbiology practice, this has been called a revolution. The system accelerated diagnostic while costs were reduced and accuracy was increased. In just a few years MALDI-TOF MS became the first-line identification tool for microorganisms. Ten years after its introduction, more than 2000 MALDI Biotyper systems are installed in laboratories which are performing routine diagnostic, and the number is still increasing. Areas covered: This article summarises changes in clinical microbiology introduced by the MALDI Biotyper and its effects, as it has been published in peer reviewed articles found in PubMed. Further, the potential of novel developments to increase the value of the system is described. Expert commentary: The MALDI Biotyper has significantly improved clinical microbiology in the area of microorganism identification. Now new developments and applications, e.g. for typing and resistance testing, might further increase its value in clinical microbiology. The systems might get the central diagnostic analyser which is getting integrated into the widely automated microbiology laboratories of the future.

  5. MALDI-MS analysis and theoretical evaluation of olanzapine as a UV laser desorption ionization (LDI) matrix.

    Science.gov (United States)

    Musharraf, Syed Ghulam; Ameer, Mariam; Ali, Arslan

    2017-01-05

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) being soft ionization technique, has become a method of choice for high-throughput analysis of proteins and peptides. In this study, we have explored the potential of atypical anti-psychotic drug olanzapine (OLZ) as a matrix for MALDI-MS analysis of peptides aided with the theoretical studies. Seven small peptides were employed as target analytes to check performance of olanzapine and compared with conventional MALDI matrix α-cyano-4-hydroxycinnamic acid (HCCA). All peptides were successfully detected when olanzapine was used as a matrix. Moreover, peptides angiotensin Ι and angiotensin ΙΙ were detected with better S/N ratio and resolution with this method as compared to their analysis by HCCA. Computational studies were performed to determine the thermochemical properties of olanzapine in order to further evaluate its similarity to MALDI matrices which were found in good agreement with the data of existing MALDI matrices. Copyright © 2016. Published by Elsevier B.V.

  6. HPLC bottom-up MS-based proteomics for mapping of specific proteins in several European spring barley varieties

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Benkovská, Dagmar; Laštovičková, Markéta; Bobálová, Janette

    2015-01-01

    Roč. 73, č. 1 (2015), s. 71-77 ISSN 0361-0470 R&D Projects: GA ČR(CZ) GPP503/12/P395 Institutional support: RVO:68081715 Keywords : barley * gel electrophoresis * MALDI-TOF/TOF MS * protein profile * RP liquid chromatography Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.492, year: 2015

  7. Comparison of the identification results of Candida species obtained by BD Phoenix™ and Maldi-TOF (Bruker Microflex LT Biotyper 3.1).

    Science.gov (United States)

    Marucco, Andrea P; Minervini, Patricia; Snitman, Gabriela V; Sorge, Adriana; Guelfand, Liliana I; Moral, Laura López

    2018-02-05

    In patients with invasive fungal infections, the accurate and rapid identification of the genus Candida is of utmost importance since antimycotic sensitivity is closely related to the species. The aim of the present study was to compare the identification results of species of the genus Candida obtained by BD Phoenix™ (Becton Dickinson [BD]) and Maldi-TOF MS (Bruker Microflex LT Biotyper 3.1). A total of 192 isolates from the strain collection belonging to the Mycology Network of the Autonomous City of Buenos Aires, Argentina, were analyzed. The observed concordance was 95%. Only 10 strains (5%) were not correctly identified by the BD Phoenix™ system. The average identification time with the Yeast ID panels was 8h 22min. The BD Phoenix™ system proved to be a simple, reliable and effective method for identifying the main species of the genus Candida. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  8. Methylobacterium Species Promoting Rice and Barley Growth and Interaction Specificity Revealed with Whole-Cell Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS Analysis.

    Directory of Open Access Journals (Sweden)

    Akio Tani

    Full Text Available Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant-microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of appropriate species that can persist on the plant is important to achieve growth promotion.

  9. Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

    Science.gov (United States)

    Zhang, Lin; Vranckx, Katleen; Janssens, Koen; Sandrin, Todd R.

    2015-01-01

    MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level. PMID:25590854

  10. Laser-induced hydrogen radical removal in UV MALDI-MS allows for the differentiation of flavonoid monoglycoside isomers.

    Science.gov (United States)

    Yamagaki, Tohru; Watanabe, Takehiro; Tanaka, Masaki; Sugahara, Kohtaro

    2014-01-01

    Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M - H](-)) and the sugar-unit lost fragment ions ([M - Sugar - H](-)). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M - H* - H](-), [M - Sugar - H* - H](-), and [M - Sugar - 2H* - H](-). It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser. The glycosyl positions depend on the extent of the hydrogen radical removals and that of the homolytic cleavage of the glycosidic bonds. Flavonoid mono-glycoside isomers were distinguished according to their TOF MS and tandem mass spectra.

  11. Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches.

    Science.gov (United States)

    Rodrigues, P; Santos, C; Venâncio, A; Lima, N

    2011-10-01

    Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification.   Aspergillus section Flavi isolates obtained from Portuguese almonds were characterized in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus and Aspergillus tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and nonaflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  12. Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches

    OpenAIRE

    Rodrigues, Paula; Venâncio, Armando; Lima, Nelson

    2011-01-01

    Section Flavi is one of the most significant Sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by MALDI-TOF ICMS, and to evaluate the discriminatory power of the various approaches in species identification. Methods and ...

  13. Evaluation of MALDI-TOF mass spectrometry and MALDI BioTyper in comparison to 16S rDNA sequencing for the identification of bacteria isolated from Arctic sea water.

    Directory of Open Access Journals (Sweden)

    Anna Maria Timperio

    Full Text Available MALDI-TOF Mass Spectrometry in association with the MALDI BioTyper 3.1 software has been evaluated for the identification and classification of 45 Arctic bacteria isolated from Kandalaksha Bay (White Sea, Russia. The high reliability of this method has been already demonstrated, in clinical microbiology, by a number of studies showing high attribution concordance with other credited analyses. Recently, it has been employed also in other branches of microbiology with controversial performance. The phyloproteomic results reported in this study were validated with those obtained by the "gold standard" 16S rDNA analysis. Concordance between the two methods was 100% at the genus level, while at the species level it was 48%. These percentages appeared to be quite high compared with other studies regarding environmental bacteria. However, the performance of MALDI BioTyper changed in relation to the taxonomical group analyzed, reflecting known identification problems related to certain genera. In our case, attribution concordance for Pseudomonas species was rather low (29%, confirming the problematic taxonomy of this genus, whereas that of strains from other genera was quite high (> 60%. Among the isolates tested in this study, two strains (Exiguobacterium oxidotolerans and Pseudomonas costantinii were misidentified by MALDI BioTyper due to absence of reference spectra in the database. Accordingly, missing spectra were acquired for the database implementation.

  14. Evaluation of MALDI-TOF mass spectrometry and MALDI BioTyper in comparison to 16S rDNA sequencing for the identification of bacteria isolated from Arctic sea water.

    Science.gov (United States)

    Timperio, Anna Maria; Gorrasi, Susanna; Zolla, Lello; Fenice, Massimiliano

    2017-01-01

    MALDI-TOF Mass Spectrometry in association with the MALDI BioTyper 3.1 software has been evaluated for the identification and classification of 45 Arctic bacteria isolated from Kandalaksha Bay (White Sea, Russia). The high reliability of this method has been already demonstrated, in clinical microbiology, by a number of studies showing high attribution concordance with other credited analyses. Recently, it has been employed also in other branches of microbiology with controversial performance. The phyloproteomic results reported in this study were validated with those obtained by the "gold standard" 16S rDNA analysis. Concordance between the two methods was 100% at the genus level, while at the species level it was 48%. These percentages appeared to be quite high compared with other studies regarding environmental bacteria. However, the performance of MALDI BioTyper changed in relation to the taxonomical group analyzed, reflecting known identification problems related to certain genera. In our case, attribution concordance for Pseudomonas species was rather low (29%), confirming the problematic taxonomy of this genus, whereas that of strains from other genera was quite high (> 60%). Among the isolates tested in this study, two strains (Exiguobacterium oxidotolerans and Pseudomonas costantinii) were misidentified by MALDI BioTyper due to absence of reference spectra in the database. Accordingly, missing spectra were acquired for the database implementation.

  15. Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds.

    Science.gov (United States)

    Torres, M J; Brandan, C Pérez; Petroselli, G; Erra-Balsells, R; Audisio, M C

    2016-01-01

    The antifungal effect of Bacillus subtilis subsp. subtilis PGPMori7 and Bacillus amyloliquefaciens PGPBacCA1 was evaluated against Macrophomina phaseolina (Tassi) Goid. Cell suspension (CS), cell-free supernatant (CFS) and the lipopeptide fraction (LF) of PGPMori7 and PGPBacCA1 were screened against three different M. phaseolina strains. CS exhibited the highest inhibitory effect (around 50%) when compared to those of CFS and LF, regardless of the fungal strain studied. The synthesis of lipopeptides was studied by UV-MALDI TOF. Chemical analysis of Bacillus metabolite synthesis revealed that surfactin and iturin were mainly produced in liquid medium. Potential fengycin was also co-produced when both Bacillus were cultivated in solid medium. In co-culture assays, the bacterial colony-fungal mycelium interface at the inhibition zone was evaluated by both scanning electron microscopy (SEM) and UV-MALDI TOF, the former to determine the structural changes on M. phaseolina cells and the latter to identify the main bioactive molecules involved in the inhibitory effect. PGPBacCA1 produced surfactin, iturin and fengycin in the inhibition zone while PGPMori7 only produced these metabolites within its colony and not in the narrow inhibition zone. Interestingly, SEM revealed that PGPBacCA1 induced damage in M. phaseolina sclerotia, generating a fungicidal effect as no growth was observed when normal growth conditions were reestablished. In turn, PGPMori7 inhibited the growth of the Macrophomina mycelium without fungal injury, resulting only in a fungistatic activity. From these results, it was determined that the two bacilli significantly inhibited the growth of an important phytopathogenic fungus by at least two different mechanisms: lipopeptide synthesis and competition among microorganisms. Copyright © 2015 Elsevier GmbH. All rights reserved.

  16. Analysis of skin derived peptides from the Cuyaba Dwarf Frog Physalaemus nattereri by off-line LC MALDI MS/MS

    DEFF Research Database (Denmark)

    Castro, Mariana S; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner

    2017-01-01

    We have investigated the potential for analysis of the complex peptide mixtures secreted from frog skin using off-line LC-MALDI MS/MS. Since only limited information about the sequence of such peptides is available, de novo sequencing followed by Blast search was needed. An automated workflow has...... for future studies of peptides secreted from frogs....

  17. MALDI-TOF mass spectrometry for the rapid identification of aetiological agents of sepsis

    Directory of Open Access Journals (Sweden)

    Roberto Degl’Innocenti

    2013-04-01

    Full Text Available Introduction: The MALDI-TOF has recently become part of the methods of microbiological investigation in many laboratories of bacteriology with advantages both practical and economical.The use of this technique for the rapid identification of the causative agents of sepsis is of strategic importance to the ability to provide the clinician with useful information for a prompt and rapid establishment of an empirical antimicrobial “targeted” therapy. Methods: It was tested a total of 343 positive blood culture bottles from 211 patients. The samples after collection were incubated in the BACTEC FX (Becton Dickinson, USA. From these bottles were taken a few milliliters of broth culture and transferred into a vacutainer tube containing gel. This was centrifuged, the supernatant was decanted, and finally recovered the bacterial suspension on the gel. With micro-organisms recovered in this way, after several washes with distilled water, was prepared a slide for microscopic examination with Gram stain, and a plate for mass spectrometry (MS-Vitek, bioMérieux, France.Then, the same samples were inoculated on solid agar media according to the protocol in use in our laboratory.The next day was checked the possible bacterial growth on solid media; we then proceeded to the identification of the colonies by Vitek MS and / or with the system Vitek2 (bioMérieux, France. Results: 258 (75.2% positive vials show concordant results between direct identification and identification after growth on agar. For 83 (24.2% positive bottles there has been full compliance with the microscopic examination but not with culture. In particular, two bottles (0.6% have given complete discordance between the direct identification and that after growth. Conclusions: The protocol we use for the direct identification of organisms responsible for sepsis, directly on positive bottles, seems to be a quick and inexpensive procedure, which in less than 60 minutes can give valuable

  18. Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    Science.gov (United States)

    Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard; Raoult, Didier

    2013-07-01

    During the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

  19. Detection of Amyloid Beta (Aβ) Oligomeric Composition Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS)

    Science.gov (United States)

    Wang, Jasmine S.-H.; Whitehead, Shawn N.; Yeung, Ken K.-C.

    2018-02-01

    The use of MALDI MS as a fast and direct method to detect the Aβ oligomers of different masses is examined in this paper. Experimental results suggest that Aβ oligomers are ionized and detected as singly charged ions, and thus, the resulting mass spectrum directly reports the oligomer size distribution. Validation experiments were performed to verify the MS data against artifacts. Mass spectra collected from modified Aβ peptides with different propensities for aggregation were compared. Generally, the relative intensities of multimers were higher from samples where oligomerization was expected to be more favorable, and vice versa. MALDI MS was also able to detect the differences in oligomeric composition before and after the incubation/oligomerization step. Such differences in sample composition were also independently confirmed with an in vitro Aβ toxicity study on primary rat cortical neurons. An additional validation was accomplished through removal of oligomers from the sample using molecular weight cutoff filters; the resulting MS data correctly reflected the removal at the expected cutoff points. The results collectively validated the ability of MALDI MS to assess the monomeric/multimeric composition of Aβ samples. [Figure not available: see fulltext.

  20. Peptidylation for the determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Tang, Feng; Cen, Si-Ying; He, Huan; Liu, Yi; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-05-23

    Determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been a great challenge in the analytical research field. Here we developed a universal peptide-based derivatization (peptidylation) strategy for the sensitive analysis of low-molecular-weight compounds by MALDI-TOF-MS. Upon peptidylation, the molecular weights of target analytes increase, thus avoiding serious matrix ion interference in the low-molecular-weight region in MALDI-TOF-MS. Since peptides typically exhibit good signal response during MALDI-TOF-MS analysis, peptidylation endows high detection sensitivities of low-molecular-weight analytes. As a proof-of-concept, we analyzed low-molecular-weight compounds of aldehydes and thiols by the developed peptidylation strategy. Our results showed that aldehydes and thiols can be readily determined upon peptidylation, thus realizing the sensitive and efficient determination of low-molecular-weight compounds by MALDI-TOF-MS. Moreover, target analytes also can be unambiguously detected in biological samples using the peptidylation strategy. The established peptidylation strategy is a universal strategy and can be extended to the sensitive analysis of various low-molecular-weight compounds by MALDI-TOF-MS, which may be potentially used in areas such as metabolomics.

  1. Limitations in SELDI-TOF MS whole serum proteomic profiling with IMAC surface to specifically detect colorectal cancer

    International Nuclear Information System (INIS)

    Wang, Qi; Gu, Jin; Shen, Jing; Li, Zhen-fu; Jie, Jian-zheng; Wang, Wen-yue; Wang, Jin; Zhang, Zhong-tao; Li, Zhi-xia; Yan, Li

    2009-01-01

    Surface enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) analysis on serum samples was reported to be able to detect colorectal cancer (CRC) from normal or control patients. We carried out a validation study of a SELDI-TOF MS approach with IMAC surface sample processing to identify CRC. A retrospective cohort of 338 serum samples including 154 CRCs, 67 control cancers and 117 non-cancerous conditions was profiled using SELDI-TOF-MS. No CRC 'specific' classifier was found. However, a classifier consisting of two protein peaks separates cancer from non-cancerous conditions with high accuracy. In this study, the SELDI-TOF-MS-based protein expression profiling approach did not perform to identify CRC. However, this technique is promising in distinguishing patients with cancer from a non-cancerous population; it may be useful for monitoring recurrence of CRC after treatment

  2. Early identification of microorganisms in blood culture prior to the detection of a positive signal in the BACTEC FX system using matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

    Science.gov (United States)

    Wang, Ming-Cheng; Lin, Wei-Hung; Yan, Jing-Jou; Fang, Hsin-Yi; Kuo, Te-Hui; Tseng, Chin-Chung; Wu, Jiunn-Jong

    2015-08-01

    Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a valuable method for rapid identification of blood stream infection (BSI) pathogens. Integration of MALDI-TOF MS and blood culture system can speed the identification of causative BSI microorganisms. We investigated the minimal microorganism concentrations of common BSI pathogens required for positive blood culture using BACTEC FX and for positive identification using MALDI-TOF MS. The time to detection with positive BACTEC FX and minimal incubation time with positive MALDI-TOF MS identification were determined for earlier identification of common BSI pathogens. The minimal microorganism concentrations required for positive blood culture using BACTEC FX were >10(7)-10(8) colony forming units/mL for most of the BSI pathogens. The minimal microorganism concentrations required for identification using MALDI-TOF MS were > 10(7) colony forming units/mL. Using simulated BSI models, one can obtain enough bacterial concentration from blood culture bottles for successful identification of five common Gram-positive and Gram-negative bacteria using MALDI-TOF MS 1.7-2.3 hours earlier than the usual time to detection in blood culture systems. This study provides an approach to earlier identification of BSI pathogens prior to the detection of a positive signal in the blood culture system using MALDI-TOF MS, compared to current methods. It can speed the time for identification of BSI pathogens and may have benefits of earlier therapy choice and on patient outcome. Copyright © 2013. Published by Elsevier B.V.

