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Sample records for maldi-tof ms identificacao

  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. [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.

  5. Applications of MALDI-TOF MS in Microbiological identification

    Directory of Open Access Journals (Sweden)

    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

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. [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.

  9. 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.

  10. 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...

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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)

  16. 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

  17. 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

  18. 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

  19. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  3. 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

  4. [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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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...

  10. [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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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...

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  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. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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...

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  15. 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.

  16. 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...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. 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…

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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 .

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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...

  10. 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...

  11. 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...

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. [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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  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. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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...

  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. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  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. 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.

    Directory of Open Access Journals (Sweden)

    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

  9. 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.

  10. 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...

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  17. 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.

  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. 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...

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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...

  12. 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.

  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. Detection of Serum Peptides in Patients with Lung Squamous Cell Carcinoma by MALDI-TOF-MS and Analysis of Their Correlation with Chemotherapy Efficacy

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    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

  15. [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.

  16. An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS

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    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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  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. 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.

  6. 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...

  7. 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.

  8. 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

  9. Superiority of SDS lysis over saponin lysis for direct bacterial identification from positive blood culture bottle by MALDI-TOF MS.

    Science.gov (United States)

    Caspar, Yvan; Garnaud, Cécile; Raykova, Mariya; Bailly, Sébastien; Bidart, Marie; Maubon, Danièle

    2017-05-01

    Fast species diagnosis has an important health care impact, as rapid and specific antibacterial therapy is of clear benefit for patient's outcome. Here, a new protocol for species identification directly from positive blood cultures is proposed. Four in-house protocols for bacterial identification by MS directly from clinical positive blood cultures evaluating two lytic agents, SDS and saponin, and two protein extraction schemes, fast (FP) and long (LP) are compared. One hundred and sixty-eight identification tests are carried out on 42 strains. Overall, there are correct identifications to the species level in 90% samples for the SDS-LP, 60% for the SDS-FP, 48% for the saponin LP, and 43% for the saponin FP. Adapted scores allowed 92, 86, 72, and 53% identification for SDS-LP, SDS-FP, saponin LP, and saponin FP, respectively. Saponin lysis is associated with a significantly lower score compared to SDS (0.87 [0.83-0.92], p-value saponin lysis and the application of this rapid and cost-effective protocol in daily routine for microbiological agents implicated in septicemia. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  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. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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...

  13. 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

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

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    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.

  15. 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.

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

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    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.

  17. 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.

  18. [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

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

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    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 .

  20. 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.

  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. 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.

  3. 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.

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

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    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.

  5. 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

  6. 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.

  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. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. [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.

  16. 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

  17. 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.

  18. 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...

  19. 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.

  20. 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

  1. 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%.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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...

  10. 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.

  11. [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.

  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. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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...

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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...

  15. 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.

  16. 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.

  17. 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...

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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 ...

  4. 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

  5. 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...

  6. MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

    Science.gov (United States)

    Rode, Tone Mari; Berget, Ingunn; Langsrud, Solveig; Møretrø, Trond; Holck, Askild

    2009-07-01

    Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions.

  7. 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.

  8. Identification of Emerging Human Mastitis Pathogens by MALDI-TOF and Assessment of Their Antibiotic Resistance Patterns

    Directory of Open Access Journals (Sweden)

    María Marín

    2017-07-01

    Full Text Available Lactational mastitis constitutes one of the main causes of undesired weaning, depriving the mother–infant pair from the benefits of breastfeeding; therefore, this condition should be considered a relevant public health issue. The role of specific microorganisms remains unclear since human milk cultures and antibiotic susceptibility testing (AST are not routinely performed, despite the fact that this would be key to ensure an early and effective diagnosis and treatment. The objective of this study was to describe the culturable microbial diversity in 647 milk samples from breastfeeding women with clinical symptoms of mastitis by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF VITEK MS technology and to analyze the antimicrobial susceptibility profiles of a collection of isolates from these samples by the VITEK 2 AST system. Staphylococcus epidermidis was the most common species isolated from mastitis samples (87.6%, while Staphylococcus aureus was detected in 22.1%. Streptococci constituted the second (68.6% most prevalent bacterial group, with Streptococcus mitis/oralis, Streptococcus salivarius, and Streptococcus parasanguinis detected with frequencies of 40.8, 36.8, and 14.4%, respectively. The antibiotic susceptibility profiles of 642 staphylococcal isolates indicated a remarkable resistance to benzylpenicillin (88.3% and erythromycin (67.3% with differences between species. A high percentage of Staphylococcus isolates were resistant to at least one antibiotic (Staphylococcus hominis, 100%; S. epidermidis, 98.2%; S. aureus, 92.9%; Staphylococcus lugdunensis, 90.5% and the percentage of multidrug-resistance (MDR isolates was noticeable (S. hominis, 81%; S. epidermidis, 64.4%; S. aureus, 11.5%; S. lugdunensis, 10.5%. In relation to streptococcal isolates (n = 524, AST revealed high or moderate percentages of resistance to erythromycin (68.7%, benzylpenicillin (63.7%, ampicillin (51.5%, and tetracycline

  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. Monitoring of barley starch amylolysis by gravitational field flow fractionation and MALDI-TOF MS

    Czech Academy of Sciences Publication Activity Database

    Mazanec, Karel; Dyčka, Filip; Bobálová, Janette

    2011-01-01

    Roč. 91, č. 15 (2011), s. 2756-2761 ISSN 0022-5142 R&D Projects: GA MŠk 1M0570; GA MŠk 1M06030; GA MŠk 2B06037 Institutional research plan: CEZ:AV0Z40310501 Keywords : barley * starch * malting process Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.436, year: 2011

  11. MALDI-TOF MS for quality control of high protein content sport supplements.

    Science.gov (United States)

    De Ceglie, Cristina; Calvano, Cosima D; Zambonin, Carlo G

    2015-06-01

    High protein content sport nutritional supplements are found as powder products containing, as ingredients, amino acids and proteins with important nutritional values as milk, soy and egg proteins. An EU Food Supplements Directive (2002) requires that supplements should be safe, both in dosages and in purity. It is important, then, to develop rapid and sensitive methods to be employed for the quality control of these substances. In this work, we apply, for the first time, matrix-assisted laser desorption ionization-mass spectrometry as a fast, reproducible and sensitive method for the quality control of sport nutritional supplements based on proteins. To this aim, several commercial egg- and/or milk-based powder products have been processed by in gel or in solution digestion and analyzed in comparison to pure standard products. This strategy allowed to assess the reliability of the indications on proteins (as caseins, whey proteins and ovalbumin) declared in the label of several sport nutritional supplements. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Cytosolic proteome profiling of aminoglycosides resistant Mycobacterium tuberculosis clinical isolates using MALDI-TOF/MS

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    2016-11-01

    Full Text Available Emergence of extremely drug resistant tuberculosis (XDR-TB is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB. Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK and kanamycin (KM resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636 and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.

  13. Cytosolic Proteome Profiling of Aminoglycosides Resistant Mycobacterium tuberculosis Clinical Isolates Using MALDI-TOF/MS.

    Science.gov (United States)

    Sharma, Divakar; Lata, Manju; Singh, Rananjay; Deo, Nirmala; Venkatesan, Krishnamurthy; Bisht, Deepa

    2016-01-01

    Emergence of extensively drug resistant tuberculosis (XDR-TB) is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB). Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK) and kanamycin (KM) resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636, and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain) of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins, respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can be prevented.

  14. Maldi-Tof /Tof-MS Reveals Elevated Serum Haptoglobin and Amyloid A in Behcet's Disease

    NARCIS (Netherlands)

    Mao, L.; Dong, H.; Yang, P.; Zhou, H.; Huang, X.; Lin, X.; Kijlstra, A.

    2008-01-01

    Behcet¿s disease (BD) is a multisystemic autoimmune disease with unclear etiology and pathogenesis. To screen aberrant serum proteins in BD, serum samples were obtained from eight male BD patients with active uveitis and eight male healthy volunteers with informed consent. The serum samples from

  15. Characterization and differentiation of aflatoxigenic species of Aspergillus section Flavi by MALDI-TOF MS

    OpenAIRE

    Rodrigues, P.; Kallow, W.; Erhard, M.; Welker, M.; Kozakiewicz, Z.; Lima, Nelson; Venâncio, Armando

    2007-01-01

    Aspergillus is a large genus, with a complex taxonomy. The genus is easily identifiedd by its characteristic conidiophore, but species identification and differentiation is complex, mainly because it is traditionally based on a range of morphological features. Aspergillus subgenus Circumdati section Flavi, also refered to as the A.dflavus group, has attracted worldwide attention for its industrial use and toxigenic potential. Sectiond Flavi is divided in two groups of species. One includes th...

  16. A polyphasic approach for black Aspergillus identification using MALDI-TOF MS.

    OpenAIRE

    Rodrigues, Paula; Soares, Célia; Santos, Cledir; Fraga, Marcelo; Venâncio, Armando; Lima, Nelson

    2009-01-01

    The Aspergillus section Nigri is among the best studied fungi, having different commercial applications, but also causing biodeterioration of commodities and food spoilage. Although being well studied, their identification is not straightforward, and, recently, new species have been described in this section1,2,3. These new species were not only separated from their relatives in the section by morphological distinction but also from molecular point of view. The concept of species is clearly a...

  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. Classification of ancient mammal individuals using dental pulp MALDI-TOF MS peptide profiling.

    Directory of Open Access Journals (Sweden)

    Thi-Nguyen-Ny Tran

    Full Text Available BACKGROUND: The classification of ancient animal corpses at the species level remains a challenging task for forensic scientists and anthropologists. Severe damage and mixed, tiny pieces originating from several skeletons may render morphological classification virtually impossible. Standard approaches are based on sequencing mitochondrial and nuclear targets. METHODOLOGY/PRINCIPAL FINDINGS: We present a method that can accurately classify mammalian species using dental pulp and mass spectrometry peptide profiling. Our work was organized into three successive steps. First, after extracting proteins from the dental pulp collected from 37 modern individuals representing 13 mammalian species, trypsin-digested peptides were used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The resulting peptide profiles accurately classified every individual at the species level in agreement with parallel cytochrome b gene sequencing gold standard. Second, using a 279-modern spectrum database, we blindly classified 33 of 37 teeth collected in 37 modern individuals (89.1%. Third, we classified 10 of 18 teeth (56% collected in 15 ancient individuals representing five mammal species including human, from five burial sites dating back 8,500 years. Further comparison with an upgraded database comprising ancient specimen profiles yielded 100% classification in ancient teeth. Peptide sequencing yield 4 and 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I in human ancient and modern dental pulp, respectively. CONCLUSIONS/SIGNIFICANCE: Mass spectrometry peptide profiling of the dental pulp is a new approach that can be added to the arsenal of species classification tools for forensics and anthropology as a complementary method to DNA sequencing. The dental pulp is a new source for collagen and other proteins for the species classification of modern and ancient mammal individuals.

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

    NARCIS (Netherlands)

    Lista, F.; Reubsaet, F.A.G.; Santis, R. de; Parchen, R.R.; Jong, A.L. de; Kieboom, J.; Laaken, A.L. van der; Voskamp-Visser, I.A.I.; Fillo, S.; Jansen, H.J. de; Plas, J. van der; Paauw, A.

    2011-01-01

    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.

  20. Classification of Ancient Mammal Individuals Using Dental Pulp MALDI-TOF MS Peptide Profiling

    Science.gov (United States)

    Tran, Thi-Nguyen-Ny; Aboudharam, Gérard; Gardeisen, Armelle; Davoust, Bernard; Bocquet-Appel, Jean-Pierre; Flaudrops, Christophe; Belghazi, Maya; Raoult, Didier; Drancourt, Michel

    2011-01-01

    Background The classification of ancient animal corpses at the species level remains a challenging task for forensic scientists and anthropologists. Severe damage and mixed, tiny pieces originating from several skeletons may render morphological classification virtually impossible. Standard approaches are based on sequencing mitochondrial and nuclear targets. Methodology/Principal Findings We present a method that can accurately classify mammalian species using dental pulp and mass spectrometry peptide profiling. Our work was organized into three successive steps. First, after extracting proteins from the dental pulp collected from 37 modern individuals representing 13 mammalian species, trypsin-digested peptides were used for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. The resulting peptide profiles accurately classified every individual at the species level in agreement with parallel cytochrome b gene sequencing gold standard. Second, using a 279–modern spectrum database, we blindly classified 33 of 37 teeth collected in 37 modern individuals (89.1%). Third, we classified 10 of 18 teeth (56%) collected in 15 ancient individuals representing five mammal species including human, from five burial sites dating back 8,500 years. Further comparison with an upgraded database comprising ancient specimen profiles yielded 100% classification in ancient teeth. Peptide sequencing yield 4 and 16 different non-keratin proteins including collagen (alpha-1 type I and alpha-2 type I) in human ancient and modern dental pulp, respectively. Conclusions/Significance Mass spectrometry peptide profiling of the dental pulp is a new approach that can be added to the arsenal of species classification tools for forensics and anthropology as a complementary method to DNA sequencing. The dental pulp is a new source for collagen and other proteins for the species classification of modern and ancient mammal individuals. PMID:21364886

  1. Discrimination of multilocus sequence typing-based Campylobacter jejuni subgroups by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Zautner, Andreas Erich; Masanta, Wycliffe Omurwa; Tareen, Abdul Malik; Weig, Michael; Lugert, Raimond; Groß, Uwe; Bader, Oliver

    2013-11-07

    Campylobacter jejuni, the most common bacterial pathogen causing gastroenteritis, shows a wide genetic diversity. Previously, we demonstrated by the combination of multi locus sequence typing (MLST)-based UPGMA-clustering and analysis of 16 genetic markers that twelve different C. jejuni subgroups can be distinguished. Among these are two prominent subgroups. The first subgroup contains the majority of hyperinvasive strains and is characterized by a dimeric form of the chemotaxis-receptor Tlp7(m+c). The second has an extended amino acid metabolism and is characterized by the presence of a periplasmic asparaginase (ansB) and gamma-glutamyl-transpeptidase (ggt). Phyloproteomic principal component analysis (PCA) hierarchical clustering of MALDI-TOF based intact cell mass spectrometry (ICMS) spectra was able to group particular C. jejuni subgroups of phylogenetic related isolates in distinct clusters. Especially the aforementioned Tlp7(m+c)(+) and ansB+/ ggt+ subgroups could be discriminated by PCA. Overlay of ICMS spectra of all isolates led to the identification of characteristic biomarker ions for these specific C. jejuni subgroups. Thus, mass peak shifts can be used to identify the C. jejuni subgroup with an extended amino acid metabolism. Although the PCA hierarchical clustering of ICMS-spectra groups the tested isolates into a different order as compared to MLST-based UPGMA-clustering, the isolates of the indicator-groups form predominantly coherent clusters. These clusters reflect phenotypic aspects better than phylogenetic clustering, indicating that the genes corresponding to the biomarker ions are phylogenetically coupled to the tested marker genes. Thus, PCA clustering could be an additional tool for analyzing the relatedness of bacterial isolates.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. Identification of uncommon oral yeasts from cancer patients by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Aslani, Narges; Janbabaei, Ghasem; Abastabar, Mahdi; Meis, Jacques F; Babaeian, Mahasti; Khodavaisy, Sadegh; Boekhout, Teun; Badali, Hamid

    2018-01-08

    Opportunistic infections due to Candida species occur frequently in cancer patients because of their inherent immunosuppression. The aim of the present study was to investigate the epidemiology of yeast species from the oral cavity of patients during treatment for oncological and haematological malignancies. MALDI-TOF was performed to identify yeasts isolated from the oral cavity of 350 cancer patients. Moreover, antifungal susceptibility testing was performed in according to CLSI guidelines (M27-A3). Among 162 yeasts and yeast-like fungi isolated from the oral cavity of cancer patients, Candida albicans was the most common species (50.6%), followed by Candida glabrata (24.7%), Pichia kudriavzevii (Candida krusei (9.9%)), Candida tropicalis (4.3%), Candida dubliniensis (3.7%), Kluyveromyces marxianus (Candida kefyr (3.7%)) and Candida parapsilosis (1%). In addition, uncommon yeast species i.e., Saprochaete capitata, Saccharomyces cerevisiae, Clavispora lusitaniae (C. lusitaniae) and Pichia kluyveri (C. eremophila) were recovered from oral lesions. Oral colonization by C. albicans, non-albicans Candida species and uncommon yeasts were as follow; 55%, 44% and 1%, whereas oral infection due to C. albicans was 33.3%, non-albicans Candida species 60.6%, and uncommon yeasts 6.1%. Poor oral hygiene and xerostomia were identified as independent risk factors associated with oral yeast colonization. The overall resistance to fluconazole was 11.7% (19/162). Low MIC values were observed for anidulafungin for all Candida and uncommon yeast species. This current study provides insight into the prevalence and susceptibility profiles of Candida species, including emerging Candida species and uncommon yeasts, isolated from the oral cavity of Iranian cancer patients. The incidence of oral candidiasis was higher amongst patients with hematological malignancies. The majority of oral infections were caused by non-albicans Candida species which were often more resistant to anti

  10. 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

  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. 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....

  13. 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).

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. MALDI-TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

    NARCIS (Netherlands)

    Ruprecht, Benjamin; Roesli, Christoph; Lemeer, Simone; Kuster, Bernhard

    2016-01-01

    Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows.

  19. 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...

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Analysis of post-operative changes in serum protein expression profiles from colorectal cancer patients by MALDI-TOF mass spectrometry: a pilot methodological study

    Directory of Open Access Journals (Sweden)

    Marsh Simon

    2010-04-01

    Full Text Available Abstract Background Mass spectrometry-based protein expression profiling of blood sera can be used to discriminate colorectal cancer (CRC patients from unaffected individuals. In a pilot methodological study, we have evaluated the changes in protein expression profiles of sera from CRC patients that occur following surgery to establish the potential of this approach for monitoring post-surgical response and possible early prediction of disease recurrence. Methods In this initial pilot study, serum specimens from 11 cancer patients taken immediately prior to surgery and at approximately 6 weeks following surgery were analysed alongside 10 normal control sera by matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS. Using a two-sided t-test the top 20 ranked protein peaks that discriminate normal from pre-operative sera were identified. These were used to classify post-operative sera by hierarchical clustering analysis (Spearman's Rank correlation and, as an independent 'test' dataset, by k-nearest neighbour and weighted voting supervised learning algorithms. Results Hierarchical cluster analysis classified post-operative sera from all six early Dukes' stage (A and B patients as normal. The remaining five post-operative sera from more advanced Dukes' stages (C1 and C2 were classified as cancer. Analysis by supervised learning algorithms similarly grouped all advanced Dukes' stages as cancer, with four of the six post-operative sera from early Dukes' stages being classified as normal (P = 0.045; Fisher's exact test. Conclusions The results of this pilot methodological study illustrate the proof-of-concept of using protein expression profiling of post-surgical blood sera from individual patients to monitor disease course. Further validation on a larger patient cohort and using an independent post-operative sera dataset would be required to evaluate the potential clinical relevance of this approach. Prospective

  5. 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.

  6. 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.

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

    Czech Academy of Sciences Publication Activity Database

    Mathis, A.; Depaquit, J.; Dvořák, V.; Tuten, H.; Banuls, A.-L.; Halada, Petr; Zapata, S.; Lehrter, V.; Hlaváčková, K.; Prudhomme, J.; Volf, P.; Sereno, D.; Kaufmann, Ch.; Pflueger, V.; Schaffner, F.

    2015-01-01

    Roč. 8, MAY 2015 (2015) ISSN 1756-3305 R&D Projects: GA ČR(CZ) GA15-04329S Institutional support: RVO:61388971 Keywords : Arthropod identification * Bruker * Centralized reference database Subject RIV: CE - Biochemistry Impact factor: 3.234, year: 2015

  8. Positionsspezifischer Nachweis und relative Quantifizierung von Modifikationen des alpha-s1-Caseins in Milch mittels MALDI-TOF-MS

    OpenAIRE

    Vollmer, Gregor

    2013-01-01

    Milch und Milchprodukte spielen eine bedeutende Rolle in der menschlichen Ernährung. Vor der Abgabe an den Verbraucher wird Milch in den allermeisten Fällen hitzebehandelt, um mikrobielle Stabilität sicherzustellen und die Haltbarkeit zu verlängern. Allerdings kann diese Hitzebehandlung die in Milch enthaltenen Nährstoffe, unter anderem Kohlenhydrate, Lipide, Proteine und Vitamine, erheblich schädigen. Vitamine werden durch starke Erhitzung zerstört, bei den Lipiden kann es zu Abbaureaktionen...

  9. Proteomic profiling of occupational medicamentosa-like dermatitis induced by trichloroethylene in serum based on MALDI-TOF MS.

    Science.gov (United States)

    Liu, Wei; Hong, Wen-Xu; Zhang, Yanfang; Huang, Peiwu; Yang, Xifei; Ren, Xiaohu; Huang, Haiyan; Liu, Jianjun

    2015-11-01

    Trichloroethylene (TCE) has long been well known as a major pollutant that affects both occupational and general environments. Occupational medicamentosa-like dermatitis induced by TCE (OMLDT) is an autoimmune disease, which has become one of the critical occupational health issues in China. In this study, we analyzed 18 OMLDT patients and 29 professional TCE contact people on serum proteomic analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and ClinProTools bioinformatics software. The intensities of 35 protein/peptide peaks were significantly different between TCE contact controls and OMLDT patients. A pattern of six peaks (m/z 1,450.33, 1,866.16, 3,262.39, 4,109.55, 5,064.85 and 5,956.57) were selected to construct a diagnostic model to discriminate the OMLDT patients from controls with sensitivity and specificity of both 93.8 %. Our findings provide an alternative proteomic approach to differentiate the OMLDT patients from TCE contact workers with high sensitivity and high specificity, which will be of potential value in clinical diagnosis for occupational disease.