  3. Direct identification from positive blood broth culture by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS

    Directory of Open Access Journals (Sweden)

    Maria Goreth Barberino

    2017-05-01

    Full Text Available Bloodstream infections (BSIs are among the most concerning bacterial infections. They are one of the leading causes of morbidity and mortality, and occur in 30–70% of critical care patients. The prompt identification of the causative microorganism can help choosing the appropriate antimicrobial therapy that will lead to better clinical outcomes. Blood culture is one of the most relevant tests for microbiological diagnosis of bacterial infections. The introduction of the MALDI-TOF microbiological diagnosis significantly decreased the time of identifying microorganisms. However, it depends on the growth on solid culture medium. In this study, 538 bottles of positive blood cultures were evaluated to test the accuracy of an in house modified protocol. The study sample consisted of 198 Gram-negative and 350 Gram-positive bacteria. In all, 460 (83.94% species were identified based on the direct plate findings. The protocol allowed the identification of 185/198 (93.43% of the Gram-negative bacteria, including aerobes, anaerobes, and non-fermenters, and 275/350 (78.85% of the Gram-positive bacteria. The proposed method has the potential to provide accurate results in comparison to the traditional method with the potential to reduce the turnaround time for the results and optimize antimicrobial therapy in BSI.

  4. Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

    Science.gov (United States)

    Clark, Andrew E.; Kaleta, Erin J.; Arora, Amit

    2013-01-01

    SUMMARY Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the “nuts and bolts” of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care. PMID:23824373

  5. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology.

    Science.gov (United States)

    Clark, Andrew E; Kaleta, Erin J; Arora, Amit; Wolk, Donna M

    2013-07-01

    Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the "nuts and bolts" of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.

  6. Screening natural antioxidants in peanut shell using DPPH-HPLC-DAD-TOF/MS methods.

    Science.gov (United States)

    Qiu, Jiying; Chen, Leilei; Zhu, Qingjun; Wang, Daijie; Wang, Wenliang; Sun, Xin; Liu, Xiaoyong; Du, Fangling

    2012-12-15

    Peanut shell, a byproduct in oil production, is rich in natural antioxidants. Here, a rapid and efficient method using DPPH-HPLC-DAD-TOF/MS was used for the first time to screen antioxidants in peanut shell. The method is based on the hypothesis that upon reaction with 1, 1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas of compounds with potential antioxidant activities in the HPLC chromatogram will be significantly reduced or disappeared, and the identity confirmation could be achieved by HPLC-DAD-TOF/MS technique. With this method, three compounds possessing potential antioxidant activities were found abundantly in the methanolic extract of peanut shell. They were identified as 5,7-dihydroxychromone, eriodictyol, and luteolin. The contents of these compounds were 0.59, 0.92, and 2.36 mg/g, respectively, and luteolin possessed the strongest radical scavenging capacity. DPPH-HPLC-DAD-TOF/MS assay facilitated rapid identification and determination of natural antioxidants in peanut shell, which may be helpful for value-added utilization of peanut processing byproducts. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. MALDI-ISD Mass Spectrometry Analysis of Hemoglobin Variants: a Top-Down Approach to the Characterization of Hemoglobinopathies

    Science.gov (United States)

    Théberge, Roger; Dikler, Sergei; Heckendorf, Christian; Chui, David H. K.; Costello, Catherine E.; McComb, Mark E.

    2015-08-01

    Hemoglobinopathies are the most common inherited disorders in humans and are thus the target of screening programs worldwide. Over the past decade, mass spectrometry (MS) has gained a more important role as a clinical means to diagnose variants, and a number of approaches have been proposed for characterization. Here we investigate the use of matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) with sequencing using in-source decay (MALDI-ISD) for the characterization of Hb variants. We explored the effect of matrix selection using super DHB or 1,5-diaminonaphthalene on ISD fragment ion yield and distribution. MALDI-ISD MS of whole blood using super DHB simultaneously provided molecular weights for the alpha and beta chains, as well as extensive fragmentation in the form of sequence defining c-, (z + 2)-, and y-ion series. We observed sequence coverage on the first 70 amino acids positions from the N- and C-termini of the alpha and beta chains in a single experiment. An abundant beta chain N-terminal fragment ion corresponding to βc34 was determined to be a diagnostic marker ion for Hb S (β6 Glu→Val, sickle cell), Hb C (β6 Glu→Lys), and potentially for Hb E (β26 Glu→Lys). The MALDI-ISD analysis of Hb S and HbSC yielded mass shifts corresponding to the variants, demonstrating the potential for high-throughput screening. Characterization of an alpha chain variant, Hb Westmead (α122 His→Gln), generated fragments that established the location of the variant. This study is the first clinical application of MALDI-ISD MS for the determination and characterization of hemoglobin variants.

  8. Chemical characterization of Azadirachta indica grafted on Melia azedarach and analyses of azadirachtin by HPLC-MS-MS (SRM) and meliatoxins by MALDI-MS.

    Science.gov (United States)

    Forim, Moacir Rossi; Cornélio, Vivian Estevam; da Silva, M Fátima das G F; Rodrigues-Filho, Edson; Fernandes, João B; Vieira, Paulo C; Matinez, Sueli Souza; Napolitano, Michael P; Yost, Richard A

    2010-01-01

    Melia azedarach adapted to cool climates was selected as rootstocks for vegetative propagation of Azadirachta indica. Cleft grafting of A. indica on M. azedarach rootstock showed excellent survival. Little is known about the chemistry of grafting. The roots, stems, leaves and seeds of this graft were examined in order to verify if grafted A. indica would produce limonoids different from those found in non-grafted plants. Intact matured fruits were also studied to verify if they were free of meliatoxins. After successive chromatographic separations the extracts afforded several limonoids. HPLC-MS/MS and MALDI-MS were used to develop sensitive methods for detecting azadirachtin on all aerial parts of this graft and meliatoxins in fruits, respectively. The stem afforded the limonoid salannin, which was previously found in the oil seeds of A. indica. Salannin is also found in the root bark of M. azedarach. Thus, the finding of salannin in this study suggests that it could have been translocated from the M. azedarach rootstock to the A. indica graft. HPLC-MS/MS analyses showed that azadirachtin was present in all parts of the fruits, stem, flowers and root, but absent in the leaves. The results of MALDI-MS analyses confirmed the absence of meliatoxins in graft fruits. This study showed that A. indica grafted onto M. azedarach rootstock produces azadirachtin, and also that its fruits are free of meliatoxins from rootstocks, confirming that this graft forms an excellent basis for breeding vigorous Neem trees in cooler regions.

  9. Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples.

    Science.gov (United States)

    Zhao, Yaju; Tang, Minmin; Liao, Qiaobo; Li, Zhoumin; Li, Hui; Xi, Kai; Tan, Li; Zhang, Mei; Xu, Danke; Chen, Hong-Yuan

    2018-04-27

    In this work, we demonstrate, for the first time, the development of a disposable MoS 2 -arrayed matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple sulfonamides (SAs). The disposable MALDI MS chip was designed and fabricated by MoS 2 array formation on a commercial indium tin oxide (ITO) glass slide. A series of SAs were analyzed, and clear deprotonated signals were obtained in negative-ion mode. Compared with MoS 2 -arrayed commercial steel plate, the prepared MALDI MS chip exhibited comparable LDI efficiency, providing a good alternative and disposable substrate for MALDI MS analysis. Furthermore, internal standard (IS) was previously deposited onto the MoS 2 array to simplify the experimental process for MALDI MS quantitation. 96 sample spots could be analyzed within 10 min in one single chip to perform quantitative analysis, recovery studies, and real foodstuff detection. Upon targeted extraction and enrichment by antibody conjugated magnetic beads, five SAs were quantitatively determined by the IS-first method with the linear range of 0.5-10 ng/mL ( R 2 > 0.990). Good recoveries and repeatability were obtained for spiked pork, egg, and milk samples. SAs in several real foodstuffs were successfully identified and quantified. The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.

  10. MALDI-MS Imaging Analysis of Fungicide Residue Distributions on Wheat Leaf Surfaces.

    Science.gov (United States)

    Annangudi, Suresh P; Myung, Kyung; Avila Adame, Cruz; Gilbert, Jeffrey R

    2015-05-05

    Improved retention and distribution of agrochemicals on plant surfaces is an important attribute in the biological activity of pesticide. Although retention of agrochemicals on plants after spray application can be quantified using traditional analytical techniques including LC or GC, the spatial distribution of agrochemicals on the plants surfaces has received little attention. Matrix assisted laser desorption/ionization (MALDI) imaging technology has been widely used to determine the distribution of proteins, peptides and metabolites in different tissue sections, but its application to environmental research has been limited. Herein, we probed the potential utility of MALDI imaging in characterizing the distribution of three commercial fungicides on wheat leaf surfaces. Using this MALDI imaging method, we were able to detect 500 ng of epoxiconazole, azoxystrobin, and pyraclostrobin applied in 1 μL drop on the leaf surfaces using MALDI-MS. Subsequent dilutions of pyraclostrobin revealed that the compound can be chemically imaged on the leaf surfaces at levels as low as 60 ng of total applied in the area of 1 μL droplet. After application of epoxiconazole, azoxystrobin, and pyraclostrobin at a field rate of 100 gai/ha in 200 L water using a track sprayer system, residues of these fungicides on the leaf surfaces were sufficiently visualized. These results suggest that MALDI imaging can be used to monitor spatial distribution of agrochemicals on leaf samples after pesticide application.

  11. S2P: A software tool to quickly carry out reproducible biomedical research projects involving 2D-gel and MALDI-TOF MS protein data.

    Science.gov (United States)

    López-Fernández, Hugo; Araújo, José E; Jorge, Susana; Glez-Peña, Daniel; Reboiro-Jato, Miguel; Santos, Hugo M; Fdez-Riverola, Florentino; Capelo, José L

    2018-03-01

    2D-gel electrophoresis is widely used in combination with MALDI-TOF mass spectrometry in order to analyze the proteome of biological samples. For instance, it can be used to discover proteins that are differentially expressed between two groups (e.g. two disease conditions, case vs. control, etc.) thus obtaining a set of potential biomarkers. This procedure requires a great deal of data processing in order to prepare data for analysis or to merge and integrate data from different sources. This kind of work is usually done manually (e.g. copying and pasting data into spreadsheet files), which is highly time consuming and distracts the researcher from other important, core tasks. Moreover, engaging in a repetitive process in a non-automated, handling-based manner is prone to error, thus threatening reliability and reproducibility. The objective of this paper is to present S2P, an open source software to overcome these drawbacks. S2P is implemented in Java on top of the AIBench framework, and relies on well-established open source libraries to accomplish different tasks. S2P is an AIBench based desktop multiplatform application, specifically aimed to process 2D-gel and MALDI-mass spectrometry protein identification-based data in a computer-aided, reproducible manner. Different case studies are presented in order to show the usefulness of S2P. S2P is open source and free to all users at http://www.sing-group.org/s2p. Through its user-friendly GUI interface, S2P dramatically reduces the time that researchers need to invest in order to prepare data for analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Seldi-tof MS Profiling of Plasma Proteins in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Shao-Pai Wu

    2006-03-01

    Conclusion: This study clearly demonstrates that the combined technology of SELDI-TOF MS and artificial intelligence is effective in distinguishing protein expression between normal and ovarian cancer plasma. The identified protein peaks may be candidate proteins for early detection of ovarian cancer or evaluation of therapeutic response.

  13. Identification of Cronobacter species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with an optimized analysis method.

    Science.gov (United States)

    Wang, Qi; Zhao, Xiao-Juan; Wang, Zi-Wei; Liu, Li; Wei, Yong-Xin; Han, Xiao; Zeng, Jing; Liao, Wan-Jin

    2017-08-01

    Rapid and precise identification of Cronobacter species is important for foodborne pathogen detection, however, commercial biochemical methods can only identify Cronobacter strains to genus level in most cases. To evaluate the power of mass spectrometry based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) for Cronobacter species identification, 51 Cronobacter strains (eight reference and 43 wild strains) were identified by both MALDI-TOF MS and 16S rRNA gene sequencing. Biotyper RTC provided by Bruker identified all eight reference and 43 wild strains as Cronobacter species, which demonstrated the power of MALDI-TOF MS to identify Cronobacter strains to genus level. However, using the Bruker's database (6903 main spectra products) and Biotyper software, the MALDI-TOF MS analysis could not identify the investigated strains to species level. When MALDI-TOF MS analysis was performed using the combined in-house Cronobacter database and Bruker's database, bin setting, and unweighted pair group method with arithmetic mean (UPGMA) clustering, all the 51 strains were clearly identified into six Cronobacter species and the identification accuracy increased from 60% to 100%. We demonstrated that MALDI-TOF MS was reliable and easy-to-use for Cronobacter species identification and highlighted the importance of establishing a reliable database and improving the current data analysis methods by integrating the bin setting and UPGMA clustering. Copyright © 2017. Published by Elsevier B.V.

  14. Comparison of MALDI-MSI and LC-MS for pharmacokinetic study of metformin

    Czech Academy of Sciences Publication Activity Database

    Strnad, Štěpán; Sýkora, D.; Cvačka, Josef; Maletínská, Lenka; Pirník, Z.; Majerčíková, Zuzana; Kuneš, Jaroslav; Vrkoslav, Vladimír

    2017-01-01

    Roč. 15, č. 1 (2017), s. 35 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : metformin * MALDI-MSI * LC-MS Subject RIV: CB - Analytical Chemistry, Separation

  15. Probing the fungicidal property of CdS quantum dots on Saccharomyces cerevisiae and Candida utilis using MALDI-MS

    Energy Technology Data Exchange (ETDEWEB)

    Manikandan, Muthu; Wu, Hui-Fen, E-mail: hwu@faculty.nsysu.edu.tw [National Sun Yat-Sen University, Department of Chemistry (China)

    2013-07-15

    For the first time, we report the successful application of inhouse synthesized CdS quantum dots (QDs) with particle sizes between 1 and 7 nm exhibiting excellent fungicidal activity based on the interactions with Saccharomyces cerevisiae and Candida utilis. The growth curves and the growth rates of both fungi were established in the presence of three varying concentrations of CdS QDs. It was observed that the CdS QDs were highly inhibitory even at the lowest concentration of 10 mg/L used in this study, while the untreated control cells followed a normal growth pattern in the cases of both Saccharomyces and Candida. MALDI-MS was applied to substantiate the observations obtained by direct cell count method. It was observed that the trend observed in the case of Saccharomyces and Candida was well-represented in the MALDI-MS spectra. This study proposes a mechanism for the first time based on MALDI-MS results, that the CdS QDs interact with the extracellular polymeric substances (EPS) and remove small molecules from EPS layer; on the other hand, it was observed that CdS QDs at all concentrations lead to enrichment of protein signals in MALDI-MS. We have substantiated these results by quantifying the EPS in the control and treated cells and also using TEM to further confirm the results.

  16. Probing the fungicidal property of CdS quantum dots on Saccharomyces cerevisiae and Candida utilis using MALDI-MS

    International Nuclear Information System (INIS)

    Manikandan, Muthu; Wu, Hui-Fen

    2013-01-01

    For the first time, we report the successful application of inhouse synthesized CdS quantum dots (QDs) with particle sizes between 1 and 7 nm exhibiting excellent fungicidal activity based on the interactions with Saccharomyces cerevisiae and Candida utilis. The growth curves and the growth rates of both fungi were established in the presence of three varying concentrations of CdS QDs. It was observed that the CdS QDs were highly inhibitory even at the lowest concentration of 10 mg/L used in this study, while the untreated control cells followed a normal growth pattern in the cases of both Saccharomyces and Candida. MALDI-MS was applied to substantiate the observations obtained by direct cell count method. It was observed that the trend observed in the case of Saccharomyces and Candida was well-represented in the MALDI-MS spectra. This study proposes a mechanism for the first time based on MALDI-MS results, that the CdS QDs interact with the extracellular polymeric substances (EPS) and remove small molecules from EPS layer; on the other hand, it was observed that CdS QDs at all concentrations lead to enrichment of protein signals in MALDI-MS. We have substantiated these results by quantifying the EPS in the control and treated cells and also using TEM to further confirm the results

  17. Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality: Effect of sample preparation on MALDI-MS of synthetic polymers.

    Science.gov (United States)

    Kooijman, Pieter C; Kok, Sander; Honing, Maarten

    2017-02-28

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides detailed and in-depth information about the molecular characteristics of synthetic polymers. To obtain the most accurate results the sample preparation parameters should be chosen to suit the sample and the aim of the experiment. Because the underlying principles of MALDI are still not fully known, a priori determination of optimal sample preparation protocols is often not possible. Employing an automated sample preparation quality assessment method recently presented by us we quantified the sample preparation quality obtained using various sample preparation protocols. Six conventional matrices with and without added potassium as a cationization agent and six ionic liquid matrices (ILMs) were assessed using poly(ethylene glycol) (PEG), polytetrahydrofuran (PTHF) and poly(methyl methacrylate) (PMMA) as samples. All sample preparation protocols were scored and ranked based on predefined quality parameters and spot-to-spot repeatability. Clearly distinctive preferences were observed in matrix identity and cationization agent for PEG, PTHF and PMMA, as the addition of an excess of potassium cationization agent results in an increased score for PMMA and a contrasting matrix-dependent effect for PTHF and PEG. The addition of excess cationization agent to sample mixtures dissipates any overrepresentation of high molecular weight polymer species. Our results show reduced ionization efficiency and similar sample deposit homogeneity for all tested ILMs, compared with well-performing conventional MALDI matrices. The results published here represent a start in the unsupervised quantification of sample preparation quality for MALDI samples. This method can select the best sample preparation parameters for any synthetic polymer sample and the results can be used to formulate hypotheses on MALDI principles. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Carole Cassagne

    Full Text Available BACKGROUND: MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory. MATERIALS AND METHODS: First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification. RESULTS: The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177 of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177, whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides. CONCLUSIONS: This work's seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting

  19. High-throughput workflow for identification of phosphorylated peptides by LC-MALDI-TOF/TOF-MS coupled to in situ enrichment on MALDI plates functionalized by ion landing

    Czech Academy of Sciences Publication Activity Database

    Krásný, Lukáš; Pompach, Petr; Strnadová, Marcela; Hynek, R.; Vališ, K.; Havlíček, Vladimír; Novák, Petr; Volný, M.