  10. MALDI-TOF MS contribution to diagnosis of melioidosis in a nonendemic country in three French travellers

    Directory of Open Access Journals (Sweden)

    V. Walewski

    2016-07-01

    Full Text Available Melioidosis is an endemic disease in Southeast Asia and northern Australia. An increasing number of cases are being reported in nonendemic countries, making the diagnosis less obvious. We discuss the identification of Burkholderia pseudomallei using matrix-assisted desorption ionization–time of flight mass spectrometry on the occasion of recent cases of imported melioidosis in French travellers.

  11. 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

  12. 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

  13. 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.

  14. Erysipelothrix rhusiopathiae bacteremia without endocarditis: rapid identification from positive blood culture by MALDI-TOF mass spectrometry. A case report and literature review

    Directory of Open Access Journals (Sweden)

    Luigi Principe

    2016-03-01

    Full Text Available Erysipelothrix rhusiopathiae is a Gram-positive bacillus that is infrequently responsible for infections in humans. Three forms have been classified: a localized cutaneous form (erysipeloid caused by traumatic penetration of E. rhusiopathiae, a generalized cutaneous form and a septicemic form. The latter type of disease has been previously associated with a high incidence of endocarditis. Here we report a case of E. rhusiopathiae bacteremia in a 74- year-old man, probably started from an erysipeloid form, in which endocarditis did not develop. This case presents some particular and uncommon features: i no correlation with animal source; ii correlation between bacteremia and erysipeloid lesion; iii absence of endocarditis. MALDI-TOF mass spectrometry allowed to obtain a rapid identification (within 4 hours from bottle positivity of E. rhusiopathiae. Together with direct antimicrobial susceptibility testing, this approach could improve the rate of appropriate therapy for bloodstream infections due to this fastidious pathogen.

  15. Analysis of hard protein corona composition on selective iron oxide nanoparticles by MALDI-TOF mass spectrometry: identification and amplification of a hidden mastitis biomarker in milk proteome.

    Science.gov (United States)

    Magro, Massimiliano; Zaccarin, Mattia; Miotto, Giovanni; Da Dalt, Laura; Baratella, Davide; Fariselli, Piero; Gabai, Gianfranco; Vianello, Fabio

    2018-05-01

    Surface active maghemite nanoparticles (SAMNs) are able to recognize and bind selected proteins in complex biological systems, forming a hard protein corona. Upon a 5-min incubation in bovine whey from mastitis-affected cows, a significant enrichment of a single peptide characterized by a molecular weight at 4338 Da originated from the proteolysis of a S1 -casein was observed. Notably, among the large number of macromolecules in bovine milk, the detection of this specific peptide can hardly be accomplished by conventional analytical techniques. The selective formation of a stable binding between the peptide and SAMNs is due to the stability gained by adsorption-induced surface restructuration of the nanomaterial. We attributed the surface recognition properties of SAMNs to the chelation of iron(III) sites on their surface by sterically compatible carboxylic groups of the peptide. The specific peptide recognition by SAMNs allows its easy determination by MALDI-TOF mass spectrometry, and a threshold value of its normalized peak intensity was identified by a logistic regression approach and suggested for the rapid diagnosis of the pathology. Thus, the present report proposes the analysis of hard protein corona on nanomaterials as a perspective for developing fast analytical procedures for the diagnosis of mastitis in cows. Moreover, the huge simplification of proteome complexity by exploiting the selectivity derived by the peculiar SAMN surface topography, due to the iron(III) distribution pattern, could be of general interest, leading to competitive applications in food science and in biomedicine, allowing the rapid determination of hidden biomarkers by a cutting edge diagnostic strategy. Graphical abstract The topography of iron(III) sites on surface active maghemite nanoparticles (SAMNs) allows the recognition of sterically compatible carboxylic groups on proteins and peptides in complex biological matrixes. The analysis of hard protein corona on SAMNs led to the

  16. 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

  17. Intact molecular characterization of cord factor (trehalose 6,6'-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Fujita, Yukiko; Naka, Takashi; McNeil, Michael R; Yano, Ikuya

    2005-10-01

    Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 microg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with alpha-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of alpha-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the

  18. 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.

  19. Clarification of the Antagonistic Effect of the Lipopeptides Produced by Bacillus amyloliquefaciens BPD1 against Pyricularia oryzae via In Situ MALDI-TOF IMS Analysis

    Directory of Open Access Journals (Sweden)

    Jen-Hung Liao

    2016-12-01

    Full Text Available This study tried to clarify the antagonistic effect of the lipopeptides secreted by Bacillus amyloliquefaciens strain BPD1 (Ba-BPD1 against Pyricularia oryzae Cavara (PO. To determine the major antifungal lipopeptides effective against PO, single and dual cultures were carried out in solid-state media. The matrix-assisted laser desorption/ionization–time of flight imaging mass spectrometry (MALDI-TOF IMS was used to identify the most effective lipopeptide in situ. Meanwhile, the morphology of pathogen fungi treated with lipopeptides was observed via the SEM. Of the three lipopeptide families, surfactin, iturin, and fengycin, the last was identified as the most effective for inhibiting mycelium growth and conidial germination of PO. The conidia and hyphae of fengycin-treated PO were shown to become deformed and tumorous under exposure. This study provides insights into the antagonistic effect of Ba-BPD1 against fungal phytopathogens. Such insights are helpful in the development of reagents for biological control applications.

  20. 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

  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. Fish proteins as targets of ferrous-catalyzed oxidation: identification of protein carbonyls by fluorescent labeling on two-dimensional gels and MALDI-TOF/TOF mass spectrometry.

    Science.gov (United States)

    Pazos, Manuel; da Rocha, Angela Pereira; Roepstorff, Peter; Rogowska-Wrzesinska, Adelina

    2011-07-27

    Protein oxidation in fish meat is considered to affect negatively the muscle texture. An important source of free radicals taking part in this process is Fenton's reaction dependent on ferrous ions present in the tissue. The aim of this study was to investigate the susceptibility of cod muscle proteins in sarcoplasmic and myofibril fractions to in vitro metal-catalyzed oxidation and to point out protein candidates that might play a major role in the deterioration of fish quality. Extracted control proteins and proteins subjected to free radicals generated by Fe(II)/ascorbate mixture were labeled with fluorescein-5-thiosemicarbazide (FTSC) to tag carbonyl groups and separated by two-dimensional gel electrophoresis. Consecutive visualization of protein carbonyl levels by capturing the FTSC signal and total protein levels by capturing the SyproRuby staining signal allowed us to quantify the relative change in protein carbonyl levels corrected for changes in protein content. Proteins were identified using MALDI-TOF/TOF mass spectrometry and homology-based searches. The results show that freshly extracted cod muscle proteins exhibit a detectable carbonylation background and that the incubation with Fe(II)/ascorbate triggers a further oxidation of both sarcoplasmic and myofibril proteins. Different proteins exhibited various degrees of sensitivity to oxidation processes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), nucleoside diphosphate kinase B (NDK), triosephosphate isomerase, phosphoglycerate mutase, lactate dehydrogenase, creatine kinase, and enolase were the sarcoplasmic proteins most vulnerable to ferrous-catalyzed oxidation. Moreover, NDK, phosphoglycerate mutase, and GAPDH were identified in several spots differing by their pI, and those forms showed different susceptibilities to metal-catalyzed oxidation, indicating that post-translational modifications may change the resistance of proteins to oxidative damage. The Fe(II)/ascorbate treatment significantly

  3. MALDI-TOF mass spectrometry analysis of small molecular weight compounds (under 10 KDa) as biomarkers of rat hearts undergoing arecoline challenge.

    Science.gov (United States)

    Chen, Tung-Sheng; Chang, Mu-Hsin; Kuo, Wei-Wen; Lin, Yueh-Min; Yeh, Yu-Lan; Day, Cecilia Hsuan; Lin, Chien-Chung; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

    2013-04-01

    Statistical and clinical reports indicate that betel nut chewing is strongly associated with progression of oral cancer because some ingredients in betel nuts are potential cancer promoters, especially arecoline. Early diagnosis for cancer biomarkers is the best strategy for prevention of cancer progression. Several methods are suggested for investigating cancer biomarkers. Among these methods, gel-based proteomics approach is the most powerful and recommended tool for investigating biomarkers due to its high-throughput. However, this proteomics approach is not suitable for screening biomarkers with molecular weight under 10 KDa because of the characteristics of gel electrophoresis. This study investigated biomarkers with molecular weight under 10 KDa in rats with arecoline challenge. The centrifuging vials with membrane (10 KDa molecular weight cut-off) played a crucial role in this study. After centrifuging, the filtrate (containing compounds with molecular weight under 10 KDa) was collected and spotted on a sample plate for MALDI-TOF mass spectrometry analysis. Compared to control, three extra peaks (m/z values were 1553.1611, 1668.2097 and 1740.1832, respectively) were found in sera and two extra peaks were found in heart tissue samples (408.9719 and 524.9961, respectively). These small compounds should play important roles and may be potential biomarker candidates in rats with arecoline. This study successfully reports a mass-based method for investigating biomarker candidates with small molecular weight in different types of sample (including serum and tissue). In addition, this reported method is more time-efficient (1 working day) than gel-based proteomics approach (5~7 working days).

  4. Performance assessment of two lysis methods for direct identification of yeasts from clinical blood cultures using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Jeddi, Fakhri; Yapo-Kouadio, Gisèle Cha; Normand, Anne-Cécile; Cassagne, Carole; Marty, Pierre; Piarroux, Renaud

    2017-02-01

    In cases of fungal infection of the bloodstream, rapid species identification is crucial to provide adapted therapy and thereby ameliorate patient outcome. Currently, the commercial Sepsityper kit and the sodium-dodecyl sulfate (SDS) method coupled with MALDI-TOF mass spectrometry are the most commonly reported lysis protocols for direct identification of fungi from positive blood culture vials. However, the performance of these two protocols has never been compared on clinical samples. Accordingly, we performed a two-step survey on two distinct panels of clinical positive blood culture vials to identify the most efficient protocol, establish an appropriate log score (LS) cut-off, and validate the best method. We first compared the performance of the Sepsityper and the SDS protocols on 71 clinical samples. For 69 monomicrobial samples, mass spectrometry LS values were significantly higher with the SDS protocol than with the Sepsityper method (P < .0001), especially when the best score of four deposited spots was considered. Next, we established the LS cut-off for accurate identification at 1.7, based on specimen DNA sequence data. Using this LS cut-off, 66 (95.6%) and 46 (66.6%) isolates were correctly identified at the species level with the SDS and the Sepsityper protocols, respectively. In the second arm of the survey, we validated the SDS protocol on an additional panel of 94 clinical samples. Ninety-two (98.9%) of 93 monomicrobial samples were correctly identified at the species level (median LS = 2.061). Overall, our data suggest that the SDS method yields more accurate species identification of yeasts, than the Sepsityper protocol. © 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.

  5. 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

  6. Rapid identification and antimicrobial susceptibility testing of positive blood cultures using MALDI-TOF MS and a modification of the standardised disc diffusion test: a pilot study.

    LENUS (Irish Health Repository)

    Fitzgerald, C

    2016-04-27

    In an era when clinical microbiology laboratories are under increasing financial pressure, there is a need for inexpensive, yet effective, rapid microbiology tests. The aim of this study was to evaluate a novel modification of standard methodology for the identification and antimicrobial susceptibility testing (AST) of pathogens in positive blood cultures, reducing the turnaround time of laboratory results by 24 h.

  7. Identification of immature stages of phlebotomine sand flies using MALDI-TOF MS and mapping of mass spectra during sand fly life cycle

    Czech Academy of Sciences Publication Activity Database

    Halada, Petr; Hlaváčková, K.; Dvořák, V.; Volf, P.

    2018-01-01

    Roč. 93, FEB 2018 (2018), s. 47-56 ISSN 0965-1748 R&D Projects: GA ČR(CZ) GA15-04329S Institutional support: RVO:61388971 Keywords : Phlebotomine sand flies * Larvae * Pupae Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.756, year: 2016

  8. Finding diabetic nephropathy biomarkers in the plasma peptidome by high-throughput magnetic bead processing and MALDI-TOF-MS analysis

    DEFF Research Database (Denmark)

    Hansen, Henning G; Overgaard, Julie; Lajer, Maria

    2010-01-01

    Diabetic nephropathy (DN) is the most common cause of end-stage renal disease and improved biomarkers would help identify high-risk individuals. The aim of this study was to discover candidate biomarkers for DN in the plasma peptidome in an in-house cross-sectional cohort (n=122) of type 1 diabet...

  9. 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

  10. Sodiation as a tool for enhancing the diagnostic value of MALDI-TOF/TOF-MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

    NARCIS (Netherlands)

    Weesepoel, Y.J.A.; Vincken, J.P.; Pop, R.M.; Liu, K.; Gruppen, H.

    2013-01-01

    The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono- and diesters. For rapid fingerprinting of these esters, matrix-assisted laser desorption ionization time of flight

  11. MALDI-TOF MS applied to apoC-III glycoforms of patients with congenital disorders affecting O-glycosylation. Comparison with two-dimensional electrophoresis

    NARCIS (Netherlands)

    Yen-Nicolay, S.; Boursier, C.; Rio, M. del; Lefeber, D.J.; Pilon, A.; Seta, N.; Bruneel, A.

    2015-01-01

    PURPOSE: The O-glycan abnormalities accompanying some congenital disorders of glycosylation, namely conserved oligomeric Golgi-congenital disorders of glycosylation (COG-CDGs) and ATP6V0A2-CDGs, are mainly detected using electrophoresis methods applied to circulating apolipoprotein C-III. The

  12. Combination of capillary isoelectric focusing in a tapered capillary with MALDI-TOF MS for rapid and reliable identification of Dickeya species from plant samples

    Czech Academy of Sciences Publication Activity Database

    Horká, Marie; Šalplachta, Jiří; Karásek, Pavel; Kubesová, Anna; Horký, J.; Matoušková, H.; Šlais, Karel; Roth, Michal

    2013-01-01

    Roč. 85, č. 14 (2013), s. 6806-6812 ISSN 0003-2700 R&D Projects: GA MV VG20112015021; GA ČR(CZ) GAP106/12/0522 Institutional support: RVO:68081715 Keywords : capillary isoelectric focusing * matrix-assisted laser desorption/ionization time-of-flight mass spectrometry * Dickeya spp. bacteria Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 5.825, year: 2013

  13. MALDI-TOF MS analysis of the self-termination products in the anionic methyl methacrylate/tert-butyl acrylate block copolymerization

    Czech Academy of Sciences Publication Activity Database

    Vlček, Petr; Čadová, Eva; Horský, Jiří; Janata, Miroslav

    2015-01-01

    Roč. 72, č. 9 (2015), s. 2227-2239 ISSN 0170-0839 Institutional support: RVO:61389013 Keywords : anionic polymerization * acrylates * block copolymer Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.371, year: 2015

  14. Prediction of Streptococcus uberis clinical mastitis risk using Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) in dairy herds.

    Science.gov (United States)

    Archer, Simon C; Bradley, Andrew J; Cooper, Selin; Davies, Peers L; Green, Martin J

    2017-09-01

    The purpose of this study was to evaluate whether the risk of Streptococcus uberis clinical mastitis at cow level could be predicted from the historical presence of specific strains of S. uberis on dairy farms. Matrix-assisted laser desorption ionization time of flight mass spectrometry was used to identify S. uberis isolates potentially capable of contagious transmission. Data were available from 10,652 cows from 52 English and Welsh dairy farms over a 14 month period, and 521 isolates of S. uberis from clinical mastitis cases were available for analysis. As well as the temporal herd history of clinical mastitis associated with particular S. uberis strains, other exposure variables included cow parity, stage of lactation, milk yield, and somatic cell count. Observations were structured longitudinally as repeated weekly measures through the study period for each cow. Data were analyzed in a Bayesian framework using multilevel logistic regression models. Similarity of mass spectral profiles between isolates of S. uberis from consecutive clinical cases of mastitis in herds was used to indicate potential for contagious phenotypic characteristics. Cross validation showed that new isolates with these characteristics could be identified with an accuracy of 90% based on bacterial protein mass spectral characteristics alone. The cow-level risk in any week of these S. uberis clinical mastitis cases increased with the presence of the same specific strains of S. uberis in other cows in the herd during the previous 2 weeks. The final statistical model indicated there would be a 2-3 fold increase in the risk of S. uberis clinical mastitis associated with particular strains if these occurred in the herd 1 and 2 weeks previously. The results suggest that specific strains of S. uberis may be involved with contagious transmission, and predictions based on their occurrence could be used as an early warning surveillance system to enhance the control of S. uberis mastitis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Efficient identification of Malassezia yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)

    NARCIS (Netherlands)

    Kolecka, A; Khayhan, K; Arabatzis, M; Velegraki, A; Kostrzewa, M; Andersson, A; Scheynius, A; Cafarchia, C; Iatta, R; Montagna, M T; Youngchim, S; Cabañes, F J; Hoopman, P; Kraak, B; Groenewald, M; Boekhout, T

    BACKGROUND: Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in

  16. Phylogenomic and MALDI-TOF MS analysis of Streptococcus sinensis HKU4T reveals a distinct phylogenetic clade in the genus Streptococcus.

    Science.gov (United States)

    Teng, Jade L L; Huang, Yi; Tse, Herman; Chen, Jonathan H K; Tang, Ying; Lau, Susanna K P; Woo, Patrick C Y

    2014-10-20

    Streptococcus sinensis is a recently discovered human pathogen isolated from blood cultures of patients with infective endocarditis. Its phylogenetic position, as well as those of its closely related species, remains inconclusive when single genes were used for phylogenetic analysis. For example, S. sinensis branched out from members of the anginosus, mitis, and sanguinis groups in the 16S ribosomal RNA gene phylogenetic tree, but it was clustered with members of the anginosus and sanguinis groups when groEL gene sequences used for analysis. In this study, we sequenced the draft genome of S. sinensis and used a polyphasic approach, including concatenated genes, whole genomes, and matrix-assisted laser desorption ionization-time of flight mass spectrometry to analyze the phylogeny of S. sinensis. The size of the S. sinensis draft genome is 2.06 Mb, with GC content of 42.2%. Phylogenetic analysis using 50 concatenated genes or whole genomes revealed that S. sinensis formed a distinct cluster with Streptococcus oligofermentans and Streptococcus cristatus, and these three streptococci were clustered with the "sanguinis group." As for phylogenetic analysis using hierarchical cluster analysis of the mass spectra of streptococci, S. sinensis also formed a distinct cluster with S. oligofermentans and S. cristatus, but these three streptococci were clustered with the "mitis group." On the basis of the findings, we propose a novel group, named "sinensis group," to include S. sinensis, S. oligofermentans, and S. cristatus, in the Streptococcus genus. Our study also illustrates the power of phylogenomic analyses for resolving ambiguities in bacterial taxonomy. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  17. 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...

  18. 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.

  19. Validation of High Resolution Melting Analysis (HRM of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification.

    Directory of Open Access Journals (Sweden)

    Hans Duyvejonck

    Full Text Available Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method, by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method to establish the presence of Candida species in three different types of clinical samples.A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by NucliSENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF.For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample.Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.

  20. 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.

  1. 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.

  2. Assessment of four protocols for rapid bacterial identification from positive blood culture pellets by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Vitek® MS).

    Science.gov (United States)

    Thomin, Jean; Aubin, Guillaume Ghislain; Foubert, Fabrice; Corvec, Stéphane

    2015-08-01

    In this study, we developed and compared four protocols to prepare a bacterial pellet from 944 positive blood cultures for direct MALDI-TOF mass spectrometry Vitek® MS analysis. Protocol 4, tested on 200 monomicrobial samples, allowed 83% of bacterial identification. This easy, fast, cheap and accurate method is promising in daily practice, especially to limit broad range antibiotic treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Methodology to detect and quantify the presence of recycled PET in bottle-grade PET blends: mass spectrometry (MALDI-TOF) and X-ray fluorescence

    International Nuclear Information System (INIS)

    Romao, Wanderson; Franco, Marcos F.; Gozzo, Fabio C.; Iglesias, Amadeu H.; Sanvido, Gustavo B.; Eberlin, Marcos N.; Bueno, Maria I.M.S.; Maretto, Danilo A.; Poppi, Ronei J.; Paoli, Marco-Aurelio de

    2009-01-01

    New methodologies were developed to detect and to quantify the presence of the bottle-grade post-consumption PET (PET pc -btg) in the bottle-grade virgin PET (PET v -btg), preventing frauds and illegal uses of recycled PET pc -btg. MALDI-MS results together with PCA (principal component analysis) was used to classify the samples into several groups: intrinsic viscosity changes; processed and not submitted to some industrial process; wt % PET pc -btg in the PET v -btg; synthesis process change (manufacturer). From these results, it was possible to create a calibration model, that differentiated between PET v -btg and PET pc -btg resins. XRF results show that some manufacturers use one or more catalysts for PET v -btg synthesis, where our prediction model is valid only when the studied resin is known. We observed also that the Fe concentration in PET increase in as a function of the recycling process. Therefore, this variable could be used, in the future work, to create chemometric models including a higher number of variables. (author)

  4. 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.