    2015-01-01

    Roč. 50, č. 6 (2015), s. 802-811 ISSN 1076-5174 R&D Projects: GA MŠk(CZ) EE2.3.20.0055; GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR GPP206/10/P018; GA ČR(CZ) GAP206/12/1150; GA ČR(CZ) GP14-21095P; GA ČR GA13-16565S; GA MŠk LH13051 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24023 Institutional support: RVO:61388971 Keywords : phosphopeptides * MALDI * enrichment Subject RIV: CE - Biochemistry Impact factor: 2.541, year: 2015

  20. A comparative study of carbocyanine dyes measured with TOF-SIMS and other mass spectrometric techniques

    International Nuclear Information System (INIS)

    Adriaensen, L.; Vangaever, F.; Gijbels, R.

    2004-01-01

    A series of cationic, zwitterionic and anionic carbocyanine dyes have been measured with TOF-SIMS under Ga + bombardment. In contrast to the cationic dyes, which give very intense molecular ion and characteristic fragment signals in the positive mode, the anionic dyes produce only a few fragment signals of low intensity. Even in the negative mode no molecular ions of the anionic dyes are seen in the recorded spectra. Actually, none of the studied molecules produces negative SIMS spectra containing molecular information. A comparative study was made between TOF-SIMS and other mass spectrometric techniques, namely, fast atom bombardment (FAB), electro spray ionization (ESI) and matrix assisted laser desorption ionization (MALDI). The measurements show that MALDI, ESI as well as FAB all give rise to spectra containing molecular ion signals, either in the positive, in the negative or in both modes. Unlike with TOF-SIMS, this observation also applies to the anionic dyes. Characteristic fragments of the dyes are present in all the recorded spectra. However, TOF-SIMS appears to induce more fragment ions in comparison with the other techniques. ESI, for instance, produces hardly any molecular fragments. Finally, the kind of fragment ions recorded depends upon the technique used, though some signals are produced by various techniques. For these carbocyanine dyes there is no clear correlation between the mass spectra obtained with TOF-SIMS and spectra obtained with the other techniques. This points to different desorption/ionization mechanisms, and makes it difficult, in practice, to make predictions on the feasibility of TOF-SIMS, starting from results of the other MS techniques

  1. MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

    DEFF Research Database (Denmark)

    Klenø, Tina Guldberg; Andreasen, Christian Maaløv; Kjeldal, Helle Ørsted

    2004-01-01

    Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increa......-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications....

  2. Direct identification from positive blood broth culture by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Barberino, Maria Goreth; Silva, Marcio de Oliveira; Arraes, Ana Carolina Palmeiras; Correia, Luís Cláudio; Mendes, Ana Verena

    Bloodstream infections (BSIs) are among the most concerning bacterial infections. They are one of the leading causes of morbidity and mortality, and occur in 30-70% of critical care patients. The prompt identification of the causative microorganism can help choosing the appropriate antimicrobial therapy that will lead to better clinical outcomes. Blood culture is one of the most relevant tests for microbiological diagnosis of bacterial infections. The introduction of the MALDI-TOF microbiological diagnosis significantly decreased the time of identifying microorganisms. However, it depends on the growth on solid culture medium. In this study, 538 bottles of positive blood cultures were evaluated to test the accuracy of an in house modified protocol. The study sample consisted of 198 Gram-negative and 350 Gram-positive bacteria. In all, 460 (83.94%) species were identified based on the direct plate findings. The protocol allowed the identification of 185/198 (93.43%) of the Gram-negative bacteria, including aerobes, anaerobes, and non-fermenters, and 275/350 (78.85%) of the Gram-positive bacteria. The proposed method has the potential to provide accurate results in comparison to the traditional method with the potential to reduce the turnaround time for the results and optimize antimicrobial therapy in BSI. Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

  3. Current status of matrix-assisted laser desorption ionisation-time of flight mass spectrometry in the clinical microbiology laboratory.

    Science.gov (United States)

    Kok, Jen; Chen, Sharon C A; Dwyer, Dominic E; Iredell, Jonathan R

    2013-01-01

    The integration of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) into many clinical microbiology laboratories has revolutionised routine pathogen identification. MALDI-TOF MS complements and has good potential to replace existing phenotypic identification methods. Results are available in a more clinically relevant timeframe, particularly in bacteraemic septic shock. Novel applications include strain typing and the detection of antimicrobial resistance, but these are not widely used. This review discusses the technical aspects, current applications, and limitations of MALDI-TOF MS.

  4. Investigation of the interaction of iron(III) complexes with dAMP by ESI-MS, MALDI-MS and potentiometric titration: insights into synthetic nuclease behavior.

    Science.gov (United States)

    Fernandes, Christiane; Oliveira Moreira, Rafaela; Lube, Leonardo M; Horn, Adolfo; Szpoganicz, Bruno; Sherrod, Stacy; Russell, David H

    2010-06-07

    We report herein the characterization by electrospray ionization (ESI) mass spectrometry (MS), matrix assisted laser desorption ionization (MALDI-MS) and potentiometric titration of three iron(III) compounds: [Fe(III)(HPClNOL)Cl2]·NO3 (1), [Cl(HPClNOL)Fe(III)-(μ-O)-Fe(III)(HPClNOL)Cl]·Cl2·H2O (2) and [(SO4)(HPClNOL)Fe(III)-(μ-O)-Fe(III)(HPClNOL)(SO4)]·6H2O (3), where HPClNOL= 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol). Despite the fact that the compounds have distinct structures in solid state and non-buffered solution, all compounds present similar ESI and MALDI mass spectra in a buffered medium (pH 7.0). At this pH, the species [(PClNOL)Fe(III)-(μ-O)-Fe(III)(PClNOL)](2+) (m/z 354) was observed for all the compounds under investigation. Potentiometric titration confirms a similar behavior for all compounds, indicating that the dihydroxo form [(OH)(HPClNOL)Fe(III)-(μ-O)-Fe(III)(HPClNOL)(OH)](2+) is the major species at pH 7.0, for all the compounds. The products of the interaction between compounds (1), (2) and (3) and dAMP (2'-deoxyadenosine-5'-monophosphate) in a buffered medium (pH 7.0) were identified by MALDI-MS/MS. The fragmentation data obtained by MS/MS allow one to identify the nature of the interaction between the iron(III) compounds and dAMP, revealing the direct interaction between the iron center and phosphate groups.

  5. Species Identification and Delineation of Pathogenic Mucorales by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

    Science.gov (United States)

    Shao, Jin; Wan, Zhe; Li, Ruoyu; Yu, Jin

    2018-04-01

    This study aimed to validate the effectiveness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification of filamentous fungi of the order Mucorales. A total of 111 isolates covering six genera preserved at the Research Center for Medical Mycology of Peking University were selected for MALDI-TOF MS analysis. We emphasized the study of 23 strains of Mucor irregularis predominantly isolated from patients in China. We first used the Bruker Filamentous Fungi library (v1.0) to identify all 111 isolates. To increase the identification rate, we created a compensatory in-house database, the Beijing Medical University (BMU) database, using 13 reference strains covering 6 species, including M. irregularis , Mucor hiemalis , Mucor racemosus , Cunninghamella bertholletiae , Cunninghamella phaeospora , and Cunninghamella echinulata All 111 isolates were then identified by MALDI-TOF MS using a combination of the Bruker library and BMU database. MALDI-TOF MS identified 55 (49.5%) and 74 (66.7%) isolates at the species and genus levels, respectively, using the Bruker Filamentous Fungi library v1.0 alone. A combination of the Bruker library and BMU database allowed MALDI-TOF MS to identify 90 (81.1%) and 111 (100%) isolates at the species and genus levels, respectively, with a significantly increased accuracy rate. MALDI-TOF MS poorly identified Mucorales when the Bruker library was used alone due to its lack of some fungal species. In contrast, this technique perfectly identified M. irregularis after main spectrum profiles (MSPs) of relevant reference strains were added to the Bruker library. With an expanded Bruker library, MALDI-TOF MS is an effective tool for the identification of pathogenic Mucorales. Copyright © 2018 American Society for Microbiology.

  6. Use of matrix assisted laser desorption ionisation-time of flight mass spectrometry in a paediatric clinical laboratory for identification of bacteria commonly isolated from cystic fibrosis patients.

    Science.gov (United States)

    Desai, Ankita Patel; Stanley, Theresa; Atuan, Maria; McKey, Jonelle; Lipuma, John J; Rogers, Beverly; Jerris, Robert

    2012-09-01

    Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) has been described as a rapid, accurate method for bacterial identification. To investigate the ability of the technique, using the unamended database supplied with the system, to identify bacteria commonly isolated in cystic fibrosis (CF) patients. Organisms commonly isolated from CF patients identified by MALDI-TOF MS were compared to conventional phenotypic and genotypic analyses. For MALDI-TOF MS, the direct colony technique was used routinely with one extraction procedure performed on a mucoid Pseudomonas aeruginosa. For 24 unique CF specimens, workload comparison and time to identification were assessed. Of 464 tested isolates, conventional (phenotypic and genotypic) identification compared to MALDI-TOF MS showed complete genus, species agreement in 92%, with genus agreement in 98%. This included 29 isolates within the Burkholderia cepacia complex. All 29 were correctly identified to the genus level and 24 of these were speciated. Time to identification with 47 bacterial isolates from 24 CF patients showed identification of 85% of isolates by MALDI-TOF MS at 48 h of incubation, compared to only 34% with conventional methods. Using the unamended database supplied with the system, MALDI-TOF MS provides rapid and reliable identification of bacteria isolated from CF specimens. Time to identification studies showed that the use of same day, same method for organism identification will decrease time to result and optimise microbiology workflow.

  7. Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

    Science.gov (United States)

    Hettick, Justin M; Green, Brett J; Buskirk, Amanda D; Kashon, Michael L; Slaven, James E; Janotka, Erika; Blachere, Francoise M; Schmechel, Detlef; Beezhold, Donald H

    2008-09-15

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI-TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI-TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI-TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI-TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.

  8. Prevalence of antimicrobial resistance and the cfiA resistance gene in Danish Bacteroides fragilis group isolates since 1973

    DEFF Research Database (Denmark)

    Ferløv-Schwensen, Simon Andreas; Sydenham, Thomas Vognbjerg; Hansen, Kia Cirkeline Møller

    2017-01-01

    Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) on the Biotyper platform. Antimicrobial resistance was determined using a disk diffusion screening method and commercial antibiotic gradient strips. Division I (cfiA-negative) and division II (cfiA-positive) B. fragilis strains were...... differentiated using MALDI-TOF MS and real-time polymerase chain reaction (PCR). RESULTS: From 1973-1980 to 2010-2015 the prevalence of antimicrobial resistance rose from 0% to 21.2%, 2.5%, and 1% for clindamycin, meropenem, and metronidazole, respectively. MALDI-TOF MS and real-time PCR identified 16 of 266 (6...... established in the recent decades in Europe. Resistance to meropenem, facilitated by expression of the cfiA resistance gene, seems to be increasing; therefore, it is imperative to monitor the occurrence of this gene, e.g. using MALDI-TOF MS....

  9. A rapid diagnostic workflow for cefotaxime-resistant Escherichia coli and Klebsiella pneumoniae detection from blood cultures by MALDI-TOF mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Elena De Carolis

    Full Text Available Nowadays, the global spread of resistance to oxyimino-cephalosporins in Enterobacteriaceae implies the need for novel diagnostics that can rapidly target resistant organisms from these bacterial species.In this study, we developed and evaluated a Direct Mass Spectrometry assay for Beta-Lactamase (D-MSBL that allows direct identification of (oxyiminocephalosporin-resistant Escherichia coli or Klebsiella pneumoniae from positive blood cultures (BCs, by using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS technology.The D-MSBL assay was performed on 93 E. coli or K. pneumoniae growing BC samples that were shortly co-incubated with cefotaxime (CTX as the indicator cephalosporin. Susceptibility and resistance defining peaks from the samples' mass spectra were analyzed by a novel algorithm for bacterial organism classification. The D-MSBL assay allowed discrimination between E. coli and K. pneumoniae that were resistant or susceptible to CTX with a sensitivity of 86.8% and a specificity of 98.2%.The proposed algorithm-based D-MSBL assay, if integrated in the routine laboratory diagnostic workflow, may be useful to enhance the establishment of appropriate antibiotic therapy and to control the threat of oxyimino-cephalosporin resistance in hospital.

  10. A rapid diagnostic workflow for cefotaxime-resistant Escherichia coli and Klebsiella pneumoniae detection from blood cultures by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    De Carolis, Elena; Paoletti, Silvia; Nagel, Domenico; Vella, Antonietta; Mello, Enrica; Palucci, Ivana; De Angelis, Giulia; D'Inzeo, Tiziana; Sanguinetti, Maurizio; Posteraro, Brunella; Spanu, Teresa

    2017-01-01

    Nowadays, the global spread of resistance to oxyimino-cephalosporins in Enterobacteriaceae implies the need for novel diagnostics that can rapidly target resistant organisms from these bacterial species. In this study, we developed and evaluated a Direct Mass Spectrometry assay for Beta-Lactamase (D-MSBL) that allows direct identification of (oxyimino)cephalosporin-resistant Escherichia coli or Klebsiella pneumoniae from positive blood cultures (BCs), by using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology. The D-MSBL assay was performed on 93 E. coli or K. pneumoniae growing BC samples that were shortly co-incubated with cefotaxime (CTX) as the indicator cephalosporin. Susceptibility and resistance defining peaks from the samples' mass spectra were analyzed by a novel algorithm for bacterial organism classification. The D-MSBL assay allowed discrimination between E. coli and K. pneumoniae that were resistant or susceptible to CTX with a sensitivity of 86.8% and a specificity of 98.2%. The proposed algorithm-based D-MSBL assay, if integrated in the routine laboratory diagnostic workflow, may be useful to enhance the establishment of appropriate antibiotic therapy and to control the threat of oxyimino-cephalosporin resistance in hospital.

  11. Contaminant screening of wastewater with HPLC-IM-qTOF-MS and LC+LC-IM-qTOF-MS using a CCS database.

    Science.gov (United States)

    Stephan, Susanne; Hippler, Joerg; Köhler, Timo; Deeb, Ahmad A; Schmidt, Torsten C; Schmitz, Oliver J

    2016-09-01

    Non-target analysis has become an important tool in the field of water analysis since a broad variety of pollutants from different sources are released to the water cycle. For identification of compounds in such complex samples, liquid chromatography coupled to high resolution mass spectrometry are often used. The introduction of ion mobility spectrometry provides an additional separation dimension and allows determining collision cross sections (CCS) of the analytes as a further physicochemical constant supporting the identification. A CCS database with more than 500 standard substances including drug-like compounds and pesticides was used for CCS data base search in this work. A non-target analysis of a wastewater sample was initially performed with high performance liquid chromatography (HPLC) coupled to an ion mobility-quadrupole-time of flight mass spectrometer (IM-qTOF-MS). A database search including exact mass (±5 ppm) and CCS (±1 %) delivered 22 different compounds. Furthermore, the same sample was analyzed with a two-dimensional LC method, called LC+LC, developed in our group for the coupling to IM-qTOF-MS. This four dimensional separation platform revealed 53 different compounds, identified over exact mass and CCS, in the examined wastewater sample. It is demonstrated that the CCS database can also help to distinguish between isobaric structures exemplified for cyclophosphamide and ifosfamide. Graphical Abstract Scheme of sample analysis and database screening.

  12. The mass spectrometry technology MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time- Of-Flight for a more rapid and economic workflow in the clinical microbiology laboratory

    Directory of Open Access Journals (Sweden)

    Simona Barnini

    2012-12-01

    Full Text Available Introduction: In order to improve the outcome of patients, reduce length of stay, costs and resources engaged in diagnostics, more rapid reports are requested to the clinical microbiologists.The purpose of this study is to assess the impact on workflow of MALDI-TOF technology, recently made available for use in routine diagnostics. Methods:The work list by the management information system is sent to the instrument MALDI-TOF, where are held at least three successive analytic sessions: the first includes bacteria isolated from CSF, blood cultures, and cases already reported as serious/urgent, the second includes all other germs isolated, the third, microorganisms that require extraction with trifluoroacetic acid (TFA or formic acid (FA for identification.The results of each session direct to the execution of different types of susceptibility testing. Results:The times of microbial identifications are reduced by 24 or 48 hours and made available to the clinician for the rational empirical therapy.The reagent costs are reduced by 40%.The subcultures were reduced by 80%, and microscopic examinations by 50%.The antibiotic susceptibility tests were immediately performed with the most appropriate method, based on the knowledge of local epidemiology and microbial species. Conclusion:The bacteriology is the less automated discipline among the clinical laboratory activities and results of diagnostic tests are poorly well-timed. The new interpretative algorithms of MALDI-TOF spectra, now available, allow the correct identification of bacteria in near real time, completely eliminating the wait is necessary for biochemical identification and guiding the operator in selecting the most appropriate antibiotic susceptibility tests. This technology makes work more rapid, economic and efficient, eliminating errors and, together with effective computerization of data, transforms the information content of the microbiological report, making it much more effective

  13. SELDI-TOF MS of quadruplicate urine and serum samples to evaluate changes related to storage conditions.

    Science.gov (United States)

    Traum, Avram Z; Wells, Meghan P; Aivado, Manuel; Libermann, Towia A; Ramoni, Marco F; Schachter, Asher D

    2006-03-01

    Proteomic profiling with SELDI-TOF MS has facilitated the discovery of disease-specific protein profiles. However, multicenter studies are often hindered by the logistics required for prompt deep-freezing of samples in liquid nitrogen or dry ice within the clinic setting prior to shipping. We report high concordance between MS profiles within sets of quadruplicate split urine and serum samples deep-frozen at 0, 2, 6, and 24 h after sample collection. Gage R&R results confirm that deep-freezing times are not a statistically significant source of SELDI-TOF MS variability for either blood or urine.

  14. Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. botulinum Neurotoxin Detection

    Directory of Open Access Journals (Sweden)

    Michael J. Perry

    2017-03-01

    Full Text Available Currently, the gold standard method for active botulinum neurotoxin (BoNT detection is the mouse bioassay (MBA. A Centers for Disease Control and Prevention-developed mass spectrometry (MS-based assay that detects active BoNT was successfully validated and implemented in a public health laboratory in clinical matrices using the Bruker MALDI-TOF MS (Matrix-assisted laser desorption ionization–time of flight mass spectrometry Biotyper. For the first time, a direct comparison with the MBA was performed to determine MS-based assay sensitivity using the Bruker MALDI Biotyper. Mice were injected with BoNT/A, /B, /E, and /F at concentrations surrounding the established MS assay limit of detection (LOD and analyzed simultaneously. For BoNT/B, /E, and /F, MS assay sensitivity was equivalent or better than the MBA at 25, 0.3, and 8.8 mLD50, respectively. BoNT/A was detected by the MBA between 1.8 and 18 mLD50, somewhat more sensitive than the MS method of 18 mLD50. Studies were performed to compare assay performance in clinical specimens. For all tested specimens, the MS method rapidly detected BoNT activity and serotype in agreement with, or in the absence of, results from the MBA. We demonstrate that the MS assay can generate reliable, rapid results while eliminating the need for animal testing.

  15. Neutral Loss Scan - Based Strategy for Integrated Identification of Amorfrutin Derivatives, New Peroxisome Proliferator-Activated Receptor Gamma Agonists, from Amorpha Fruticosa by UPLC-QqQ-MS/MS and UPLC-Q-TOF-MS.

    Science.gov (United States)

    Chen, Chu; Xue, Ying; Li, Qing-Miao; Wu, Yan; Liang, Jian; Qing, Lin-Sen

    2018-04-01

    Amorfrutins with a 2-hydroxybenzoic acid core structure are promising natural PPARγ agonists with potent antidiabetic activity. Owing to the complex matrix and low concentration in botanical material, the identification of unknown amorfrutins remains a challenge. In the present study, a combined application of UPLC-Q-TOF-MS and UPLC-QqQ-MS was developed to discover unknown amorfrutins from fruits of Amorpha fruticosa. First, reference compounds of amorfrutin A (AA), amorfrutin B (AB), and 2-carboxy-3,5-dihydroxy-4-geranylbibenzyl (AC) were analyzed using UPLC-Q-TOF-MS to reveal the characteristic fragment ions and the possible neutral loss. Second, the extract of A. fruticosa was separated and screened by UPLC-QqQ-MS using neutral loss scan to find out suspect compounds associated with the specified neutral fragment Δm/z 44. Third, the extract was re-analyzed by UPLC-Q-TOF-MS to obtain the exact mass of quasi-molecular ion and fragment ions of each suspect compound, and to subsequently calculate their corresponding molecular formulas. Finally, according to the molecular formula of suspect compound and its fragment ions and comparing with literature data, structure elucidation of four unidentified amorfrutins was achieved. The results indicated that the combination of QqQ-MS neutral loss scan and Q-TOF-MS molecular formula calculation was proven to be a powerful tool for unknown natural product identification, and this strategy provides an effective solution to discover natural products or metabolites of trace content. Graphical Abstract ᅟ.

  16. Preparation of positive blood cultures for direct MALDI-ToF MS identification.

    Science.gov (United States)

    Robinson, Andrew M; Ussher, James E

    2016-08-01

    MALDI-ToF MS can be used to identify microorganisms directly from blood cultures. This study compared two methods of sample preparation. Similar levels of genus- (91% vs 90%) and species-level identifications (79% vs 74%) were obtained with differential centrifugation and SDS methods. The SDS method is faster and requires minimal handling. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Analysis of Wheat Prolamins, the Causative Agents of Celiac Sprue, Using Reversed Phase High Performance Liquid Chromatography (RP-HPLC and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS

    Directory of Open Access Journals (Sweden)

    Jaime H. Mejías

    2014-04-01

    Full Text Available Wheat prolamins, commonly known as “gluten”, are a complex mixture of 71–78 proteins, which constitute ~80% of the proteins in the wheat grains and supply 50% of the global dietary protein demand. Prolamins are also responsible for numerous gluten-induced disorders and determine the unique visco-elastic properties of the wheat dough. These properties necessitate the reliable determination of the prolamin composition in wheat grains and their derived products. Therefore, this study examined the impact of HPLC conditions, including column type, column temperature, flow rate, and the gradient of polar and non-polar solvents in the mobile phase, to improve the analytical resolution of prolamins. The following conditions were found optimal for analyses: column temperature 60 °C, flow rate 1.0 mL/min and an elution gradient of 20%–60% of 0.1% trifluoroacetic acid + acetonitrile in 60 min. For further improvement of resolution, gliadin and glutenin extracts were analyzed using MALDI-TOF-MS in combination with HPLC fractionation. Two semi-quantitative methods, densitometry of stained polyacrylamide gels and HPLC, were used to determine relative prolamin quantities and the correspondence between the methods was established. The combinatorial gluten analyses approach developed during the present study was used to analyze prolamin profiles of wheat transformants expressing DEMETER silencing artificial microRNA, and the results are discussed.

  18. Rapid detection of porins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry

    Directory of Open Access Journals (Sweden)

    Yanyan eHU

    2015-08-01

    Full Text Available The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of E. coli and eight strains of K. pneumoniae were conducted for both SDS-PAGE and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the corrospondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000-m/z, 37,000-m/z and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC and OmpF with molecular weight of approximately ca. 38 kDa, 40 kDa and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000-m/z and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 kDa and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.

  19. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of fungal rhinosinusitis pathogens.

    Science.gov (United States)

    Huang, Yanfei; Wang, Jinglin; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Lu, Xinxin

    2017-03-01

    Filamentous fungi are among the most important pathogens, causing fungal rhinosinusitis (FRS). Current laboratory diagnosis of FRS pathogens mainly relies on phenotypic identification by culture and microscopic examination, which is time consuming and expertise dependent. Although matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has been employed to identify various fungi, its efficacy in the identification of FRS fungi is less clear. A total of 153 FRS isolates obtained from patients were analysed at the Clinical Laboratory at the Beijing Tongren Hospital affiliated to the Capital Medical University, between January 2014 and December 2015. They were identified by traditional phenotypic methods and Bruker MALDI-TOF MS (Bruker, Biotyper version 3.1), respectively. Discrepancies between the two methods were further validated by sequencing. Among the 153 isolates, 151 had correct species identification using MALDI-TOF MS (Bruker, Biot 3.1, score ≥2.0 or 2.3). MALDI-TOF MS enabled identification of some very closely related species that were indistinguishable by conventional phenotypic methods, including 1/10 Aspergillus versicolor, 3/20 Aspergillus flavus, 2/30 Aspergillus fumigatus and 1/20 Aspergillus terreus, which were misidentified by conventional phenotypic methods as Aspergillus nidulans, Aspergillus oryzae, Aspergillus japonicus and Aspergillus nidulans, respectively. In addition, 2/2 Rhizopus oryzae and 1/1 Rhizopus stolonifer that were identified only to the genus level by the phenotypic method were correctly identified by MALDI-TOF MS. MALDI-TOF MS is a rapid and accurate technique, and could replace the conventional phenotypic method for routine identification of FRS fungi in clinical microbiology laboratories.

  20. Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of clinically important yeast species.

    Science.gov (United States)

    Stevenson, Lindsay G; Drake, Steven K; Shea, Yvonne R; Zelazny, Adrian M; Murray, Patrick R

    2010-10-01

    We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid identification of yeast species. Using Bruker Daltonics MALDI BioTyper software, we created a spectral database library with m/z ratios of 2,000 to 20,000 Da for 109 type and reference strains of yeast (44 species in 8 genera). The database was tested for accuracy by use of 194 clinical isolates (23 species in 6 genera). A total of 192 (99.0%) of the clinical isolates were identified accurately by MALDI-TOF MS. The MALDI-TOF MS-based method was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

  1. Mass spectrometry

    DEFF Research Database (Denmark)

    Nyvang Hartmeyer, Gitte; Jensen, Anne Kvistholm; Böcher, Sidsel

    2010-01-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently being introduced for the rapid and accurate identification of bacteria. We describe 2 MALDI-TOF MS identification cases - 1 directly on spinal fluid and 1 on grown bacteria. Rapidly obtained...

  2. Fast methods of fungal and bacterial identification. MALDI-TOF mass spectrometry, chromogenic media.

    Science.gov (United States)

    Siller-Ruiz, María; Hernández-Egido, Sara; Sánchez-Juanes, Fernando; González-Buitrago, José Manuel; Muñoz-Bellido, Juan Luis

    2017-05-01

    MALDI-TOF mass spectrometry is now a routine resource in Clinical Microbiology, because of its speed and reliability in the identification of microorganisms. Its performance in the identification of bacteria and yeasts is perfectly contrasted. The identification of mycobacteria and moulds is more complex, due to the heterogeneity of spectra within each species. The methodology is somewhat more complex, and expanding the size of species libraries, and the number of spectra of each species, will be crucial to achieve greater efficiency. Direct identification from blood cultures has been implemented, since its contribution to the management of severe patients is evident, but its application to other samples is more complex. Chromogenic media have also contributed to the rapid diagnosis in both bacteria and yeast, since they accelerate the diagnosis, facilitate the detection of mixed cultures and allow rapid diagnosis of resistant species. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  3. Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry improves appropriateness of antibiotic treatment of bacteremia.

    Directory of Open Access Journals (Sweden)

    Anne L M Vlek

    Full Text Available Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS allows the identification of microorganisms directly from positive blood culture broths. Use of the MALDI-TOF MS for rapid identification of microorganisms from blood culture broths can reduce the turnaround time to identification and may lead to earlier appropriate treatment of bacteremia. During February and April 2010, direct MALDI-TOF MS was routinely performed on all positive blood cultures. During December 2009 and March 2010 no direct MALDI-TOF MS was used. Information on antibiotic therapy was collected from the hospital and intensive care units' information systems from all positive blood cultures during the study period. In total, 253 episodes of bacteremia were included of which 89 during the intervention period and 164 during the control period. Direct performance of MALDI-TOF MS on positive blood culture broths reduced the time till species identification by 28.8-h and was associated with an 11.3% increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64.0% in the control period versus 75.3% in the intervention period (p0.01. Routine implementation of this technique increased the proportion of patients on adequate antimicrobial treatment within 24 hours.

  4. Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry improves appropriateness of antibiotic treatment of bacteremia.

    Science.gov (United States)

    Vlek, Anne L M; Bonten, Marc J M; Boel, C H Edwin

    2012-01-01

    Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification of microorganisms directly from positive blood culture broths. Use of the MALDI-TOF MS for rapid identification of microorganisms from blood culture broths can reduce the turnaround time to identification and may lead to earlier appropriate treatment of bacteremia. During February and April 2010, direct MALDI-TOF MS was routinely performed on all positive blood cultures. During December 2009 and March 2010 no direct MALDI-TOF MS was used. Information on antibiotic therapy was collected from the hospital and intensive care units' information systems from all positive blood cultures during the study period. In total, 253 episodes of bacteremia were included of which 89 during the intervention period and 164 during the control period. Direct performance of MALDI-TOF MS on positive blood culture broths reduced the time till species identification by 28.8-h and was associated with an 11.3% increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64.0% in the control period versus 75.3% in the intervention period (p0.01)). Routine implementation of this technique increased the proportion of patients on adequate antimicrobial treatment within 24 hours.

  5. Rapid identification of bacteria from bioMérieux BacT/ALERT blood culture bottles by MALDI-TOF MS.

    Science.gov (United States)

    Haigh, J D; Green, I M; Ball, D; Eydmann, M; Millar, M; Wilks, M

    2013-01-01

    Several studies have reported poor results when trying to identify microorganisms directly from the bioMérieux BacT/ALERT blood culture system using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry. The aim of this study is to evaluate two new methods, Sepsityper and an enrichment method for direct identification of microorganisms from this system. For both methods the samples were processed using the Bruker Microflex LT mass spectrometer (Biotyper) using the Microflex Control software to obtain spectra. The results from direct analysis were compared with those obtained by subculture and subsequent identification. A total of 350 positive blood cultures were processed simultaneously by the two methods. Fifty-three cultures were polymocrobial or failed to grow any organism on subculture, and these results were not included as there was either no subculture result, or for polymicrobial cultures it was known that the Biotyper would not be able to distinguish the constituent organisms correctly. Overall, the results showed that, contrary to previous reports, it is possible to identify bacteria directly from bioMérieux blood culture bottles, as 219/297 (74%) correct identifications were obtained using the Bruker Sepsityper method and 228/297 (77%) were obtained for the enrichment method when there is only one organism was present. Although the enrichment method was simpler, the reagent costs for the Sepsityper method were approximately pound 4.00 per sample compared to pound 0.50. An even simpler and cheaper method, which was less labour-intensive and did not require further reagents, was investigated. Seventy-seven specimens from positive signalled blood cultures were analysed by inoculating prewarmed blood agar plates and analysing any growth after 1-, 2- and 4-h periods of incubation at 37 degrees C, by either direct transfer or alcohol extraction. This method gave the highest number of correct identifications, 66/77 (86

  6. Detection and mapping of illicit drugs and their metabolites in fingermarks by MALDI MS and compatibility with forensic techniques

    Science.gov (United States)

    Groeneveld, G.; de Puit, M.; Bleay, S.; Bradshaw, R.; Francese, S.

    2015-06-01

    Despite the proven capabilities of Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) in laboratory settings, research is still needed to integrate this technique into current forensic fingerprinting practice. Optimised protocols enabling the compatible application of MALDI to developed fingermarks will allow additional intelligence to be gathered around a suspect’s lifestyle and activities prior to the deposition of their fingermarks while committing a crime. The detection and mapping of illicit drugs and metabolites in latent fingermarks would provide intelligence that is beneficial for both police investigations and court cases. This study investigated MALDI MS detection and mapping capabilities for a large range of drugs of abuse and their metabolites in fingermarks; the detection and mapping of a mixture of these drugs in marks, with and without prior development with cyanoacrylate fuming or Vacuum Metal Deposition, was also examined. Our findings indicate the versatility of MALDI technology and its ability to retrieve chemical intelligence either by detecting the compounds investigated or by using their ion signals to reconstruct 2D maps of fingermark ridge details.

  7. Proteomic analysis of phosphoproteins sensitive to a phosphatidylinositol 3-kinase inhibitor, ZSTK474, by using SELDI-TOF MS

    Directory of Open Access Journals (Sweden)

    Yamori Takao

    2009-03-01

    Full Text Available Abstract Background Phosphoproteins play important roles in a vast series of biological processes. Recent proteomic technologies offer the comprehensive analyses of phosphoproteins. Recently, we demonstrated that surface-enhanced laser desorption/ionization time of flight mass (SELDI-TOF MS would detect phosphoproteins quantitatively, which was a new application of SELDI-TOF MS. Results We combined immobilized metal affinity chromatography (IMAC with SELDI-TOF MS. After SELDI-TOF MS analysis of IMAC-enrichment phosphoproteins from A549 cancer cells, a series of protein peaks at 12.9, 12.8, 12.7 and 12.6 kDa was obtained in a mass spectrum. The peak intensities of these proteins decreased after a phosphatase treatment and, interestingly, they also decreased when the cells were pre-treated with a novel phosphatidylinositol 3-kinase (PI3K inhibitor, ZSTK474, suggesting that these proteins were ZSTK474-sensitive phosphoproteins. Identity of the phosphoproteins, which were predicted as the multi-phosphorylated forms of 4E-binding protein 1 (4E-BP1 with the aid of TagIdent algorithm, was confirmed by immunoprecipitation and subsequent SELDI-TOF MS analysis. 4E-BP1 is a downstream component of the PI3K/Akt/mTOR pathway and it regulates protein synthesis. We also investigated the effect of ZSTK474 on 4E-BP1 phosphorylation using phospho-specific antibodies. ZSTK474, which have little inhibitory activity for mTOR, inhibited phosphorylation of Ser65, Thr70 and Thr37/46 in 4E-BP1. In contrast, rapamycin, an inhibitor of mTOR, blocked phosphorylation only of Ser65 and Thr70. These results suggest that ZSTK474 and rapamycin inhibited the phosphorylation of 4E-BP1 in a different manner. Conclusion We identified a group of ZSTK474-sensitive phosphoproteins as the multi-phosphorylated form of 4E-BP1 by combining IMAC, SELDI-TOF MS and antibodies.