  5. The solubilisation of boar sperm membranes by different detergents - a microscopic, MALDI-TOF MS, 31P NMR and PAGE study on membrane lysis, extraction efficiency, lipid and protein composition

    Directory of Open Access Journals (Sweden)

    Müller Karin

    2009-11-01

    Full Text Available Abstract Background Detergents are often used to isolate proteins, lipids as well as "detergent-resistant membrane domains" (DRMs from cells. Different detergents affect different membrane structures according to their physico-chemical properties. However, the effects of different detergents on membrane lysis of boar spermatozoa and the lipid composition of DRMs prepared from the affected sperm membranes have not been investigated so far. Results Spermatozoa were treated with the selected detergents Pluronic F-127, sodium cholate, CHAPS, Tween 20, Triton X-100 and Brij 96V. Different patterns of membrane disintegration were observed by light and electron microscopy. In accordance with microscopic data, different amounts of lipids and proteins were released from the cells by the different detergents. The biochemical methods to assay the phosphorus and cholesterol contents as well as 31P NMR to determine the phospholipids were not influenced by the presence of detergents since comparable amounts of lipids were detected in the organic extracts from whole cell suspensions after exposure to each detergent. However, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry applied to identify phospholipids was essentially disturbed by the presence of detergents which exerted particular suppression effects on signal intensities. After separation of the membrane fractions released by detergents on a sucrose gradient only Triton X-100 and sodium cholate produced sharp turbid DRM bands. Only membrane solubilisation by Triton X-100 leads to an enrichment of cholesterol, sphingomyelin, phosphatidylinositol and phosphatidylethanolamine in a visible DRM band accompanied by a selective accumulation of proteins. Conclusion The boar sperm membranes are solubilised to a different extent by the used detergents. Particularly, the very unique DRMs isolated after Triton X-100 exposure are interesting candidates for further studies regarding the architecture of sperm.

  6. OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay

    NARCIS (Netherlands)

    Paauw, A.; Trip, H.; Niemcewicz, M.; Sellek, R.; Heng, J.M.E.; Mars-Groenendijk, R.H.; Jong, A.L. de; Majchrzykiewicz-Koehorst, J.A.; Olsen, J.S.; Tsivtsivadze, E.

    2014-01-01

    Background Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an

  7. Rapid Detection and Identification of Candidemia by Direct Blood Culturing on Solid Medium by Use of Lysis-Centrifugation Method Combined with Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

    Science.gov (United States)

    Idelevich, Evgeny A.; Grünastel, Barbara

    2016-01-01

    ABSTRACT Candida sepsis is a life-threatening condition with increasing prevalence. In this study, direct blood culturing on solid medium using a lysis-centrifugation procedure enabled successful Candida species identification by matrix-assisted laser desorption–ionization time of flight mass spectrometry on average 3.8 h (Sabouraud agar) or 7.4 h (chocolate agar) before the positivity signal for control samples in Bactec mycosis-IC/F or Bactec Plus aerobic/F bottles, respectively. Direct culturing on solid medium accelerated candidemia diagnostics compared to that with automated broth-based systems. PMID:27795344

  8. Rapid Detection and Identification of Candidemia by Direct Blood Culturing on Solid Medium by Use of Lysis-Centrifugation Method Combined with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Idelevich, Evgeny A; Grünastel, Barbara; Becker, Karsten

    2017-01-01

    Candida sepsis is a life-threatening condition with increasing prevalence. In this study, direct blood culturing on solid medium using a lysis-centrifugation procedure enabled successful Candida species identification by matrix-assisted laser desorption-ionization time of flight mass spectrometry on average 3.8 h (Sabouraud agar) or 7.4 h (chocolate agar) before the positivity signal for control samples in Bactec mycosis-IC/F or Bactec Plus aerobic/F bottles, respectively. Direct culturing on solid medium accelerated candidemia diagnostics compared to that with automated broth-based systems. Copyright © 2016 American Society for Microbiology.

  9. 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/.

  10. 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.

  11. Emerging and Future Applications of Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry in the Clinical Microbiology Laboratory: A Report of the Association for Molecular Pathology.

    Science.gov (United States)

    Doern, Christopher D; Butler-Wu, Susan M

    2016-11-01

    The performance of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) for routine bacterial and yeast identification as well as direct-from-blood culture bottle identification has been thoroughly evaluated in the peer-reviewed literature. Microbiologists are now moving beyond these methods to apply MS to other areas of the diagnostic process. This review discusses the emergence of advanced matrix-assisted laser desorption ionization time-of-flight MS applications, including the identification of filamentous fungi and mycobacteria and the current and future state of antimicrobial resistance testing. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  12. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system.

    Directory of Open Access Journals (Sweden)

    Alexandra Machen

    Full Text Available Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS. After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012 category agreement of antimicrobials tested, with 3.6% (36/1012 minor error, 1.7% (7/1012 major error, and 1.3% (13/1012 very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001. Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.

  13. Same Day Identification and Full Panel Antimicrobial Susceptibility Testing of Bacteria from Positive Blood Culture Bottles Made Possible by a Combined Lysis-Filtration Method with MALDI-TOF VITEK Mass Spectrometry and the VITEK2 System

    Science.gov (United States)

    Machen, Alexandra; Drake, Tim; Wang, Yun F. (Wayne)

    2014-01-01

    Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (p<0.00001). Thus, the same-day results of microorganism identification and antimicrobial susceptibility testing directly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship. PMID:24551067

  14. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system.

    Science.gov (United States)

    Machen, Alexandra; Drake, Tim; Wang, Yun F Wayne

    2014-01-01

    Rapid identification and antimicrobial susceptibility testing of microorganisms causing bloodstream infections or sepsis have the potential to improve patient care. This proof-of-principle study evaluates the Lysis-Filtration Method for identification as well as antimicrobial susceptibility testing of bacteria directly from positive blood culture bottles in a clinical setting. A total of 100 non-duplicated positive blood cultures were tested and 1012 microorganism-antimicrobial combinations were assessed. An aliquot of non-charcoal blood culture broth was incubated with lysis buffer briefly before being filtered and washed. Microorganisms recovered from the filter membrane were first identified by using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight VITEK® Mass Spectrometry (VITEK MS). After quick identification from VITEK MS, filtered microorganisms were inoculated to VITEK®2 system for full panel antimicrobial susceptibility testing analysis. Of 100 bottles tested, the VITEK MS resulted in 94.0% correct organism identification to the species level. Compared to the conventional antimicrobial susceptibility testing methods, direct antimicrobial susceptibility testing from VITEK®2 resulted in 93.5% (946/1012) category agreement of antimicrobials tested, with 3.6% (36/1012) minor error, 1.7% (7/1012) major error, and 1.3% (13/1012) very major error of antimicrobials. The average time to identification and antimicrobial susceptibility testing was 11.4 hours by using the Lysis-Filtration method for both VITEK MS and VITEK®2 compared to 56.3 hours by using conventional methods (pdirectly from positive blood culture can be achieved and can be used for appropriate antibiotic therapy and antibiotic stewardship.

  15. 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.

  16. 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.

  17. Identification of Leishmania by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry Using a Free Web-Based Application and a Dedicated Mass-Spectral Library.

    Science.gov (United States)

    Lachaud, Laurence; Fernández-Arévalo, Anna; Normand, Anne-Cécile; Lami, Patrick; Nabet, Cécile; Donnadieu, Jean Luc; Piarroux, Martine; Djenad, Farid; Cassagne, Carole; Ravel, Christophe; Tebar, Silvia; Llovet, Teresa; Blanchet, Denis; Demar, Magalie; Harrat, Zoubir; Aoun, Karim; Bastien, Patrick; Muñoz, Carmen; Gállego, Montserrat; Piarroux, Renaud

    2017-10-01

    Human leishmaniases are widespread diseases with different clinical forms caused by about 20 species within the Leishmania genus. Leishmania species identification is relevant for therapeutic management and prognosis, especially for cutaneous and mucocutaneous forms. Several methods are available to identify Leishmania species from culture, but they have not been standardized for the majority of the currently described species, with the exception of multilocus enzyme electrophoresis. Moreover, these techniques are expensive, time-consuming, and not available in all laboratories. Within the last decade, mass spectrometry (MS) has been adapted for the identification of microorganisms, including Leishmania However, no commercial reference mass-spectral database is available. In this study, a reference mass-spectral library (MSL) for Leishmania isolates, accessible through a free Web-based application (mass-spectral identification [MSI]), was constructed and tested. It includes mass-spectral data for 33 different Leishmania species, including species that infect humans, animals, and phlebotomine vectors. Four laboratories on two continents evaluated the performance of MSI using 268 samples, 231 of which were Leishmania strains. All Leishmania strains, but one, were correctly identified at least to the complex level. A risk of species misidentification within the Leishmania donovani , L. guyanensis , and L. braziliensis complexes was observed, as previously reported for other techniques. The tested application was reliable, with identification results being comparable to those obtained with reference methods but with a more favorable cost-efficiency ratio. This free online identification system relies on a scalable database and can be implemented directly in users' computers. Copyright © 2017 American Society for Microbiology.

  18. Identification of some chemical constituents of Indigofera hirsuta Linn. (Fabaceae) by HPLC-ESI-MS (TOF) and evaluation of the anti radical activity; Identificacao de alguns constituintes quimicos de Indigofera hirsuta Linn. (Fabaceae) por de alguns constituintes quimicos de Indigofera hirsuta Linn. (Fabaceae) por CLAE-IES-EM (TOF) e avaliacao da atividade antirradicalar

    Energy Technology Data Exchange (ETDEWEB)

    Moura, Adriana Candido da Silva; Vilegas, Wagner; Santos, Lourdes Campaner dos [Universidade Estadual Paulista (UNESP), Araraquara, SP (Brazil). Inst. de Quimica.Dept. de Quimica Organica

    2011-07-01

    A rapid analytical approach, suitable to characterize the compounds present in the aqueous and methanol extracts prepared from the aerial parts of Indigofera hirsute, was developed. The method based on high-performance liquid chromatography coupled to mass spectrometry, electrospray positive ionization and detection by time of flight (HPLC-ESI-MS-TOF) identified, tryptophan, uracil, rutin, kempferol-3-O{beta}{sub -}-D-glucopyranoside, gallic acid and methyl gallate. The antiradical activity of this extract was evaluated using DPPH assay, with gallic acid as antiradical pattern. The study revealed the antiradical activity of methyl galatte (EC{sub 50} = 5 {+-} 0.3 {mu}g mL{sup -1}) gallic acid (EC{sub 50} = 5 {+-} 0.2 {mu}g mL{sup -1}) and rutin (EC{sub 50} = 21.6 {+-} 0.6 {mu}g m L{sup -1}), isolated from methanol extract (EC{sub 50} = 67.7 {+-} 0.9 {mu}g mL{sup -1}), which showed strong antiradical activity. (author)

  19. Tyrosine residues modification studied by MALDI-TOF mass spectrometry

    International Nuclear Information System (INIS)

    Santrucek, Jiri; Strohalm, Martin; Kadlcik, Vojtech; Hynek, Radovan; Kodicek, Milan

    2004-01-01

    Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding

  20. Evaluation of VITEK mass spectrometry (MS), a matrix-assisted laser desorption ionization time-of-flight MS system for identification of anaerobic bacteria.