  8. Use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analyser in a diagnostic microbiology laboratory in a developing country

    Directory of Open Access Journals (Sweden)

    Atang Bulane

    2017-12-01

    Objective: We compared MALDI-TOF MS against two commercial systems, MicroScan Walkaway and VITEK 2 MS. Methods: Over a three-month period from July 2013 to September 2013, a total of 227 bacteria and yeasts were collected from an academic microbiology laboratory (N = 121; 87 Gramnegatives, seven Gram-positives, 27 yeasts and other laboratories (N = 106; 35 Gram-negatives, 34 Gram-positives, 37 yeasts. Sixty-five positive blood cultures were initially processed with Bruker Sepsityper kit for direct identification. Results: From the 65 blood culture bottles, four grew more than one bacterial pathogen and MALDI-TOF MS identified only one isolate. The blood cultures yielded 21 Gram-negatives, 43 Gram-positives and one Candida. There were 21 Escherirchia coli isolates which were reported by the MALDI-TOF MS as E. coli/Shigella. Of the total 292 isolates, discrepant results were found for one bacterial and three yeast isolates. Discrepant results were resolved by testing with the API system with MALDI-TOF MS showing 100% correlation. Conclusion: The MALDI-TOF MS proved to be very useful for rapid and reliable identification of bacteria and yeasts directly from blood cultures and after culture of other specimens. The difference in time to identification was significant for all isolates. However, for positive blood cultures with minimal sample preparation time there was a massive difference in turn-around time with great appreciation by clinicians.

  9. Toxicological screening of basic drugs in whole blood using UPLC-TOF-MS

    DEFF Research Database (Denmark)

    Dalsgaard, Petur Weihe; Rasmussen, Brian Schou; Müller, Irene Breum

    2012-01-01

    Ultra performance liquid chromatography (UPLC) coupled with time-of-flight (TOF) mass spectrometry (MS) was established for toxicological screening of basic drugs in whole blood and tested on authentic samples. Whole blood samples (0.2 ml) were extracted using a Gilson apparatus equipped with Bond...

  10. Optimization of Sample Preparation and Instrumental Parameters for the Rapid Analysis of Drugs of Abuse in Hair samples by MALDI-MS/MS Imaging

    Science.gov (United States)

    Flinders, Bryn; Beasley, Emma; Verlaan, Ricky M.; Cuypers, Eva; Francese, Simona; Bassindale, Tom; Clench, Malcolm R.; Heeren, Ron M. A.

    2017-08-01

    Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) has been employed to rapidly screen longitudinally sectioned drug user hair samples for cocaine and its metabolites using continuous raster imaging. Optimization of the spatial resolution and raster speed were performed on intact cocaine contaminated hair samples. The optimized settings (100 × 150 μm at 0.24 mm/s) were subsequently used to examine longitudinally sectioned drug user hair samples. The MALDI-MS/MS images showed the distribution of the most abundant cocaine product ion at m/z 182. Using the optimized settings, multiple hair samples obtained from two users were analyzed in approximately 3 h: six times faster than the standard spot-to-spot acquisition method. Quantitation was achieved using longitudinally sectioned control hair samples sprayed with a cocaine dilution series. A multiple reaction monitoring (MRM) experiment was also performed using the `dynamic pixel' imaging method to screen for cocaine and a range of its metabolites, in order to differentiate between contaminated hairs and drug users. Cocaine, benzoylecgonine, and cocaethylene were detectable, in agreement with analyses carried out using the standard LC-MS/MS method. [Figure not available: see fulltext.

  11. Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS

    Energy Technology Data Exchange (ETDEWEB)

    Senyuva, Hamide Z. [Ankara Test and Analysis Laboratory, Scientific and Technological Research Council of Turkey, Ankara 06330 (Turkey)], E-mail: hamide.senyuva@tubitak.gov.tr; Gilbert, John [Central Science Laboratory, Sand Hutton, York YO41 1LZ (United Kingdom); Oztuerkoglu, Sebnem [Ankara Test and Analysis Laboratory, Scientific and Technological Research Council of Turkey, Ankara 06330 (Turkey)

    2008-06-09

    A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.

  12. Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization-time of flight mass spectrometry and cydB real-time quantitative PCR.

    Science.gov (United States)

    Pizauro, Lucas J L; de Almeida, Camila C; Soltes, Glenn A; Slavic, Durda; Rossi-Junior, Oswaldo D; de Ávila, Fernando A; Zafalon, Luiz F; MacInnes, Janet I

    2017-05-01

    Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Comparison of HPLC/MS and MALDI-MS for characterizing triacylglycerols in insects: Specie-specific composition of lipids in fat bodies of bumblebee males

    Czech Academy of Sciences Publication Activity Database

    Kofroňová, Edita; Cvačka, Josef; Vrkoslav, Vladimír; Hanus, Robert; Jiroš, Pavel; Kindl, Jiří; Hovorka, Oldřich; Valterová, Irena

    2009-01-01

    Roč. 877, č. 30 (2009), s. 3878-3884 ISSN 1570-0232 R&D Projects: GA ČR GA203/09/0139; GA ČR GA203/09/1446; GA MŠk 2B06007 Institutional research plan: CEZ:AV0Z40550506 Keywords : Bumblebee * fat body * triacylglycerols * HPLC/APCI-MS * MALDI-MS Subject RIV: CC - Organic Chemistry Impact factor: 2.777, year: 2009

  14. Analysis of hazardous biological material by MALDI mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    KL Wahl; KH Jarman; NB Valentine; MT Kingsley; CE Petersen; ST Cebula; AJ Saenz

    2000-03-21

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) has become a valuable tool for analyzing microorganisms. The speed with which data can be obtained from MALDI-MS makes this a potentially important tool for biological health hazard monitoring and forensic applications. The excitement in the mass spectrometry community in this potential field of application is evident by the expanding list of research laboratories pursuing development of MALDI-MS for bacterial identification. Numerous research groups have demonstrated the ability to obtain unique MALDI-MS spectra from intact bacterial cells and bacterial cell extracts. The ability to differentiate strains of the same species has been investigated. Reproducibility of MALDI-MS spectra from bacterial species under carefully controlled experimental conditions has also been demonstrated. Wang et al. have reported on interlaboratory reproducibility of the MALDI-MS analysis of several bacterial species. However, there are still issues that need to be addressed, including the careful control of experimental parameters for reproducible spectra and selection of optimal experimental parameters such as solvent and matrix.

  15. Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

    OpenAIRE

    Barnini, S; Ghelardi, Emilia; Brucculeri, V; Morici, Paola; Lupetti, Antonella

    2015-01-01

    Background Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identif...

  16. A simple algorithm improves mass accuracy to 50-100 ppm for delayed extraction linear MALDI-TOF mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hack, Christopher A.; Benner, W. Henry

    2001-10-31

    A simple mathematical technique for improving mass calibration accuracy of linear delayed extraction matrix assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF MS) spectra is presented. The method involves fitting a parabola to a plot of Dm vs. mass data where Dm is the difference between the theoretical mass of calibrants and the mass obtained from a linear relationship between the square root of m/z and ion time of flight. The quadratic equation that describes the parabola is then used to correct the mass of unknowns by subtracting the deviation predicted by the quadratic equation from measured data. By subtracting the value of the parabola at each mass from the calibrated data, the accuracy of mass data points can be improved by factors of 10 or more. This method produces highly similar results whether or not initial ion velocity is accounted for in the calibration equation; consequently, there is no need to depend on that uncertain parameter when using the quadratic correction. This method can be used to correct the internally calibrated masses of protein digest peaks. The effect of nitrocellulose as a matrix additive is also briefly discussed, and it is shown that using nitrocellulose as an additive to a CHCA matrix does not significantly change initial ion velocity but does change the average position of ions relative to the sample electrode at the instant the extraction voltage is applied.

  17. Profiling LC-DAD-ESI-TOF MS method for the determination of phenolic metabolites from avocado (Persea americana).

    Science.gov (United States)

    Hurtado-Fernández, Elena; Carrasco-Pancorbo, Alegría; Fernández-Gutiérrez, Alberto

    2011-03-23

    A powerful HPLC-DAD-ESI-TOF MS method was established for the efficient identification of the chemical constituents in the methanolic extracts of avocado (Persea americana). Separation and detection conditions were optimized by using a standard mix containing 39 compounds belonging to phenolic acids and different categories of flavonoids, analytes that could be potentially present in the avocado extracts. Optimum LC separation was achieved on a Zorbax Eclipse Plus C18 analytical column (4.6×150 mm, 1.8 μm particle size) by gradient elution with water+acetic acid (0.5%) and acetonitrile as mobile phases, at a flow rate of 1.6 mL/min. The detection was carried out by ultraviolet-visible absorption and ESI-TOF MS. The developed method was applied to the study of 3 different varieties of avocado, and 17 compounds were unequivocally identified with standards. Moreover, around 25 analytes were tentatively identified by taking into account the accuracy and isotopic information provided by TOF MS.

  18. Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

    Science.gov (United States)

    Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter

    1997-12-01

    In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.

  19. Capillary electrophoresis-MALDI interface based on inkjet technology

    Science.gov (United States)

    Vannatta, Michael W.; Whitmore, Colin D.; Dovichi, Norman J.

    2010-01-01

    An ink jet printer valve and nozzle were used to deliver matrix and sample from an electrophoresis capillary onto a MALDI plate. The system was evaluated by separation of a set of standard peptides. That separation generated up to 40,000 theoretical plates in less than three minutes. Detection limits were 500 amol using an ABI TOF-TOF instrument and 2 fmol for an ABI Q-TOF instrument. Over 70% coverage was obtained for the tryptic digest of α-lactalbumin in less than 2.5 minutes. PMID:19960472

  20. Blood group genotyping: the power and limitations of the Hemo ID Panel and MassARRAY platform.

    Science.gov (United States)

    McBean, Rhiannon S; Hyland, Catherine A; Flower, Robert L

    2015-01-01

    Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS), is a sensitive analytical method capable of resolving DNA fragments varying in mass by a single nucleotide. MALDI-TOF MS is applicable to blood group genotyping, as the majority of blood group antigens are encoded by single nucleotide polymorphisms. Blood group genotyping by MALDI-TOF MS can be performed using a panel (Hemo ID Blood Group Genotyping Panel, Agena Bioscience Inc., San Diego, CA) that is a set of genotyping assays that predict the phenotype for 101 antigens from 16 blood group systems. These assays involve three fundamental stages: multiplex target-specific polymerase chain reaction amplification, allele-specific single base primer extension, and MALDI-TOFMS analysis using the MassARRAY system. MALDI-TOF MS-based genotyping has many advantages over alternative methods including high throughput, high multiplex capability, flexibility and adaptability, and the high level of accuracy based on the direct detection method. Currently available platforms for MALDI-TOF MS-based genotyping are not without limitations, including high upfront instrumentation costs and the number of non-automated steps. The Hemo ID Blood Group Genotyping Panel, developed and optimized in a collaboration between the vendor and the Blood Transfusion Service of the Swiss Red Cross in Zurich, Switzerland, is not yet widely utilized, although several laboratories are currently evaluating the MassARRAY system for blood group genotyping. Based on the accuracy and other advantages offered by MALDITOF MS analysis, in the future, this method is likely to become widely adopted for blood group genotyping, in particular, for population screening.

  1. Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Aspergillus Species Directly from Growth on Solid Agar Media

    Directory of Open Access Journals (Sweden)

    Ying Li

    2017-06-01

    Full Text Available We evaluated the accuracy of the Bruker Biotyper matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS system at identifying clinical isolates of Aspergillus species that were grown on agar media. A total of 381 non-duplicate Aspergillus isolates representing 21 different Aspergillus species identified by molecular analysis were included in this study. The Bruker Biotyper MALDI-TOF MS system was able to identify 30.2% (115/381 of the isolates to the species level (score values of ≥2.000 and 49.3% to the genus level (score values of 1.700–1.999. When the identification cutoff value was lowered from ≥2.000 to ≥1.700, the species-level identification rate increased to 79.5% with a slight rise of false identification from 2.6 to 5.0%. From another aspect, a correct species-level identification rate of 89% could be reached by the Bruker Biotyper MALDI-TOF MS system regardless of the score values obtained. The Bruker Biotyper MALDI-TOF MS system had a moderate performance in identification of Aspergillus directly inoculated on solid agar media. Continued expansion of the Bruker Biotyper MALDI-TOF MS database and adoption of alternative cutoff values for interpretation are required to improve the performance of the system for identifying highly diverse species of clinically encountered Aspergillus isolates.

  2. Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Aspergillus Species Directly from Growth on Solid Agar Media.

    Science.gov (United States)

    Li, Ying; Wang, He; Zhao, Yu-Pei; Xu, Ying-Chun; Hsueh, Po-Ren

    2017-01-01

    We evaluated the accuracy of the Bruker Biotyper matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) system at identifying clinical isolates of Aspergillus species that were grown on agar media. A total of 381 non-duplicate Aspergillus isolates representing 21 different Aspergillus species identified by molecular analysis were included in this study. The Bruker Biotyper MALDI-TOF MS system was able to identify 30.2% (115/381) of the isolates to the species level (score values of ≥2.000) and 49.3% to the genus level (score values of 1.700-1.999). When the identification cutoff value was lowered from ≥2.000 to ≥1.700, the species-level identification rate increased to 79.5% with a slight rise of false identification from 2.6 to 5.0%. From another aspect, a correct species-level identification rate of 89% could be reached by the Bruker Biotyper MALDI-TOF MS system regardless of the score values obtained. The Bruker Biotyper MALDI-TOF MS system had a moderate performance in identification of Aspergillus directly inoculated on solid agar media. Continued expansion of the Bruker Biotyper MALDI-TOF MS database and adoption of alternative cutoff values for interpretation are required to improve the performance of the system for identifying highly diverse species of clinically encountered Aspergillus isolates.

  3. MALDI TOF imaging mass spectrometry in clinical pathology: a valuable tool for cancer diagnostics (review).

    Science.gov (United States)

    Kriegsmann, Jörg; Kriegsmann, Mark; Casadonte, Rita

    2015-03-01

    Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) is an evolving technique in cancer diagnostics and combines the advantages of mass spectrometry (proteomics), detection of numerous molecules, and spatial resolution in histological tissue sections and cytological preparations. This method allows the detection of proteins, peptides, lipids, carbohydrates or glycoconjugates and small molecules.Formalin-fixed paraffin-embedded tissue can also be investigated by IMS, thus, this method seems to be an ideal tool for cancer diagnostics and biomarker discovery. It may add information to the identification of tumor margins and tumor heterogeneity. The technique allows tumor typing, especially identification of the tumor of origin in metastatic tissue, as well as grading and may provide prognostic information. IMS is a valuable method for the identification of biomarkers and can complement histology, immunohistology and molecular pathology in various fields of histopathological diagnostics, especially with regard to identification and grading of tumors.

  4. Proteomics approaches for identification of tumor relevant protein targets in pulmonary squamous cell carcinoma by 2D-DIGE-MS.

    Directory of Open Access Journals (Sweden)

    Hao Lihong

    Full Text Available Potential markers for progression of pulmonary squamous cell carcinoma (SCC were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS, and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF. The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P ≤ 0.05 in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.

  5. Cost Savings Realized by Implementation of Routine Microbiological Identification by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

    Science.gov (United States)

    Tran, Anthony; Alby, Kevin; Kerr, Alan; Jones, Melissa; Gilligan, Peter H

    2015-08-01

    Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is an emerging technology for rapid identification of bacterial and fungal isolates. In comparison to conventional methods, this technology is much less labor intensive and can provide accurate and reliable results in minutes from a single isolated colony. We compared the cost of performing the bioMérieux Vitek MALDI-TOF MS with conventional microbiological methods to determine the amount saved by the laboratory by converting to the new technology. Identification costs for 21,930 isolates collected between April 1, 2013, and March 31, 2014, were directly compared for MALDI-TOF MS and conventional methodologies. These isolates were composed of commonly isolated organisms, including commonly encountered aerobic and facultative bacteria and yeast but excluding anaerobes and filamentous fungi. Mycobacterium tuberculosis complex and rapidly growing mycobacteria were also evaluated for a 5-month period during the study. Reagent costs and a total cost analysis that included technologist time in addition to reagent expenses and maintenance service agreement costs were analyzed as part of this study. The use of MALDI-TOF MS equated to a net savings of $69,108.61, or 87.8%, in reagent costs annually compared to traditional methods. When total costs are calculated to include technologist time and maintenance costs, traditional identification would have cost $142,532.69, versus $68,886.51 with the MALDI-TOF MS method, resulting in a laboratory savings of $73,646.18, or 51.7%, annually by adopting the new technology. The initial cost of the instrument at our usage level would be offset in about 3 years. MALDI-TOF MS not only represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates, it also provides a significant cost savings for the laboratory. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. The importance of matrix-assisted laser desorption ionization–time of flight mass spectrometry for correct identification of Clostridium difficile isolated from chromID C. difficile chromogenic agar

    Directory of Open Access Journals (Sweden)

    Jonathan H.K. Chen

    2017-10-01

    Full Text Available The clinical workflow of using chromogenic agar and matrix-assisted laser desorption ionization time-of-fight mass spectrometry (MALDI-TOF MS for Clostridium difficile identification was evaluated. The addition of MALDI-TOF MS identification after the chromID C. difficile chromogenic agar culture could significantly improve the diagnostic accuracy of C. difficile.