    Science.gov (United States)

    Lee, Wonmok; Kim, Myungsook; Yong, Dongeun; Jeong, Seok Hoon; Lee, Kyungwon; Chong, Yunsop

    2015-01-01

    By conventional methods, the identification of anaerobic bacteria is more time consuming and requires more expertise than the identification of aerobic bacteria. Although the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems are relatively less studied, they have been reported to be a promising method for the identification of anaerobes. We evaluated the performance of the VITEK MS in vitro diagnostic (IVD; 1.1 database; bioMérieux, France) in the identification of anaerobes. We used 274 anaerobic bacteria isolated from various clinical specimens. The results for the identification of the bacteria by VITEK MS were compared to those obtained by phenotypic methods and 16S rRNA gene sequencing. Among the 249 isolates included in the IVD database, the VITEK MS correctly identified 209 (83.9%) isolates to the species level and an additional 18 (7.2%) at the genus level. In particular, the VITEK MS correctly identified clinically relevant and frequently isolated anaerobic bacteria to the species level. The remaining 22 isolates (8.8%) were either not identified or misidentified. The VITEK MS could not identify the 25 isolates absent from the IVD database to the species level. The VITEK MS showed reliable identifications for clinically relevant anaerobic bacteria.

  1. Identification of clinical yeasts by Vitek MS system compared with API ID 32 C.

    Science.gov (United States)

    Durán-Valle, M Teresa; Sanz-Rodríguez, Nuria; Muñoz-Paraíso, Carmen; Almagro-Moltó, María; Gómez-Garcés, José Luis

    2014-05-01

    We performed a clinical evaluation of the Vitek MS matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) system with the commercial database version 2.0 for rapid identification of medically important yeasts as compared with the conventional phenotypic method API ID 32 C. We tested 161 clinical isolates, nine isolates from culture collections and five reference strains. In case of discrepant results or no identification with one or both methods, molecular identification techniques were employed. Concordance between both methods was observed with 160/175 isolates (91.42%) and misidentifications by both systems occurred only when taxa were not included in the respective databases, i.e., one isolate of Candida etchellsii was identified as C. globosa by Vitek MS and two isolates of C. orthopsilosis were identified as C. parapsilosis by API ID 32 C. Vitek MS could not identify nine strains (5.14%) and API ID 32 C did not identify 13 (7.42%). Vitek MS was more reliable than API ID 32 C and reduced the time required for the identification of clinical isolates to only a few minutes.

  2. 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

  3. 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

  4. Performance of two resin-containing blood culture media in detection of bloodstream infections and in direct matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) broth assays for isolate identification: clinical comparison of the BacT/Alert Plus and Bactec Plus systems.

    Science.gov (United States)

    Fiori, Barbara; D'Inzeo, Tiziana; Di Florio, Viviana; De Maio, Flavio; De Angelis, Giulia; Giaquinto, Alessia; Campana, Lara; Tanzarella, Eloisa; Tumbarello, Mario; Antonelli, Massimo; Sanguinetti, Maurizio; Spanu, Teresa

    2014-10-01

    We compared the clinical performances of the BacT/Alert Plus (bioMérieux) and Bactec Plus (Becton Dickinson) aerobic and anaerobic blood culture (BC) media with adsorbent polymeric beads. Patients ≥ 16 years old with suspected bloodstream infections (BSIs) were enrolled in intensive care units and infectious disease wards. A single 40-ml blood sample was collected from each and used to inoculate (10 ml/bottle) one set of BacT/Alert Plus cultures and one set of Bactec Plus cultures, each set consisting of one aerobic and one anaerobic bottle. Cultures were incubated ≤ 5 days in the BacT/Alert 3D and Bactec FX instruments, respectively. A total of 128 unique BSI episodes were identified based on the recovery of clinically significant growth in 212 aerobic cultures (106 BacT/Alert and 106 Bactec) and 151 anaerobic cultures (82 BacT/Alert and 69 Bactec). The BacT/Alert aerobic medium had higher recovery rates for Gram-positive cocci (P = 0.024), whereas the Bactec aerobic medium was superior for recovery of Gram-negative bacilli (P = 0.006). BacT/Alert anaerobic medium recovery rates exceeded those of the Bactec anaerobic medium for total organisms (P = 0.003), Gram-positive cocci (P = 0.013), and Escherichia coli (P = 0.030). In terms of capacity for diagnosing the 128 septic episodes, the BacT/Alert and Bactec sets were comparable, although the former sets diagnosed more BSIs caused by Gram-positive cocci (P = 0.008). They also allowed earlier identification of coagulase-negative staphylococcal growth (mean, 2.8 h; P = 0.003) and growth in samples from patients not on antimicrobial therapy that yielded positive results (mean, 1.3 h; P direct matrix-assisted laser desorption ionization-time of flight mass spectrometry assay of BC broths. The BacT/Alert Plus media line appears to be a reliable, timesaving tool for routine detection of BSIs in the population we studied, although further studies are needed to evaluate their performance in other settings. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. 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.

  6. 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...

  7. 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.

  8. 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.

  9. Multicenter study evaluating the Vitek MS system for identification of medically important yeasts.

    Science.gov (United States)

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

    2013-07-01

    The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

  10. Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system.

    Science.gov (United States)

    Garner, O; Mochon, A; Branda, J; Burnham, C-A; Bythrow, M; Ferraro, M; Ginocchio, C; Jennemann, R; Manji, R; Procop, G W; Richter, S; Rychert, J; Sercia, L; Westblade, L; Lewinski, M

    2014-04-01

    Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  11. 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.

  12. Multicenter Evaluation of the Vitek MS v3.0 System for the Identification of Filamentous Fungi.

    Science.gov (United States)

    Rychert, Jenna; Slechta, E Sue; Barker, Adam P; Miranda, Edwin; Babady, N Esther; Tang, Yi-Wei; Gibas, Connie; Wiederhold, Nathan; Sutton, DeAnna; Hanson, Kimberly E

    2018-02-01

    Invasive fungal infections are an important cause of morbidity and mortality affecting primarily immunocompromised patients. While fungal identification to the species level is critical to providing appropriate therapy, it can be slow and laborious and often relies on subjective morphological criteria. The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has the potential to speed up and improve the accuracy of identification. In this multicenter study, we evaluated the accuracy of the Vitek MS v3.0 system in identifying 1,601 clinical mold isolates compared to identification by DNA sequence analysis and supported by morphological and phenotypic testing. Among the 1,519 isolates representing organisms in the v3.0 database, 91% ( n = 1,387) were correctly identified to the species level. An additional 27 isolates (2%) were correctly identified to the genus level. Fifteen isolates were incorrectly identified, due to either a single incorrect identification ( n = 13) or multiple identifications from different genera ( n = 2). In those cases, when a single identification was provided that was not correct, the misidentification was within the same genus. The Vitek MS v3.0 was unable to identify 91 (6%) isolates, despite repeat testing. These isolates were distributed among all the genera. When considering all isolates tested, even those that were not represented in the database, the Vitek MS v3.0 provided a single correct identification 98% of the time. These findings demonstrate that the Vitek MS v3.0 system is highly accurate for the identification of common molds encountered in the clinical mycology laboratory. Copyright © 2018 American Society for Microbiology.

  13. Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

    Science.gov (United States)

    Richter, S S; Sercia, L; Branda, J A; Burnham, C-A D; Bythrow, M; Ferraro, M J; Garner, O B; Ginocchio, C C; Jennemann, R; Lewinski, M A; Manji, R; Mochon, A B; Rychert, J A; Westblade, L F; Procop, G W

    2013-12-01

    This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l'Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer's instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7% of the 965 isolates tested, with 83.8% correct to the species level and 12.8% limited to a genus-level identification. There was no identification for 1.7% of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

  14. 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.

  15. Pediatric MS

    Science.gov (United States)

    ... Pediatric MS Share this page Facebook Twitter Email Pediatric MS Pediatric MS Pediatric MS Support Pediatric Providers ... system through the Pediatric MS Support Group . Treating pediatric MS In 2018 the U.S. Food and Drug ...

  16. Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues† †Electronic supplementary information (ESI) available: Synthesis of TEG–BA, Gluen–BA, mGluen–BA and Gluen–Ph; 1H NMR, 13C NMR, MALDI-TOF MS, electronic absorption, and CD spectra; zeta potential distributions; SLS plots; DLS histograms; and related experimental procedures. See DOI: 10.1039/c5sc00524h Click here for additional data file.

    Science.gov (United States)

    Mogaki, Rina

    2015-01-01

    Water-soluble bioadhesive polymers bearing multiple guanidinium ion (Gu+) pendants at their side-chain termini (Gluen–BA, n = 10 and 29) that were conjugated with benzamidine (BA) as a trypsin inhibitor were developed. The Gluen–BA molecules are supposed to adhere to oxyanionic regions of the trypsin surface, even in buffer, via a multivalent Gu+/oxyanion salt-bridge interaction, such that their BA group properly blocks the substrate-binding site. In fact, Glue10–BA and Glue29–BA exhibited 35- and 200-fold higher affinities for trypsin, respectively, than a BA derivative without the glue moiety (TEG–BA). Most importantly, Glue10–BA inhibited the protease activity of trypsin 13-fold more than TEG–BA. In sharp contrast, mGlue27–BA, which bears 27 Gu+ units along the main chain and has a 5-fold higher affinity than TEG–BA for trypsin, was inferior even to TEG–BA for trypsin inhibition. PMID:28706668

  17. Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

    Science.gov (United States)

    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; Westblade, Lars F.; Ferraro, Mary Jane; Branda, John A.

    2013-01-01

    Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting. PMID:23658261

  18. Multicenter evaluation of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Gram-positive aerobic bacteria.

    Science.gov (United States)

    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; Westblade, Lars F; Ferraro, Mary Jane; Branda, John A

    2013-07-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

  19. Identification of uncommon oral yeasts from cancer patients by MALDI-TOF mass spectrometry

    NARCIS (Netherlands)

    Aslani, Narges; Janbabaei, Ghasem; Abastabar, Mahdi; Meis, Jacques F.; Babaeian, Mahasti; Khodavaisy, Sadegh; Boekhout, Teun; Badali, Hamid

    2018-01-01

    BACKGROUND: Opportunistic infections due to Candida species occur frequently in cancer patients because of their inherent immunosuppression. The aim of the present study was to investigate the epidemiology of yeast species from the oral cavity of patients during treatment for oncological and

  20. A high throughput screening assay for identifying glycation inhibitors on MALDI-TOF target.

    Science.gov (United States)

    Zhang, Qiuting; Tu, Zongcai; Wang, Hui; Fan, Liangliang; Huang, Xiaoqin; Xiao, Hui

    2015-03-01

    The Maillard reaction plays an important role in the food industry, however, the deleterious effects generated by the advanced glycation end-products (AGEs) have been well recognized. Many efforts have been made to seek new AGE inhibitors, in particular those natural ones without adverse effect. We have developed a rapid, mass spectrometry based, on-plate screening assay for novel AGE inhibitors. The glycation reaction, inhibition feedback as well as the subsequent MALDI mass spectrometric analysis occurred on one single MALDI plate. At 1:10 M ratio of peptide to sugar, as little as 4h incubation time allowed the screening test to be ready for analysis. DSP, inhibition and IC50 were calculated to evaluate selected inhibitors and resulting inhibition efficiencies were consistent with available references. We demonstrated that this method provide a potential high throughput screening assay to analyze and identify the anti-glycation agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Comparison of ViTEK 2, MALDI-TOF and Partial Sequencing of 16S ...