  7. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?

    Science.gov (United States)

    van Belkum, Alex; Welker, Martin; Pincus, David; Charrier, Jean Philippe; Girard, Victoria

    2017-11-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microbial species in clinical microbiology laboratories. MALDI-TOF-MS has swiftly become the new gold-standard method owing to its key advantages of simplicity and robustness. However, as with all new methods, adoption of the MALDI-TOF MS approach is still not widespread. Optimal sample preparation has not yet been achieved for several applications, and there are continuing discussions on the need for improved database quality and the inclusion of additional microbial species. New applications such as in the field of antimicrobial susceptibility testing have been proposed but not yet translated to the level of ease and reproducibility that one should expect in routine diagnostic systems. Finally, during routine identification testing, unexpected results are regularly obtained, and the best methods for transmitting these results into clinical care are still evolving. We here discuss the success of MALDI-TOF MS in clinical microbiology and highlight fields of application that are still amenable to improvement. © The Korean Society for Laboratory Medicine.

  8. Rapid analysis of the main components of the total glycosides of Ranunculus japonicus by UPLC/Q-TOF-MS.

    Science.gov (United States)

    Rui, Wen; Chen, Hongyuan; Tan, Yuzhi; Zhong, Yanmei; Feng, Yifan

    2010-05-01

    A rapid method for the analysis of the main components of the total glycosides of Ranunculus japonicus (TGOR) was developed using ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The separation analysis was performed on a Waters Acquity UPLC system and the accurate mass of molecules and their fragment ions were determined by Q-TOF MS. Twenty compounds, including lactone glycosides, flavonoid glycosides and flavonoid aglycones, were identified and tentatively deduced on the basis of their elemental compositions, MS/MS data and relevant literature. The results demonstrated that lactone glycosides and flavonoids were the main constituents of TGOR. Furthermore, an effective and rapid pattern was established allowing for the comprehensive and systematic characterization of the complex samples.

  9. [Application of mass spectrometry in mycology].

    Science.gov (United States)

    Quiles Melero, Inmaculada; Peláez, Teresa; Rezusta López, Antonio; Garcia-Rodríguez, Julio

    2016-06-01

    MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) is becoming an essential tool in most microbiology laboratories. At present, by using a characteristic fungal profile obtained from whole cells or through simple extraction protocols, MALDI-TOF MS allows the identification of pathogenic fungi with a high performance potential. This methodology decreases the laboratory turnaround time, optimizing the detection of mycoses. This article describes the state-of-the-art of the use of MALDI-TOF MS for the detection of human clinical fungal pathogens in the laboratory and discusses the future applications of this technology, which will further improve routine mycological diagnosis. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

  10. Direct detection of the plant pathogens Burkholderia glumae, Burkholderia gladioli pv. gladioli, and Erwinia chrysanthemi pv. zeae in infected rice seedlings using matrix assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Kajiwara, Hideyuki

    2016-01-01

    The plant pathogens Burkholderia glumae, Burkholderia gladioli pv. gladioli, and Erwinia chrysanthemi pv. zeae were directly detected in extracts from infected rice seedlings by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method did not require culturing of the pathogens on artificial medium. In the MALDI-TOF MS analysis, peaks originating from bacteria were found in extracts from infected rice seedlings. The spectral peaks showed significantly high scores, in spite of minor differences in spectra. The spectral peaks originating from host plant tissues did not affect this direct MALDI-TOF MS analysis for the rapid identification of plant pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. MALDI-TOF mass spectrometry imaging reveals molecular level changes in ultrahigh molecular weight polyethylene joint implants in correlation with lipid adsorption.

    Science.gov (United States)

    Fröhlich, Sophie M; Archodoulaki, Vasiliki-Maria; Allmaier, Günter; Marchetti-Deschmann, Martina

    2014-10-07

    Ultrahigh molecular weight polyethylene (PE-UHMW), a material with high biocompatibility and excellent mechanical properties, is among the most commonly used materials for acetabular cup replacement in artificial joint systems. It is assumed that the interaction with synovial fluid in the biocompartment leads to significant changes relevant to material failure. In addition to hyaluronic acid, lipids are particularly relevant for lubrication in an articulating process. This study investigates synovial lipid adsorption on two different PE-UHMW materials (GUR-1050 and vitamin E-doped) in an in vitro model system by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry imaging (MSI). Lipids were identified by high performance thin layer chromatography (HP-TLC) and tandem mass spectrometry (MS/MS) analysis, with an analytical focus on phospholipids and cholesterol, both being species of high importance for lubrication. Scanning electron microscopy (SEM) analysis was applied in the study to correlate molecular information with PE-UHMW material qualities. It is demonstrated that lipid adsorption preferentially occurs in rough or oxidized polymer regions. Polymer modifications were colocalized with adsorbed lipids and found with high density in regions identified by SEM. Explanted, the in vivo polymer material showed comparable and even more obvious polymer damage and lipid adsorption when compared with the static in vitro model. A three-dimensional reconstruction of MSI data from consecutive PE-UHMW slices reveals detailed information about the diffusion process of lipids in the acetabular cup and provides, for the first time, a promising starting point for future studies correlating molecular information with commonly used techniques for material analysis (e.g., Fourier-transform infrared spectroscopy, nanoindentation).

  12. Species identification of Streptococcus bovis group isolates causing bacteremia

    DEFF Research Database (Denmark)

    Agergaard, Charlotte N; Knudsen, Elisa; Dargis, Rimtas

    2017-01-01

    This study compared two MALDI-TOF MS systems (Biotyper and VITEK MS) on clinical Streptococcus bovis group isolates (n=66). The VITEK MS gave fewer misidentifications and a higher rate of correct identifications than the Biotyper. Only the identification of S. lutetiensis by the VITEK MS was reli......This study compared two MALDI-TOF MS systems (Biotyper and VITEK MS) on clinical Streptococcus bovis group isolates (n=66). The VITEK MS gave fewer misidentifications and a higher rate of correct identifications than the Biotyper. Only the identification of S. lutetiensis by the VITEK MS...

  13. The Exploration of Peptide Biomarkers in Malignant Pleural Effusion of Lung Cancer Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Jing Xu

    2017-01-01

    Full Text Available Background. Diagnoses of malignant pleural effusion (MPE are a crucial problem in clinics. In our study, we compared the peptide profiles of MPE and tuberculosis pleural effusion (TPE to investigate the value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS in diagnosis of MPE. Material and Methods. The 46 MPE and 32 TPE were randomly assigned to training set and validation set. Peptides were isolated by weak cation exchange magnetic beads and peaks in the m/z range of 800–10000 Da were analyzed. Comparing the peptide profile between 30 MPE and 22 TPE samples in training set by ClinProTools software, we screened the specific biomarkers and established a MALDI-TOF-MS classification of MPE. Finally, the other 16 MPE and 10 TPE were included to verify the model. We additionally determined carcinoembryonic antigen (CEA in MPE and TPE samples using electrochemiluminescent immunoassay method. Results. Five peptide peaks (917.37 Da, 4469.39 Da, 1466.5 Da, 4585.21 Da, and 3216.87 Da were selected to separate MPE and TPE by MALDI-TOF-MS. The sensitivity, specificity, and accuracy of the classification were 93.75%, 100%, and 96.15%, respectively, after blinded test. The sensitivity of CEA was significantly lower than MALDI-TOF-MS classification (P=0.035. Conclusions. The results indicate MALDI-TOF-MS is a potential method for diagnosing MPE.

  14. The use of Gram stain and matrix-assisted laser desorption ionization time-of-flight mass spectrometry on positive blood culture: synergy between new and old technology.

    Science.gov (United States)

    Fuglsang-Damgaard, David; Nielsen, Camilla Houlberg; Mandrup, Elisabeth; Fuursted, Kurt

    2011-10-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is promising as an alternative to more costly and cumbersome methods for direct identifications in blood cultures. We wanted to evaluate a simplified pre-treatment method for using MALDI-TOF-MS directly on positive blood cultures using BacT/Alert blood culture system, and to test an algorithm combining the result of the initial microscopy with the result suggested by MALDI-TOF-MS. Using the recommended cut-off score of 1.7 the best results were obtained among Gram-negative rods with correct identifications in 91% of Enterobacteriaceae, 83% in aerobic/non-fermentative Gram-negative rods, whereas results were more modest among Gram-positive cocci with correct identifications in 52% of Staphylococci, 54% in Enterococci and only 20% in Streptococci. Combining the results of Gram stain with the top reports by MALDI-TOF-MS, increased the sensitivity from 91% to 93% in the score range from 1.5 to 1.7 and from 48% to 85% in the score range from 1.3 to 1.5. Thus, using this strategy and accepting a cut-off at 1.3 instead of the suggested 1.7, overall sensitivity could be increased from 88.1% to 96.3%. MALDI-TOF-MS is an efficient method for direct routine identification of bacterial isolates in blood culture, especially when combined with the result of the Gram stain. © 2011 The Authors. APMIS © 2011 APMIS.

  15. Integrated quantitative and qualitative workflow for in vivo bioanalytical support in drug discovery using hybrid Q-TOF-MS.

    Science.gov (United States)

    Ranasinghe, Asoka; Ramanathan, Ragu; Jemal, Mohammed; D'Arienzo, Celia J; Humphreys, W Griffith; Olah, Timothy V

    2012-03-01

    UHPLC coupled with orthogonal acceleration hybrid quadrupole-TOF (Q-TOF)-MS is an emerging technique offering new strategies for the efficient screening of new chemical entities and related molecules at the early discovery stage within the pharmaceutical industry. In the first part of this article, we examine the main instrumental parameters that are critical for the integration of UHPLC-Q-TOF technology to existing bioanalytical workflows, in order to provide simultaneous quantitative and qualitative bioanalysis of samples generated following in vivo studies. Three modern Q-TOF mass spectrometers, including Bruker maXis™, Agilent 6540 and Sciex TripleTOF™ 5600, all interfaced with UHPLC systems, are evaluated in the second part of the article. The scope of this work is to demonstrate the potential of Q-TOF for the analysis of typical small molecules, therapeutic peptides (molecular weight <6000 Da), and enzymatically (i.e., trypsin, chymotrypsin and pepsin) cleaved peptides from larger proteins. This work focuses mainly on full-scan TOF data obtained under ESI conditions, the major mode of TOF operation in discovery bioanalytical research, where the compounds are selected based on their pharmacokinetic/pharmacodynamic behaviors using animal models prior to selecting a few desirable candidates for further development. Finally, important emerging TOF technologies that could potentially benefit bioanalytical research in the semi-quantification of metabolites without synthesized standards are discussed. Particularly, the utility of captive spray ionization coupled with TripleTOF 5600 was evaluated for improving sensitivity and providing normalized MS response for drugs and their metabolites. The workflow proposed compromises neither the efficiency, nor the quality of pharmacokinetic data in support of early drug discovery programs.

  16. Validation of LC–TOF-MS Screening for Drugs, Metabolites, and Collateral Compounds in Forensic Toxicology Specimens

    Science.gov (United States)

    Guale, Fessessework; Shahreza, Shahriar; Walterscheid, Jeffrey P.; Chen, Hsin-Hung; Arndt, Crystal; Kelly, Anna T.; Mozayani, Ashraf

    2013-01-01

    Liquid chromatography time-of-flight mass spectrometry (LC–TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic “Spice/K2” cannabinoids and cathinone “bath salt” designer drugs. The extract was applied to LC–TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC–TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework. PMID:23118149

  17. Validation of LC-TOF-MS screening for drugs, metabolites, and collateral compounds in forensic toxicology specimens.

    Science.gov (United States)

    Guale, Fessessework; Shahreza, Shahriar; Walterscheid, Jeffrey P; Chen, Hsin-Hung; Arndt, Crystal; Kelly, Anna T; Mozayani, Ashraf

    2013-01-01

    Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic "Spice/K2" cannabinoids and cathinone "bath salt" designer drugs. The extract was applied to LC-TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC-TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework.

  18. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    Science.gov (United States)

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  19. UPLC-Q-TOF/MS based metabolomic profiling of serum and urine of hyperlipidemic rats induced by high fat diet

    Directory of Open Access Journals (Sweden)

    Qiong Wu

    2014-12-01

    Full Text Available Hyperlipidemia is considered to be a high lipid level in blood, can induce metabolic disorders and dysfunctions of the body, and results in some severe complications. Therefore, hunting for some metabolite markers and clarifying the metabolic pathways in vivo will be an important strategy in the treatment and prevention of hyperlipidemia. In this study, a rat model of hyperlipidemia was constructed according to histopathological data and biochemical parameters, and the metabolites of serum and urine were analyzed by UPLC-Q-TOF/MS. Combining pattern recognition and statistical analysis, 19 candidate biomarkers were screened and identified. These changed metabolites indicated that during the development and progression of hyperlipidemia, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism were mainly disturbed, which are reported to be closely related to diabetes, cardiovascular diseases, etc. This study demonstrated that a UPLC-Q-TOF/MS based metabolomic approach is useful to profile the alternation of endogenous metabolites of hyperlipidemia. Keywords: UPLC-Q-TOF/MS, Hyperlipidemia, Metabolomic, Pattern recognition

  20. Revealing the glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1, serotype Ogawa with the BSA protein carrier using LC-ESI-QqTOF-MS/MS

    Science.gov (United States)

    Jahouh, Farid; Saksena, Rina; Kováč, Pavol; Banoub, Joseph

    2012-01-01

    In this manuscript, we present the determination of glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein. PMID:22791257

  1. Exploring the "intensity fading" phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

    DEFF Research Database (Denmark)

    Yanes, Oscar; Aviles, Francesc X; Roepstorff, Peter

    2007-01-01

    the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding...... molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs...

  2. Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.

    Science.gov (United States)

    Chen, Jonathan H K; Ho, Pak-Leung; Kwan, Grace S W; She, Kevin K K; Siu, Gilman K H; Cheng, Vincent C C; Yuen, Kwok-Yung; Yam, Wing-Cheong

    2013-06-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and fungi was recently introduced in microbiology laboratories. This technology could greatly improve the clinical management of patients and guidance for chemotherapy. In this study, we used a commercial MALDI Sepsityper extraction method to evaluate the performance of two commercial MALDI-TOF MS systems, the Vitek MS IVD (bioMérieux) and the Microflex LT Biotyper (Bruker Daltonics) for direct bacterial identification in positive blood cultures. In 181 monomicrobial cultures, both systems generated genus to species level identifications for >90% of the specimens (Biotyper, 177/181 [97.8%]; Vitek MS IVD, 167/181 [92.3%]). Overall, the Biotyper system generated significantly more accurate identifications than the Vitek MS IVD system (P = 0.016; 177 versus 167 out of 181 specimens). The Biotyper system identified the minority species among polymicrobial blood cultures. We also compared the performance of an in-house extraction method with that of the Sepsityper on both MALDI-TOF MS systems. The in-house method generated more correct identifications at the genus level than the Sepsityper (96.7% versus 93.5%) on the Biotyper system, whereas the two methods exhibited the same performance level (88.0% versus 88.0%) on the Vitek MS IVD system. Our study confirmed the practical advantages of MALDI-TOF MS, and our in-house extraction method reduced the reagent cost to $1 per specimen, with a shorter turnaround time of 3 h, which is highly cost-effective for a diagnostic microbiology service.

  3. Identification of Candida species isolated from vulvovaginitis using matrix assisted laser desorption ionization-time of flight mass spectrometry.

    Science.gov (United States)

    Alizadeh, Majid; Kolecka, Anna; Boekhout, Teun; Zarrinfar, Hossein; Ghanbari Nahzag, Mohamad A; Badiee, Parisa; Rezaei-Matehkolaei, Ali; Fata, Abdolmajid; Dolatabadi, Somayeh; Najafzadeh, Mohammad J

    2017-12-01

    Vulvovaginal candidiasis (VVC) is a common problem in women. The purpose of this study was to identify Candida isolates by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) from women with vulvovaginitis that were referred to Ghaem Hospital, Mashhad, Iran. This study was conducted on 65 clinical samples isolated from women that were referred to Ghaem Hospital. All specimens were identified using phenotyping techniques, such as microscopy and culture on Sabouraud dextrose agar and corn meal agar. In addition, all isolates were processed for MALDI-TOF MS identification. Out of the 65 analyzed isolates, 61 (94%) samples were recognized by MALDI-TOF MS. However, the remaining four isolates (6%) had no reliable identification. According to the results, C. albicans (58.5%) was the most frequently isolated species, followed by C. tropicalis (16.9%), C. glabrata (7.7%), C. parapsilosis (7.7%), and guilliermondii (3.1%). As the findings indicated, MALDI TOF MS was successful in the identification of clinical Candida species. C. albicans was identified as the most common Candida species isolated from the women with VVC. Moreover, C. tropicalis was the most common species among the non- albicans Candida species.

  4. Identifying modifications in RNA by MALDI mass spectrometry

    DEFF Research Database (Denmark)

    Douthwaite, Stephen; Kirpekar, Finn

    2007-01-01

    as RNA modifications added in cell-free in vitro systems. MALDI-MS is particularly useful in cases in which other techniques such as those involving primer extension or chromatographic analyses are not practicable. To date, MALDI-MS has been used to localize rRNA modifications that are involved......Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass...... spectra produced by MALDI are relatively straightforward to interpret, because they are dominated by singly charged ions, making it possible to analyze complex mixtures of RNA oligonucleotides ranging from trinucleotides up to 20-mers. Analysis of modifications within much longer RNAs, such as ribosomal...

  5. Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS.

    Science.gov (United States)

    Zhou, Hui; Warren, Peter G; Froehlich, John W; Lee, Richard S

    2014-07-01

    Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.

  6. A Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (LC-Q-TOF MS) Study for Analyzing 35 Corticosteroid Compounds: Elucidation of MS/MS Fragmentation Pathways

    International Nuclear Information System (INIS)

    Noh, Eunyoung; Yoon, Chang-Yong; Lee, Ji Hyun; Baek, Sun-Young; Do, Jung-Ah; Lee, Jung-min; Oh, Han Bin

    2016-01-01

    Corticosteroids have been often found to be added to a dietary supplement for the purpose of illegally improving the effect of their products. Thus, it is imperative to develop or improve a method that enables one to rapidly and reliably analyze corticosteroids in health or dietary supplements, for the safety management purpose. In the present study, results from liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) experiments for the selected 35 corticosteroid compounds are presented, which can be useful for the qualitative screening of corticosteroids in health or dietary supplements. Specifically, retention times, accurate mass data of the protonated steroids, m/z values of major fragment ions are given for the 35 corticosteroids. Further, fragmentation pathways for the selected steroids are also suggested. Based on the suggested fragmentation pathways, it was shown that an unknown steroid compound can be readily identified using the knowledge of a group of unique and specific common skeletal fragments. The high selectivity and sensitivity of the LC-Q-TOF-MS/MS results combined with the knowledge of the fragmentation pathways can offer a new opportunity for rapid and accurate screening of corticosteroids, thus preventing health-related incidents involving adulterated products and clamping down on illegally circulated health products.

  7. A Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (LC-Q-TOF MS) Study for Analyzing 35 Corticosteroid Compounds: Elucidation of MS/MS Fragmentation Pathways

    Energy Technology Data Exchange (ETDEWEB)

    Noh, Eunyoung; Yoon, Chang-Yong; Lee, Ji Hyun; Baek, Sun-Young; Do, Jung-Ah [Ministry of Food and Drug Safety, Cheongju (Korea, Republic of); Lee, Jung-min; Oh, Han Bin [Sogang University, Seoul (Korea, Republic of)

    2016-07-15

    Corticosteroids have been often found to be added to a dietary supplement for the purpose of illegally improving the effect of their products. Thus, it is imperative to develop or improve a method that enables one to rapidly and reliably analyze corticosteroids in health or dietary supplements, for the safety management purpose. In the present study, results from liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) experiments for the selected 35 corticosteroid compounds are presented, which can be useful for the qualitative screening of corticosteroids in health or dietary supplements. Specifically, retention times, accurate mass data of the protonated steroids, m/z values of major fragment ions are given for the 35 corticosteroids. Further, fragmentation pathways for the selected steroids are also suggested. Based on the suggested fragmentation pathways, it was shown that an unknown steroid compound can be readily identified using the knowledge of a group of unique and specific common skeletal fragments. The high selectivity and sensitivity of the LC-Q-TOF-MS/MS results combined with the knowledge of the fragmentation pathways can offer a new opportunity for rapid and accurate screening of corticosteroids, thus preventing health-related incidents involving adulterated products and clamping down on illegally circulated health products.

  8. Functional proteomics with new mass spectrometric and bioinformatics tools

    International Nuclear Information System (INIS)

    Kesners, P.W.A.

    2001-01-01

    A comprehensive range of mass spectrometric tools is required to investigate todays life science applications and a strong focus is on addressing the needs of functional proteomics. Application examples are given showing the streamlined process of protein identification from low femtomole amounts of digests. Sample preparation is achieved with a convertible robot for automated 2D gel picking, and MALDI target dispensing. MALDI-TOF or ESI-MS subsequent to enzymatic digestion. A choice of mass spectrometers including Q-q-TOF with multipass capability, MALDI-MS/MS with unsegmented PSD, Ion Trap and FT-MS are discussed for their respective strengths and applications. Bioinformatics software that allows both database work and novel peptide mass spectra interpretation is reviewed. The automated database searching uses either entire digest LC-MS n ESI Ion Trap data or MALDI MS and MS/MS spectra. It is shown how post translational modifications are interactively uncovered and de-novo sequencing of peptides is facilitated

  9. Development of a Direct Headspace Collection Method from Arabidopsis Seedlings Using HS-SPME-GC-TOF-MS Analysis

    Directory of Open Access Journals (Sweden)

    Kazuki Saito

    2013-04-01

    Full Text Available Plants produce various volatile organic compounds (VOCs, which are thought to be a crucial factor in their interactions with harmful insects, plants and animals. Composition of VOCs may differ when plants are grown under different nutrient conditions, i.e., macronutrient-deficient conditions. However, in plants, relationships between macronutrient assimilation and VOC composition remain unclear. In order to identify the kinds of VOCs that can be emitted when plants are grown under various environmental conditions, we established a conventional method for VOC profiling in Arabidopsis thaliana (Arabidopsis involving headspace-solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (HS-SPME-GC-TOF-MS. We grew Arabidopsis seedlings in an HS vial to directly perform HS analysis. To maximize the analytical performance of VOCs, we optimized the extraction method and the analytical conditions of HP-SPME-GC-TOF-MS. Using the optimized method, we conducted VOC profiling of Arabidopsis seedlings, which were grown under two different nutrition conditions, nutrition-rich and nutrition-deficient conditions. The VOC profiles clearly showed a distinct pattern with respect to each condition. This study suggests that HS-SPME-GC-TOF-MS analysis has immense potential to detect changes in the levels of VOCs in not only Arabidopsis, but other plants grown under various environmental conditions.

  10. Structural study of synthetic polymers by MALDI-TOFMS; MALDI-TOFMS ni yoru gosei kobunshi no kozo kaiseki

    Energy Technology Data Exchange (ETDEWEB)

    Noguchi, K.; Hirayama, K. [Ajinomoto Co. Inc., Tokyo (Japan)

    1998-08-01

    As observation results on the time-dependent change in the ring-opening reaction of novolac epoxy resin with acetic acid by MALDI-TOFMS, the epoxy ring was opened with reaction time, the hydroxy group formed by the ring-opening reaction was acetylated, those components were measured. In the case of the FABMS/MS observation of materials and the reaction products, the estimation structure could be confirmed from the measured results of MALDI-TOFMS. In the polymerization of bisphenol A epoxy resin with N, N`-dimethylethylenediamine, it was observed by MALDI-TOFMS that many kinds of polymers with high molecular weight were formed with an increase of reaction time. In this case, the LSIMS/MS observation of materials and the reaction products was carried out, the estimation structure could be confirmed from the measured results of MALDI-TOFMS. 19 refs., 8 figs.

  11. Prediction of fetal lung maturity using the lecithin/sphingomyelin (L/S) ratio analysis with a simplified sample preparation, using a commercial microtip-column combined with mass spectrometric analysis.

    Science.gov (United States)

    Kwak, Ho-Seok; Chung, Hee-Jung; Choi, Young Sik; Min, Won-Ki; Jung, So Young

    2015-07-01

    Fetal lung maturity is estimated using the lecithin/sphingomyelin ratio (L/S ratio) in amniotic fluid and it is commonly measured with thin-layer chromatography (TLC). The TLC method is time consuming and technically difficult; however, it is widely used because there is no alternative. We evaluated a novel method for measuring the L/S ratio, which involves a tip-column with a cation-exchange resin and mass spectrometry. Phospholipids in the amniotic fluid were extracted using methanol and chloroform. Choline-containing phospholipids such as lecithin and sphingomyelin were purified by passing them through the tip-column. LC-MS/MS and MALDI-TOF were used to directly analyze the purified samples. The L/S ratio by mass spectrometry was calculated from the sum peak intensity of the six lecithin, and that of sphingomyelin 34:1. In 20 samples, the L/S ratio determined with TLC was significantly correlated with that obtained by LC-MS/MS and MALDI-TOF. There was a 100% concordance between the L/S ratio by TLC and that by LC-MS/MS (kappa value=1.0). The concordance between the L/S ratio by TLC and that by MALDI-TOF was also 100% (kappa value=1.0). Our method provides a faster, simpler, and more reliable assessment of fetal lung maturity. The L/S ratio measured by LC-MS/MS and MALDI-TOF offers a compelling alternative method to traditional TLC. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    De Carolis, E; Posteraro, B; Lass-Flörl, C; Vella, A; Florio, A R; Torelli, R; Girmenia, C; Colozza, C; Tortorano, A M; Sanguinetti, M; Fadda, G

    2012-05-01

    Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

  13. Rapid identification and simultaneous analysis of multiple constituents from Rheum tanguticum Maxim. ex Balf. by UPLC/Q-TOF-MS.

    Science.gov (United States)

    Gao, Liang-Liang; Guo, Tao; Xu, Xu-Dong; Yang, Jun-Shan

    2017-07-01

    Rhubarb contains biologically active compounds such as anthraquinones, anthrones, stilbenes and tannins. A rapid and efficient UPLC/Q-TOF-MS/MS method was developed and applied towards identifying the constituents of Rheum tanguticum Maxim. ex Balf. for the first time. Chemical constituents were separated and investigated by UPLC/Q-TOF-MS/MS in the negative ion mode. The ESI-MS 2 fragmentation pathways of four types of compounds were interpreted, providing a very useful guidance for the characterisation of different types of compounds. Based on the exact mass information, fragmentation characteristic and LC retention time of 7 reference standards, 30 constituents were tentatively identified from the methanol extract of R. tanguticum. Among them, seven compounds were described for the first time from R. tanguticum and two from the genus Rheum were described for the first time. The analytical tool used here is valuable for the rapid separation and identification of multiple and minor constituents in methanol extracts of R. tanguticum.

  14. Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for caspofungin susceptibility testing of Candida and Aspergillus species.

    Science.gov (United States)

    De Carolis, Elena; Vella, Antonietta; Florio, Ada R; Posteraro, Patrizia; Perlin, David S; Sanguinetti, Maurizio; Posteraro, Brunella

    2012-07-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility.

  15. Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

    KAUST Repository

    Janjanam, Jagadeesh; Jamwal, Manu; Singh, Surender V.; Kumar, Saravanan; Panigrahi, Aswini Kumar; Hariprasad, Gururao; Jena, Manoj Kumar; Anand, Vijay R.; Kumar, Sudarshan Suresh; Kaushik, Jai Kumar; Dang, Ajaykumar; Mukesh, Manishi; Mishra, Bishnu Prasad; Srinivasan, Alagiri; Reddy, Vanga Siva Belum; Mohanty, Ashok Kumar

    2013-01-01

    in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134

  16. Nanoscale Affinity Chip Interface for Coupling Inhibition SPR Immunosensor Screening with Nano-LC TOF MS

    NARCIS (Netherlands)

    Marchesini, G.R.; Buijs, J.; Haasnoot, W.; Hooijerink, H.; Jansson, O.; Nielen, M.W.F.

    2008-01-01

    The on-line nanoscale coupling of a surface plasmon resonance (SPR)-based inhibition biosensor immunoassay (iBIA) for the screening of low molecular weight molecules with nano-liquid-chromatography electrospray ionization time-of-flight mass spectrometry (nano-LC ESI TOF MS) for identification is

  17. Preclinical pharmacokinetic evaluation and metabolites identification of methyl salicylate-2-O-β-d-lactoside in rats using LC-MS/MS and Q-TOF-MS methods.

    Science.gov (United States)

    Zhang, Dan; Huang, Chao; Xin, Wenyu; Ma, Xiaowei; Zhang, Weiku; Zhang, Tiantai; Du, Guanhua

    2015-05-10

    Methyl salicylate-2-O-β-d-lactoside (MSL) is a natural salicylate derivative from the traditional Chinese medicine of Gaultheria yunnanensis (Franch.) Rehder (G. yunnanensis). As a non-steroidal anti-inflammatory drug (NSAID), MSL exerts a significant anti-arthritis effect but hardly has any gastrointestinal toxicity. In this paper, the pharmacokinetics, distribution, excretion and identification of MSL and its metabolites are described following rat oral and intravenous administration. The biological samples were quantified by UPLC-MS/MS and the metabolites in urine and feces were identified by using Q-TOF-MS. These results will support future investigations leading to clinical development of this drug. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Surface-MALDI mass spectrometry in biomaterials research

    DEFF Research Database (Denmark)

    Griesser, H.J.; Kingshott, P.; McArthur, S.L.

    2004-01-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new...... surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological...... solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established...

  19. Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives

    DEFF Research Database (Denmark)

    Brun, Sophie; Madrid, Hugo; Gerrits Van Den Ende, Bert

    2013-01-01

    The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species-complexes of morp......The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species...... trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades...... of A. solani, and the third included all strains of A. tomatophila, as well as all but one strain of A. solani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely...

  20. An in-house assay is superior to Sepsityper for direct matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification of yeast species in blood cultures.

    Science.gov (United States)

    Bidart, Marie; Bonnet, Isabelle; Hennebique, Aurélie; Kherraf, Zine Eddine; Pelloux, Hervé; Berger, François; Cornet, Muriel; Bailly, Sébastien; Maubon, Danièle

    2015-05-01

    We developed an in-house assay for the direct identification, by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, of yeasts in blood culture. Sixty-one representative strains from 12 species were analyzed in spiked blood cultures. Our assay accurately identified 95 of 107 (88.8%) positive blood cultures and outperformed the commercial Sepsityper kit (81.7% identification). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Integration of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in blood culture diagnostics: a fast and effective approach.

    Science.gov (United States)

    Klein, Sabrina; Zimmermann, Stefan; Köhler, Christine; Mischnik, Alexander; Alle, Werner; Bode, Konrad A

    2012-03-01

    Sepsis is a major cause of mortality in hospitalized patients worldwide, with lethality rates ranging from 30 to 70 %. Sepsis is caused by a variety of different pathogens, and rapid diagnosis is of outstanding importance, as early and adequate antimicrobial therapy correlates with positive clinical outcome. In recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has become a powerful tool in microbiological diagnostics. The direct identification of micro-organisms in a positive blood culture by MALDI-TOF MS can shorten the diagnostic procedure significantly. Therefore, the aim of the present study was to evaluate whether identification rates could be improved by using the new Sepsityper kit from Bruker Daltonics for direct isolation and identification of bacteria from positive blood cultures by MALDI-TOF MS compared with the use of conventional separator gel columns, and to integrate the MALDI-TOF MS-based identification method into the routine course of blood culture diagnostics in the setting of a microbiological laboratory at a university hospital in Germany. The identification of Gram-negative bacteria by MALDI-TOF MS was significantly better using the Sepsityper kit compared with a separator gel tube-based method (99 and 68 % correct identification, respectively). For Gram-positive bacteria, only 73 % were correctly identified by MALDI-TOF with the Sepsityper kit and 59 % with the separator gel tube assay. A major problem of both methods was the poor identification of Gram-positive grape-like clustered cocci. As differentiation of Staphylococcus aureus from coagulase-negative staphylococci is of clinical importance, a PCR was additionally established that was capable of identifying S. aureus directly from positive blood cultures, thus closing this diagnostic gap. Another benefit of the PCR approach is the possibility of directly detecting the genes responsible for meticillin

  2. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for the identification of clinical filamentous fungi.

    Science.gov (United States)

    Huang, Yanfei; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Wang, Jinglin; Lu, Xinxin

    2017-07-01

    Infections caused by filamentous fungi have become a health concern, and require rapid and accurate identification in order for effective treatment of the pathogens. To compare the performance of two MALDI-TOF MS systems (Bruker Microflex LT and Xiamen Microtyper) in the identification of filamentous fungal species. A total of 374 clinical filamentous fungal isolates sequentially collected in the Clinical Laboratory at the Beijing Tongren Hospital between January 2014 and December 2015 were identified by traditional phenotypic methods, Bruker Microflex LT and Xiamen Microtyper MALDI-TOF MS, respectively. The discrepancy between these methods was resolved by sequencing for definitive identification. Bruker Microflex LT and Xiamen Microtyper had similar correct species ID (98.9 vs. 99.2%), genus ID (99.7 vs. 100%), mis-ID (0.3 vs. 0%) and no ID (0 vs. 0). The rate of correct species identification by both MALDI-TOF MS (98.9 and 99.2%, respectively) was much higher compared with phenotypic approach (91.9%). Both MALDI-TOF MS systems provide accurate identification of clinical filamentous fungi compared with conventional phenotypic method, and have the potential to replace identification for routine identification of these fungi in clinical mycology laboratories. Both systems have similar performance in the identification of clinical filamentous fungi.

  3. Thermoresponsive Arrays Patterned via Photoclick Chemistry: Smart MALDI Plate for Protein Digest Enrichment, Desalting, and Direct MS Analysis.