    African Journals Online (AJOL)

    All the strains were susceptible to Vancomycin, Linezolid and Rifampicin while they were all resistant to Penicillin, Fusidic acid, and Trimethoprim. Brevibacterium epidermidis were generally resistant to Erythromycin and Clindamycin while B. iodinum and B. oceani were susceptible. Conclusion - 16S rRNA identification is ...

  2. Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives

    NARCIS (Netherlands)

    Brun, S.; Madrid, H.; Gerritis van den Ende, B.; Andersen, B.; Marinach-Patrice, C.; Mazier, D.; de Hoog, G.S.

    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

  3. Polyphasic approach including maldi-tof mass spectrometry to characterise aflatoxigenic species of aspergillus section flavi

    OpenAIRE

    Rodrigues, Paula; Santos, Cledir; Kozakiewicz, Zofia; Venâncio, Armando; Lima, Nelson

    2009-01-01

    Aflatoxins are toxic compounds which are produced as secondary metabolites by the fungi Aspergillus flavus, A. parasiticus and A. nomius growing on a variety of food products and are known to be carcinogenic, mutagenic, teratogenic and immunosuppressive1,2. Aspergillus is a large genus, with a complex taxonomy. The genus is easily identified by its characteristic conidiophore, but species identification and differentiation is complex, mainly because it is traditionally based on a range of mor...

  4. Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

    Czech Academy of Sciences Publication Activity Database

    Arnoštová, P.; Jedelsky, P. L.; Soukup, Tomáš; Žurmanová, J.

    2011-01-01

    Roč. 2011, - (2011), e634253 ISSN 1110-7243 R&D Projects: GA AV ČR IAAX01110901; GA ČR(CZ) GA304/08/0256 Institutional research plan: CEZ:AV0Z50110509 Keywords : cardiac MyHC isoforms * MyHC isoform mobility * effect of thyroid hormones * mass spectrometry * SDS-PAGE and western blot Subject RIV: ED - Physiology Impact factor: 2.436, year: 2011

  5. Determination of Elizabethkingia Diversity by MALDI-TOF Mass Spectrometry and Whole-Genome Sequencing

    DEFF Research Database (Denmark)

    Eriksen, Helle Brander; Gumpert, Heidi; Faurholt, Cecilie Haase

    2017-01-01

    In a hospital-acquired infection with multidrug-resistant Elizabethkingia, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene analysis identified the pathogen as Elizabethkingia miricola. Whole-genome sequencing, genus-level core genome analysis, and in...

  6. MALDI-TOF ICMS: avanços na identificação de fungos

    OpenAIRE

    Santos, Cledir; Lima, Nelson

    2011-01-01

    O sistema mais antigo pam a classificação das espécies de fungos, que incluem fungos filamentosos e levedums, são baseados em dados morfulógicos, principalmente naqueles ligados às estrutums reprodutivas. No entanto, este método de classificação apresenta limitações criticas, tais como as cultums de fungos que não desenvolvem estrutums reprodutivas, ou a semelbança morfológica entre membros de espécies diferentes. A incorpomção de testes bioquímicos e moleculares em taxonomia de fung...

  7. SEC-MALDI-TOF mass spectral characterization of a hyperbranched polyether prepared via melt transetherification

    NARCIS (Netherlands)

    Jayakannan, M.; Dongen, van J.L.J.; Behera, G.Ch.; Ramakrishnan, S.

    2002-01-01

    An AB2 monomer, 1-(2-hydroxyethoxy)-3,5-bis-(methoxymethyl)-2,4,6-trimethylbenzene, was synthesized from mesitol and melt-polycondensed in the presence of an acid catalyst via a transetherification process at 145-150 °C to yield a soluble, moderately high molecular weight hyperbranched polyether.

  8. 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.

  9. Identification / characterisation and authentication of microbial strains by MALDI-TOF ICMS

    OpenAIRE

    Santos, C.; Lima, Nelson

    2011-01-01

    The identification of species is an important goal in microbial taxonomy. Information about each microorganism (e.g. morphological description, physiological and biochemical properties, molecular biology sequencings, ecological roles, and societal risks or benefits) is key element in this process. Identifications can been a long and seemingly never-ended process with frequent revisions of the taxonomic schemes. The application of sound tolls to smooth the progress of identifica...

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

    NARCIS (Netherlands)

    Paauw, A.; Jonker, D.; Roeselers, G.; Heng, J.M.E.; Mars-Groenendijk, R.H.; Trip, H.; Molhoek, E.M.; Jansen, H.J.; Plas, J. van der; Jong, A.L. de; Majchrzykiewicz-Koehorst, J.A.; Speksnijder, A.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

  11. MALDI-TOF mass spectrometric detection of multiplex single base extended primers

    DEFF Research Database (Denmark)

    Mengel-From, Jonas; Sanchez Sanchez, Juan Jose; Børsting, Claus

    2004-01-01

    of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin...

  12. 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....

  13. CASE REPORT

    African Journals Online (AJOL)

    Blood results showed C-Reactive. Protein 395 mg/L ... Blood cultures were repeatedly negative as were tests for ... extension from infected tissues nearby or direct inoculation into the ... (MALDI-TOF MS) is another helpful method for identifying.

  14. White Spot Syndrome Virus infection in Penaeus monodon is ...

    Indian Academy of Sciences (India)

    2013-11-06

    assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the host ..... and early feature of cell death in different cell types triggered to die with different suicidal stimuli (Ucker et al. 2012).

  15. The microbial epidemiology of breast implant infections in a regional referral centre for plastic and reconstructive surgery in the south of France

    Directory of Open Access Journals (Sweden)

    Piseth Seng

    2015-06-01

    Conclusions: The microbiological epidemiology was noted by an increasing the proportion of Gram-negative bacteria and anaerobic bacteria detected with the advent of MALDI-TOF MS and molecular identification for diagnosis.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Principal component analysis of normalized full spectrum mass spectrometry data in multiMS-toolbox: An effective tool to identify important factors for classification of different metabolic patterns and bacterial strains.

    Science.gov (United States)

    Cejnar, Pavel; Kuckova, Stepanka; Prochazka, Ales; Karamonova, Ludmila; Svobodova, Barbora

    2018-06-15

    Explorative statistical analysis of mass spectrometry data is still a time-consuming step. We analyzed critical factors for application of principal component analysis (PCA) in mass spectrometry and focused on two whole spectrum based normalization techniques and their application in the analysis of registered peak data and, in comparison, in full spectrum data analysis. We used this technique to identify different metabolic patterns in the bacterial culture of Cronobacter sakazakii, an important foodborne pathogen. Two software utilities, the ms-alone, a python-based utility for mass spectrometry data preprocessing and peak extraction, and the multiMS-toolbox, an R software tool for advanced peak registration and detailed explorative statistical analysis, were implemented. The bacterial culture of Cronobacter sakazakii was cultivated on Enterobacter sakazakii Isolation Agar, Blood Agar Base and Tryptone Soya Agar for 24 h and 48 h and applied by the smear method on an Autoflex speed MALDI-TOF mass spectrometer. For three tested cultivation media only two different metabolic patterns of Cronobacter sakazakii were identified using PCA applied on data normalized by two different normalization techniques. Results from matched peak data and subsequent detailed full spectrum analysis identified only two different metabolic patterns - a cultivation on Enterobacter sakazakii Isolation Agar showed significant differences to the cultivation on the other two tested media. The metabolic patterns for all tested cultivation media also proved the dependence on cultivation time. Both whole spectrum based normalization techniques together with the full spectrum PCA allow identification of important discriminative factors in experiments with several variable condition factors avoiding any problems with improper identification of peaks or emphasis on bellow threshold peak data. The amounts of processed data remain still manageable. Both implemented software utilities are available

  6. 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...

  7. 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

  8. Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC–ESI-MS response for the bioanalysis of small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Hussain, Shah; Pezzei, Cornelia [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Güzel, Yüksel [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); ADSI-Austrian Drug Screening Institute, Innrain 66a, 6020 Innsbruck (Austria); Rainer, Matthias [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Huck, Christian W., E-mail: Christian.W.Huck@uibk.ac.at [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Bonn, Günther K. [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); ADSI-Austrian Drug Screening Institute, Innrain 66a, 6020 Innsbruck (Austria)

    2014-12-10

    Highlights: • A novel sample preparation technique for isolation of small molecules from human plasma. • Effectiveness of zirconium silicate for the removal of residual proteins after protein precipitation. • Abolishing the consumption of salts for the depletion of residual proteins after protein precipitation. • More than 99.6% removal of plasma proteins. - Abstract: An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization

  9. Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC–ESI-MS response for the bioanalysis of small molecules

    International Nuclear Information System (INIS)

    Hussain, Shah; Pezzei, Cornelia; Güzel, Yüksel; Rainer, Matthias; Huck, Christian W.; Bonn, Günther K.

    2014-01-01

    Highlights: • A novel sample preparation technique for isolation of small molecules from human plasma. • Effectiveness of zirconium silicate for the removal of residual proteins after protein precipitation. • Abolishing the consumption of salts for the depletion of residual proteins after protein precipitation. • More than 99.6% removal of plasma proteins. - Abstract: An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. MALDI-TOF and 13C NMR Analysis of Tannin–Furanic–Polyurethane Foams Adapted for Industrial Continuous Lines Application

    Directory of Open Access Journals (Sweden)

    Maria Cecilia Basso

    2014-12-01

    Full Text Available Mixed phenolic-polyurethane-type rigid foams were developed using tannin-furfuryl alcohol natural materials co-reacted with polymeric isocyanate in the proportions imposed by the limitations inherent to continuous industrial plants for polyurethane foams. A variety of different copolymerization oligomers formed. Urethanes appeared to have been formed with two flavonoid tannin sites, mainly at the flavonoid hydroxyl group at C3, but also, although less, on the phenolic hydroxyl groups of the flavonoid oligomers. Urethanes are also formed with (i glyoxal in the formulation, be it pre-reacted or not with the tannin; (ii with phenolsulfonic acid and (iii with furfural. This latter one, however, greatly favors reaction with the A-ring of the flavonoids through a methylene bridge rather than reaction with the isocyanate groups to form urethanes. All of the materials appeared to have co-reacted in a manner to form urethane and methylene bridges between all of the main components used. Thus, the tannin, the furfuryl alcohol, the isocyanate, the glyoxal and even the phenol sulfonic acid hardener formed a number of mixed species linked by the two bridge types. Several mixed species comprised of 2, 3 and even 4 co-reacted different components have been observed.