    Science.gov (United States)

    Meng, Xiao; Hu, Junjie; Chao, Zhicong; Liu, Ying; Ju, Huangxian; Cheng, Quan

    2018-01-10

    Sample desalting and concentration are crucial steps before matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis. Current sample pretreatment approaches require tedious fabrication and operation procedures, which are unamenable to high-throughput analysis and also result in sample loss. Here, we report the development of a smart MALDI substrate for on-plate desalting, enrichment, and direct MS analysis of protein digests based on thermoresponsive, hydrophilic/hydrophobic transition of surface-grafted poly(N-isopropylacrylamide) (PNIPAM) microarrays. Superhydrophilic 1-thioglycerol microwells are first constructed on alkyne-silane-functionalized rough indium tin oxide substrates based on two sequential thiol-yne photoclick reactions, whereas the surrounding regions are modified with hydrophobic 1H,1H,2H,2H-perfluorodecanethiol. Surface-initiated atom-transfer radical polymerization is then triggered in microwells to form PNIPAM arrays, which facilitate sample loading and enrichment of protein digests by concentrating large-volume samples into small dots and achieving on-plate desalting through PNIPAM configuration change at elevated temperature. The smart MALDI plate shows high performance for mass spectrometric analysis of cytochrome c and neurotensin in the presence of 1 M urea and 100 mM NaHCO 3 , as well as improved detection sensitivity and high sequence coverage for α-casein and cytochrome c digests in femtomole range. The work presents a versatile sample pretreatment platform with great potential for proteomic research.

  4. Screening for illicit and medicinal drugs in whole blood using fully automated SPE and UHPLC-TOF-MS with data-independent acquisition

    DEFF Research Database (Denmark)

    Pedersen, Anders Just; Dalsgaard, Petur Weihe; Rode, Andrej Jaroslav

    2013-01-01

    , butylone, cathine, fentanyl, LSD, mCPP, MDPV, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and PMMA. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in detection of common drugs of abuse as well as new designer-drugs and more rarely occurring drugs. Thus, TOF...

  5. De novo quence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue-green alga Aphanizomenon flos-aquae

    DEFF Research Database (Denmark)

    Rinalducci, Sara; Roepstorff, Peter; Zolla, Lello

    2009-01-01

    isothiocyanate (SPITC) and MALDI-TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20...... of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI- and ESI-MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon...

  6. Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Caspofungin Susceptibility Testing of Candida and Aspergillus Species

    Science.gov (United States)

    De Carolis, Elena; Vella, Antonietta; Florio, Ada R.; Posteraro, Patrizia; Perlin, David S.; Posteraro, Brunella

    2012-01-01

    Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility. PMID:22535984

  7. Simultaneously tracing the geographical origin and presence of bovine milk in Italian water buffalo Mozzarella cheese using MALDI-TOF data of casein signature peptides.

    Science.gov (United States)

    Caira, Simonetta; Pinto, Gabriella; Nicolai, Maria Adalgisa; Chianese, Lina; Addeo, Francesco

    2016-08-01

    Water buffalo (WB) casein (CN) and curd samples from indigenous Italian and international breeds were examined with the objective of identifying signature peptides that could function as an indicator to determine the origin of their milk products. CN in complex mixtures were digested with trypsin, and peptide fragments were subsequently identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The unique presence of a β-CN A variant and an internally deleted αs1-CN (f35-42) variant in international WB milk samples was ascertained by identifying signature tryptic peptides from either dephosphorylated or native CN. Four signature unphosphorylated peptides derived from β-CN A, i.e. (f49-68) Asn(68) (2223.6 Da), (f1-28) Ser(10) (3169.4 Da), (f1-29) Ser(10) (3297.4 Da) and (f33-48) Thr(41) (1982 Da) and two from αs1-CN (f35-42) deleted fragments, i.e. (f23-34) Met(31) (1415.7 Da) and (f43-58) Val(44) (1752.7 Da), were identified. Two signature casein phosphopeptides (CPPs), i.e. β-CN (f1-28) 4P (3489.1 Da) and β-CN (f33-48) 1P (2062.0 Da), were identified in the tryptic hydrolysate of native casein or curd and cheese samples using in-batch hydroxyapatite (HA) chromatography. All these fragments functioned as analytical surrogates of two αs1- and β-casein variants that specifically occur in the milk of international WB breeds. Furthermore, the bovine peptide β-CN (f1-28) 4P had a distinct and lower molecular mass compared with the WB counterpart and functioned as a species-specific marker for all breeds of WB. Advantages of this analytical approach are that (i) peptides are easier to separate than proteins, (ii) signature peptide probes originating from specific casein variants allow for the targeting of all international WB milk, curd and cheese samples and (iii) bovine and WB casein in mixtures can be simultaneously determined in protected designation of origin (PDO) "Mozzarella di Bufala Campana" cheese

  8. Dietary keto-acid feed-back on pituitary activity in gilthead sea bream

    DEFF Research Database (Denmark)

    Ibarz, Antoni; Costa, Rita; Harrison, Adrian Paul

    2010-01-01

    bream pituitary proteome was generated. Proteins with a modified expression between Controls and AKG treated fish were further analysed by MALDI-TOF/TOF-MS and liquid chromatography combined with a nanoelectrospray (LC-MS/MS). The main changes in the proteome induced by AKG treatment were grouped...

  9. [Dynamic variation of components in exocarp of Juglans mandshurica with browning based on UPLC-Q-TOF/MS].

    Science.gov (United States)

    Sun, Guo-Dong; Huo, Jin-Hai; Xie, Rong-Juan; Wang, Wei-Ming

    2017-08-01

    To analyze the dynamic changes in components in exocarp of Juglans mandshurica at different browning periods. Twenty-six batches of exocarp of J. mandshurica samples from thirteen browning periods were assessed by UPLC-Q-TOF-MS/MS. The formula of different compounds were determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/masterview1.0 software. Then their structures were determined by analysis of MS/MS fragment or comparison with standard substances and references. The contents of chemical components were changed significantly in different browning periods and twenty five compounds were identified or inferred. Of the 13 naphthoquinone compounds, the contents of 6 compounds with similar parent nucleus as juglone and 3 naphthoquinone glycosides compounds were decreased significantly, and 4 naphthoquinone derivatives such as regiolone were produced; the contents of four flavones and two phenolic acids compounds were decreased significantly; and the contents of 6 diarylheptanoids compounds were increased significantly. UPLC-Q-TOF/MS method can be used to identify and analyze the chemical constituents from exocarp of J. mandshurica rapidly and accurately, and analyze the rules of dynamic changes, to reveal the browning of Chinese medicinal materials and its effects on compositions of fruits and vegetables. Copyright© by the Chinese Pharmaceutical Association.

  10. Major phytopathogens and strains from cocoa (Theobroma cacao L.) are differentiated by MALDI-MS lipid and/or peptide/protein profiles.

    Science.gov (United States)

    Dos Santos, Fábio Neves; Tata, Alessandra; Belaz, Kátia Roberta Anacleto; Magalhães, Dilze Maria Argôlo; Luz, Edna Dora Martins Newman; Eberlin, Marcos Nogueira

    2017-03-01

    Phytopathogens are the main disease agents that promote attack of cocoa plantations in all tropical countries. The similarity of the symptoms caused by different phytopathogens makes the reliable identification of the diverse species a challenge. Correct identification is important in the monitoring and management of these pests. Here we show that matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis is able to rapidly and reliably differentiate cocoa phytopathogens, namely Moniliophthora perniciosa, Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C. paradoxa, and C. fimbriata. MALDI-MS reveals unique peptide/protein and lipid profiles which differentiate these phytopathogens at the level of genus, species, and single strain coming from different hosts or cocoa tissues collected in several plantations/places. This fast methodology based on molecular biomarkers is also shown to be sufficiently reproducible and selective and therefore seems to offer a suitable tool to guide the correct application of sanitary defense approaches for infected cocoa plantations. International trading of cocoa plants and products could also be efficiently monitored by MALDI-MS. It could, for instance, prevent the entry of new phytopathogens into a country, e.g., as in the case of Moniliophthora roreri fungus that is present in all cocoa plantations of countries bordering Brazil, but that has not yet attacked Brazilian plantations. Graphical Abstract Secure identification of phytopathogens attacking cocoa plantations has been demonstrated via typical chemical profiles provided by mass spectrometric screening.

  11. Detection of Bacteriocins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

    OpenAIRE

    Rose, Natisha L.; Sporns, Peter; McMullen, Lynn M.

    1999-01-01

    The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of bacteriocins was investigated. A 30-s water wash of the sample on the MALDI-TOF MS probe was effective in removing contaminants of the analyte. This method was used for rapid detection of nisin, pediocin, brochocin A and B, and enterocin A and B from culture supernatants and for detection of enterocin B throughout its purification.

  12. Disulfiram generates a stable N,N-diethylcarbamoyl adduct on Cys-125 of rat hemoglobin beta-chains in vivo

    DEFF Research Database (Denmark)

    Erve, J C; Jensen, Ole Nørregaard; Valentine, H S

    2000-01-01

    residues each. MALDI-TOF MS analysis of two new globin species from DSF-treated rats collected by HPLC revealed increments of 99 Da above the mass of the unmodified chains (beta-2 and beta-3). In a separate experiment, the globin mixture was digested for 2 h with Glu-C and reanalyzed by MALDI-TOF MS....... Results showed a peptide at m/z 2716.3 having a mass 99 Da higher than a known Cys-containing peptide. Subsequently, the Glu-C digest was analyzed using Q-TOF tandem MS, enabling observation of the +4 charge state of the peptide with m/z 2716.3. This peptide was fragmented to produce y-sequence ions...

  13. Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of molds of the Fusarium genus.

    Science.gov (United States)

    Triest, David; Stubbe, Dirk; De Cremer, Koen; Piérard, Denis; Normand, Anne-Cécile; Piarroux, Renaud; Detandt, Monique; Hendrickx, Marijke

    2015-02-01

    The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity. Copyright © 2015

  14. Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Molds of the Fusarium Genus

    Science.gov (United States)

    Stubbe, Dirk; De Cremer, Koen; Piérard, Denis; Normand, Anne-Cécile; Piarroux, Renaud; Detandt, Monique; Hendrickx, Marijke

    2014-01-01

    The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity. PMID:25411180

  15. Diagnostic performance of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry in blood bacterial infections: a systematic review and meta-analysis.

    Science.gov (United States)

    Scott, Jamie S; Sterling, Sarah A; To, Harrison; Seals, Samantha R; Jones, Alan E

    2016-07-01

    Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has shown promise in decreasing time to identification of causative organisms compared to traditional methods; however, the utility of MALDI-TOF MS in a heterogeneous clinical setting is uncertain. To perform a systematic review on the operational performance of the Bruker MALDI-TOF MS system and evaluate published cut-off values compared to traditional blood cultures. A comprehensive literature search was performed. Studies were included if they performed direct MALDI-TOF MS analysis of blood culture specimens in human patients with suspected bacterial infections using the Bruker Biotyper software. Sensitivities and specificities of the combined studies were estimated using a hierarchical random effects linear model (REML) incorporating cut-off scores of ≥1.7 and ≥2.0. Fifty publications were identified, with 11 studies included after final review. The estimated sensitivity utilising a cut-off of ≥2.0 from the combined studies was 74.6% (95% CI = 67.9-89.3%), with an estimated specificity of 88.0% (95% CI = 74.8-94.7%). When assessing a cut-off of ≥1.7, the combined sensitivity increases to 92.8% (95% CI = 87.4-96.0%), but the estimated specificity decreased to 81.2% (95% CI = 61.9-96.6%). In this analysis, MALDI-TOF MS showed acceptable sensitivity and specificity in bacterial speciation with the current recommended cut-off point compared to blood cultures; however, lowering the cut-off point from ≥2.0 to ≥1.7 would increase the sensitivity of the test without significant detrimental effect on the specificity, which could improve clinician confidence in their results.

  16. Metabonomics research of diabetic nephropathy and type 2 diabetes mellitus based on UPLC-oaTOF-MS system

    International Nuclear Information System (INIS)

    Zhang Jie; Yan Lijuan; Chen Wengui; Lin Lin; Song Xiuyu; Yan Xiaomei; Hang Wei; Huang Benli

    2009-01-01

    Ultra performance liquid chromatography (UPLC) coupled with orthogonal acceleration time-of-flight (oaTOF) mass spectrometry has showed great potential in diabetes research. In this paper, a UPLC-oaTOF-MS system was employed to distinguish the global serum profiles of 8 diabetic nephropathy (DN) patients, 33 type 2 diabetes mellitus (T2DM) patients and 25 healthy volunteers, and tried to find potential biomarkers. The UPLC system produced information-rich chromatograms with typical measured peak widths of 4 s, generating peak capacities of 225 in 15 min. Furthermore, principal component analysis (PCA) was used for group differentiation and marker selection. As shown in the scores plot, the distinct clustering between the patients and controls was observed, and DN and T2DM patients were also separated into two individual groups. Several compounds were tentatively identified based on accurate mass, isotopic pattern and MS/MS information. In addition, significant changes in the serum level of leucine, dihydrosphingosine and phytoshpingosine were noted, indicating the perturbations of amino acid metabolism and phospholipid metabolism in diabetic diseases, which having implications in clinical diagnosis and treatment.

  17. Metabonomics research of diabetic nephropathy and type 2 diabetes mellitus based on UPLC-oaTOF-MS system

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Jie, E-mail: jiezhang@dicp.ac.cn [Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, 361005 Xiamen (China); Yan Lijuan [XiaMen Entry-Exit Inspection and Quarantine Bureau, 361012 Xiamen (China); Chen Wengui [First Hospital of Xiamen, 361003 Xiamen (China); Lin Lin [Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, 361005 Xiamen (China); Song Xiuyu [First Hospital of Xiamen, 361003 Xiamen (China); Yan Xiaomei [Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, 361005 Xiamen (China); Hang Wei, E-mail: weihang@xmu.edu.cn [Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, 361005 Xiamen (China); Huang Benli [Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, 361005 Xiamen (China)

    2009-09-14

    Ultra performance liquid chromatography (UPLC) coupled with orthogonal acceleration time-of-flight (oaTOF) mass spectrometry has showed great potential in diabetes research. In this paper, a UPLC-oaTOF-MS system was employed to distinguish the global serum profiles of 8 diabetic nephropathy (DN) patients, 33 type 2 diabetes mellitus (T2DM) patients and 25 healthy volunteers, and tried to find potential biomarkers. The UPLC system produced information-rich chromatograms with typical measured peak widths of 4 s, generating peak capacities of 225 in 15 min. Furthermore, principal component analysis (PCA) was used for group differentiation and marker selection. As shown in the scores plot, the distinct clustering between the patients and controls was observed, and DN and T2DM patients were also separated into two individual groups. Several compounds were tentatively identified based on accurate mass, isotopic pattern and MS/MS information. In addition, significant changes in the serum level of leucine, dihydrosphingosine and phytoshpingosine were noted, indicating the perturbations of amino acid metabolism and phospholipid metabolism in diabetic diseases, which having implications in clinical diagnosis and treatment.

  18. N-glycosylation of Colorectal Cancer Tissues

    Science.gov (United States)

    Balog, Crina I. A.; Stavenhagen, Kathrin; Fung, Wesley L. J.; Koeleman, Carolien A.; McDonnell, Liam A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

    2012-01-01

    Colorectal cancer is the third most common cancer worldwide with an annual incidence of ∼1 million cases and an annual mortality rate of ∼655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers. PMID:22573871

  19. N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation.

    Science.gov (United States)

    Balog, Crina I A; Stavenhagen, Kathrin; Fung, Wesley L J; Koeleman, Carolien A; McDonnell, Liam A; Verhoeven, Aswin; Mesker, Wilma E; Tollenaar, Rob A E M; Deelder, André M; Wuhrer, Manfred

    2012-09-01

    Colorectal cancer is the third most common cancer worldwide with an annual incidence of ~1 million cases and an annual mortality rate of ~655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers.

  20. Evaluation of Bruker Biotyper and Vitek MS for the identification of Candida tropicalis on different solid culture media.

    Science.gov (United States)

    Wang, He; Li, Ying; Fan, Xin; Chiueh, Tzong-Shi; Xu, Ying-Chun; Hsueh, Po-Ren

    2017-11-11

    The aim of this study was to investigate the performance of the Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Vitek MS systems for identification of genetically-confirmed blood isolates of Candida tropicalis that had been grown on several types of culture media commonly used for primary fungal isolation. Isolates included 105 from the National China Hospital Invasive Fungal Surveillance Net program (CHIF-NET) and 120 from National Taiwan University Hospital (NTUH). Culture media tested for CHIF-NET isolates included trypticase soy agar supplemented with 5% sheep blood (BAP), Sabouraud dextrose agar (SDA-C), CHROMagar, China blue agar (CBA), chocolate agar supplemented with vancomycin (CAP-VA), and MacConkey agar (MAC). Culture media used for NTUH isolates included BAP, SDA, CHROMagar, eosin methylene blue (EMB), inhibitory mold agar (IMA), Mycosel agar, and cornmeal agar (CMA). The Bruker Biotyper correctly identified all CHIF-NET isolates to the species level on all six agar media tested and correctly identified the majority of NTUH isolates with the exception of isolates grown on SDA (85.8%) and CMA (52.5%). The Vitek MS system correctly identified all CHIF-NET isolates to the species level with the exception of isolates grown on CHROMagar (84.8%), and correctly identified the majority of NTUH isolates with the exception of isolates grown on SDA (51.7%), Mycosel agar (57.5%), and CMA (9.2%) for NTUH isolates. Clinical microbiologists should be aware that different culture media can affect the performance of the Bruker Biotyper MALDI-TOF MS and Vitek MS systems in identifying C. tropicalis. Copyright © 2017. Published by Elsevier B.V.