  19. Possible mistranslation of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF and Orbitrap mass spectrometry

    Science.gov (United States)

    RATIONALE: Shiga toxin-producing Escherichia coli (STEC) are often subjected to DNA damaging antibiotics during culturing in order to elicit the bacterial SOS response and up-regulation of bacteriophage-encoded proteins including Shiga toxin (Stx). However, such antibiotic exposure and stress may al...

  20. Multilocus phylogeny and MALDI-TOF analysis of the plant-pathogenic species Alternaria dauci and relatives

    NARCIS (Netherlands)

    Brun, S.; Madrid, H.; Gerrits van den Ende, A.H.G.; Andersen, B.; Marinach-Patrice, C.; Mazier, D.; de Hoog, G.S.

    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

  1. Immunoproteomic analysis of proteins from unsporulated Oocysts of Eimeria tenella in MALDI TOF/TOF tandem mass spectrometry

    Science.gov (United States)

    Immunoproteomic approaches were conducted to identify antigenic proteins from the total proteins of unsporulated oocysts of Eimeria tenella (E. tenella). Approximately 101 protein spots were recognized by chicken sera infected experimentally with E. tenella. Fourty-six spots of unsporulated oocysts ...

  2. Utilization of the linear mode of MALDI-TOF mass spectrometry in the study of glycation during the malting process

    Czech Academy of Sciences Publication Activity Database

    Laštovičková, Markéta; Mazanec, Karel; Smětalová, Dagmar; Bobálová, Janette

    2010-01-01

    Roč. 116, č. 3 (2010), s. 245-250 ISSN 0046-9750 R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : barley * brewing * glycation Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.897, year: 2010

  3. Profiling the triacylglyceride contents in bat integumentary lipids by preparative thin layer chromatography and MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Pannkuk, Evan L; Risch, Thomas S; Savary, Brett J

    2013-09-05

    The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

  4. Enumeration of sugars and sugar alcohols hydroxyl groups by aqueous-based acetylation and MALDI-TOF mass spectrometry

    Science.gov (United States)

    A method is described for enumerating hydroxyl groups on analytes in aqueous media is described, and applied to some common polyalcohols (erythritol, mannitol, and xylitol) and selected carbohydrates. The analytes were derivatized in water with vinyl acetate in presence of sodium phosphate buffer. ...

  5. 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.

  6. [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.

  7. 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.

  8. Characterization of synthetic peptides by mass spectrometry

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Mirza, Osman Asghar; Højrup, Peter

    2015-01-01

    Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI......-TOF-MS and LC-MS of synthetic peptides....

  9. 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...

  10. Comparison of two matrix-assisted laser desorption ionisation-time of flight mass spectrometry methods for the identification of clinically relevant anaerobic bacteria

    NARCIS (Netherlands)

    Veloo, A. C. M.; Knoester, M.; Degener, J. E.; Kuijper, E. J.

    2011-01-01

    Two commercially available MALDI-TOF MS systems, Bruker MS and Shimadzu MS, were compared for the identification of clinically relevant anaerobic bacteria. A selection of 79 clinical isolates, representing 19 different genera, were tested and compared with identification obtained by 16S rRNA gene

  11. 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.

  12. 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.

  13. 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.

  14. Detection and quantification of neurotensin in human brain tissue by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

    DEFF Research Database (Denmark)

    Gobom, J; Kraeuter, K O; Persson, R

    2000-01-01

    A method was developed for mass spectrometric detection of neurotensin (NT)-like immunoreactivity and quantification of NT in human brain tissue. The method is based on immunoprecipitation followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF......-MS). The identity of the major component of the immunoprecipitates as neurotensin was confirmed by fragment ion analysis on an electrospray ionization quadrupole time-of-flight instrument. MALDI-TOF-MS quantification of NT was achieved using stable-isotope-labeled NT as the internal standard, yielding an error...

  15. Species identification of clinical isolates of anaerobic bacteria: a comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems

    DEFF Research Database (Denmark)

    Justesen, Ulrik Stenz; Holm, Anette; Knudsen, Elisa

    2011-01-01

    We compared two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Shimadzu/SARAMIS and Bruker) on a collection of consecutive clinically important anaerobic bacteria (n = 290). The Bruker system had more correct identifications to the species level...... (67.2% versus 49.0%), but also more incorrect identifications (7.9% versus 1.4%). The system databases need to be optimized to increase identification levels. However, MALDI-TOF MS in its present version seems to be a fast and inexpensive method for identification of most clinically important...

  16. 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

  17. Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

    DEFF Research Database (Denmark)

    Truhlar, Stephanie M E; Cervantes, Carla F; Torpey, Justin W

    2008-01-01

    . MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot...

  18. A 50 SNP-multiplex mass spectrometry assay for human identification

    DEFF Research Database (Denmark)

    Wächter, Andrea; Mengel-From, Jonas; Børsting, Claus

    2008-01-01

    We developed a 50 SNP-multiplex assay for detection on a MALDI-TOF MS platform based on the SNPs in the 52 SNP-multiplex assay recently developed by the SNPforID Consortium. After PCR amplification, the products were purified on Qiagen columns and used as templates in one single base extension (SBE...... primers were extended with biotin labelled ddNTPs and purified on avidin beads ensuring that only the extended SBE primers were isolated and spotted on the MALDI-TOF anchor target. Detection of the 50 extended primers from the SBE reaction was performed in a mass range between 3000 and 10,000 m/z...

  19. Relapsing-Remitting MS (RRMS)

    Medline Plus

    Full Text Available ... Area Donate Donate Search v What Is MS? Definition of MS What Causes MS? Who Gets MS? ... Support Personal Stories d What Is MS? d Definition of MS Myelin Immune-Mediated Disease T Cells ...

  20. Comparative study using phenotypic, genotypic and proteomics methods for identification of coagulase-negative staphylococci

    NARCIS (Netherlands)

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

    2012-01-01

    Five methods were compared to determine the best technique for accurate identification of coagulase-negative staphylococci (CoNS) (n=142 strains). MALDI-TOF MS showed the best results for rapid and accurate CoNS differentiation (correct identity in 99.3%). An alternative to this approach could be

  1. Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Benkovská, Dagmar; Macejová, D.; Bialesova, L.; Hunakova, L.; Brtko, J.; Bobálová, Janette

    2015-01-01

    Roč. 232, č. 1 (2015), s. 226-232 ISSN 0378-4274 R&D Projects: GA MŠk(CZ) 7AMB12SK151 Institutional support: RVO:68081715 Keywords : retinoic acid * polyacrylamide gel electrophoresis * MALDI TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.522, year: 2015

  2. Chemical characterization of carbohydrate-based biosurfactants

    Science.gov (United States)

    High-yield, glycolipid-based biosurfactants are of increasing interest for use in environmentally benign cleaning or emulsifying agents. We have developed a MALDI-TOF/MS screen for the rapid analysis of several types of biosurfactants, including various acylated rhamnolipids in Pseudomonas extracts...

  3. Carbohydrate-based renewable biosurfactants: Rhamnolipids, sophorolipids, and novel liamocins

    Science.gov (United States)

    High-yield, glycolipid-based biosurfactants are of increasing interest for use in environmentally benign cleaning or emulsifying agents. We have developed a MALDI-TOF/MS screen for the rapid analysis of several types of biosurfactants, including various acylated rhamnolipids in Pseudomonas extracts...

  4. 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.

  5. A SIMPLE AND RAPID MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME OF FLIGHT MASS SPECTROMETRY METHOD TO SCREEN FISH PLASMA SAMPLES FOR ESTROGEN-RESPONSIVE BIOMARKERS

    Science.gov (United States)

    In this study, we describe and evaluate the performance of a simple and rapid mass spectral method for screening fish plasma for estrogen-responsive biomarkers using matrix assisted laster desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) couopled with a short...

  6. Identification of differentially expressed proteins in response to Pb ...

    African Journals Online (AJOL)

    In response to Pb, a total of 76 proteins, out of the 95 differentially expressed proteins, were subjected to MALDI-TOF-MS Of these, 46 identities were identified by PMF and 19 identities were identified by microsequencing. Basic metabolisms such as photosynthesis, photorespiration and protein biosynthesis in C. roseus ...

  7. First case of breakthrough pneumonia due to Aspergillus nomius in a patient with acute myeloid leukemia.

    Science.gov (United States)

    Caira, Morena; Posteraro, Brunella; Sanguinetti, Maurizio; de Carolis, Elena; Leone, Giuseppe; Pagano, Livio

    2012-10-01

    We report the first known case of a breakthrough pulmonary infection caused by Aspergillus nomius in an acute myeloid leukemia patient receiving caspofungin therapy. The isolate was identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and sequencing-based methods. The organism was found to be fully susceptible, in vitro, to echinocandin antifungal agents.

  8. Dynamic labeling of diagnostically significant microbial cells in cerebrospinal fluid by red chromophoric non-ionogenic surfactant for capillary electrophoresis separations

    Czech Academy of Sciences Publication Activity Database

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

    2012-01-01

    Roč. 728, MAY 30 (2012), s. 86-92 ISSN 0003-2670 R&D Projects: GA MV VG20112015021; GA AV ČR IAAX00310701 Keywords : electormigration techniques * MALDI - TOF MS * Monilinia spp. Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.387, year: 2012

  9. Selection of potential probiotic Lactobacillus strains of human origin for use in dairy industry

    Czech Academy of Sciences Publication Activity Database

    Španová, A.; Dráb, V.; Turková, K.; Špano, M.; Burdychová, R.; Šedo, O.; Šrůtková, Dagmar; Rada, V.; Rittich, B.

    2015-01-01

    Roč. 241, č. 6 (2015), s. 861-869 ISSN 1438-2377 R&D Projects: GA MŠk 2B06053 Institutional support: RVO:61388971 Keywords : Lactobacillus * Identification * MALDI - TOF MS Subject RIV: EE - Microbiology, Virology Impact factor: 1.433, year: 2015

  10. Current trends in microbial diagnostics based on mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Havlíček, Vladimír; Lemr, Karel; Schug, K. A.

    2013-01-01

    Roč. 85, č. 2 (2013), s. 790-797 ISSN 0003-2700 R&D Projects: GA ČR(CZ) GAP206/12/1150; GA MŠk(CZ) ME10013 Institutional support: RVO:61388971 Keywords : DESORPTION IONIZATION-TIME * MALDI - TOF -MS * CLINICAL MICROBIOLOGY Subject RIV: EE - Microbiology, Virology Impact factor: 5.825, year: 2013

  11. First clinical description of Eggerthia catenaformis bacteremia in a patient with dental abscess

    DEFF Research Database (Denmark)

    Kordjian, Hayarpi H; Schultz, Joyce D J H; Rosenvinge, Flemming Schønning

    2015-01-01

    We present a case of Eggerthia catenaformis bacteremia originating from a dental abscess and imitating necrotizing fasciitis in a previously healthy adult. The isolates were easily identified by MALDI-TOF MS. The clinical course, surgical and antibiotic treatment as well as the successful outcome...

  12. Effects of Preslaughter Stress Levels