Matrix Optical Absorption in UV-MALDI MS.

Science.gov (United States)

Robinson, Kenneth N; Steven, Rory T; Bunch, Josephine

2018-03-01

In ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI MS) matrix compound optical absorption governs the uptake of laser energy, which in turn has a strong influence on experimental results. Despite this, quantitative absorption measurements are lacking for most matrix compounds. Furthermore, despite the use of UV-MALDI MS to detect a vast range of compounds, investigations into the effects of laser energy have been primarily restricted to single classes of analytes. We report the absolute solid state absorption spectra of the matrix compounds α-cyano-4-hydroxycinnamic acid (CHCA), para-nitroaniline (PNA), 2-mercaptobenzothiazole (MBT), 2,5-dihydroxybenzoic acid (2,5-DHB), and 2,4,6-trihydroxyacetophenone (THAP). The desorption/ionization characteristics of these matrix compounds with respect to laser fluence was investigated using mixed systems of matrix with either angiotensin II, PC(34:1) lipid standard, or haloperidol, acting as representatives for typical classes of analyte encountered in UV-MALDI MS. The first absolute solid phase spectra for PNA, MBT, and THAP are reported; additionally, inconsistencies between previously published spectra for CHCA are resolved. In light of these findings, suggestions are made for experimental optimization with regards to matrix and laser wavelength selection. The relationship between matrix optical cross-section and wavelength-dependant threshold fluence, fluence of maximum ion yield, and R, a new descriptor for the change in ion intensity with fluence, are described. A matrix cross-section of 1.3 × 10 -17 cm -2 was identified as a potential minimum for desorption/ionization of analytes. Graphical Abstract ᅟ.

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

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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

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

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Susane Chang

2016-02-01

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

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

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Ismail Lafri

2016-01-01

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

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

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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

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

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Lista Florigio

2011-12-01

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

Determination of post-culture processing with carbohydrates by MALDI-MS and TMS derivatization GC-MS.

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Wunschel, David S; Wahl, Karen L; Melville, Angela M; Sorensen, Christina M; Colburn, Heather A; Valentine, Nancy B; Stamper, Casey L

2011-10-15

Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC-MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC-MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone - PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC-MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC-MS was

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

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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.

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...

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

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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.

Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries.

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Chin, Jefferson; Wood, Elizabeth; Peters, Grace S; Drexler, Dieter M

2016-02-01

In the early stages of drug discovery, high-throughput screening (HTS) of compound libraries against pharmaceutical targets is a common method to identify potential lead molecules. For these HTS campaigns to be efficient and successful, continuous quality control of the compound collection is necessary and crucial. However, the large number of compound samples and the limited sample amount pose unique challenges. Presented here is a proof-of-concept study for a novel process flow for the quality control screening of small-molecule compound libraries that consumes only minimal amounts of samples and affords compound-specific molecular data. This process employs an acoustic sample deposition (ASD) technique for the offline sample preparation by depositing nanoliter volumes in an array format onto microscope glass slides followed by matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis. An initial study of a 384-compound array employing the ASD-MALDI-MS workflow resulted in a 75% first-pass positive identification rate with an analysis time of <1 s per sample. © 2015 Society for Laboratory Automation and Screening.

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

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Reginaldo Lima-Neto

2014-06-01

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

Reagent Precoated Targets for Rapid In-Tissue Derivatization of the Anti-Tuberculosis Drug Isoniazid Followed by MALDI Imaging Mass Spectrometry

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Manier, M. Lisa; Reyzer, Michelle L.; Goh, Anne; Dartois, Veronique; Via, Laura E.; Barry, Clifton E.; Caprioli, Richard M.

2011-08-01

Isoniazid (INH) is an important component of front-line anti-tuberculosis therapy with good serum pharmacokinetics but unknown ability to penetrate tuberculous lesions. However, endogenous background interferences hinder our ability to directly analyze INH in tissues. Chemical derivatization has been successfully used to measure isoniazid directly from tissue samples using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). MALDI targets were pretreated with trans-cinnamaldehyde (CA) prior to mounting tissue slices. Isoniazid present in the tissues was efficiently derivatized and the INH-CA product measured by MS/MS. Precoating of MALDI targets allows the tissues to be directly thaw-mounted and derivatized, thus simplifying the preparation. A time-course series of tissues from tuberculosis infected/INH dosed animals were assayed and the MALDI MS/MS response correlates well with the amount of INH determined to be in the tissues by high-performance liquid chromatography (HPLC)-MS/MS.

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

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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.

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

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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.

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

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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.

MALDI-Imaging Mass Spectrometry of Ochratoxin A and Fumonisins in Mold-Infected Food.

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Hickert, Sebastian; Cramer, Benedikt; Letzel, Matthias C; Humpf, Hans-Ulrich

2016-09-06

Mycotoxins are toxic secondary metabolites produced by various fungi. Their distribution within contaminated material is of high interest to obtain insight into infection mechanisms and the possibility of reducing contamination during food processing. Various vegetable foodstuffs were infected with fungi of the genera Fusarium and Aspergillus. The localization of the produced mycotoxins was studied by matrix assisted laser desorption ionization time of flight imaging mass spectrometry (MALDI-MSI) of cryosections obtained from infected material. The results were confirmed by HPLC-electrospray ionization triple quadrupole mass spectrometry (HPLC/MS/MS). The mycotoxins ochratoxin A (OTA) and fumonisins of the B- and C-series (FB 1 , FB 2 , FB 3 , FB 4 , FC 2/3 , and FC 4 ) as well as partially hydrolyzed fumonisins (pHFB 1 , pHFB 2 , pHFB 3 , pHFC 1 , and pHFC 2/3 ) could successfully be detected by MALDI-IMS in mold-infested foodstuffs. The toxins are distributed differently in the material: OTA is co-localized with visible fungal spoilage while fumonisins could be detected throughout the whole sample. This work shows the applicability of MALDI-Imaging Mass Spectrometry (MALDI-MSI) to mycotoxin analysis. It has been demonstrated that the analyzed mycotoxins are differently distributed within moldy foodstuffs. These findings show the potential of MALDI-MSI for the localization of these hazardous compounds in various plant tissues. This article is protected by copyright. All rights reserved.

An On-Target Desalting and Concentration Sample Preparation Protocol for MALDI-MS and MS/MS Analysis

DEFF Research Database (Denmark)

Zhang, Xumin; Wang, Quanhui; Lou, Xiaomin

2012-01-01

2DE coupled with MALDI-MS is one of the most widely used and powerful analytic technologies in proteomics study. The MALDI sample preparation method has been developed and optimized towards the combination of simplicity, sample-cleaning, and sample concentration since its introduction. Here we...... present a protocol of the so-called Sample loading, Matrix loading, and on-target Wash (SMW) method which fulfills the three criteria by taking advantage of the AnchorChip™ targets. Our method is extremely simple and no pre-desalting or concentration is needed when dealing with samples prepared from 2DE...

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.

A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF

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Laëtitia Théron

2016-10-01

Full Text Available Mass spectrometry imaging (MSI is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.

Introduction of a 20 kHz Nd:YVO4 laser into a hybrid quadrupole time-of-flight mass spectrometer for MALDI-MS imaging.

Science.gov (United States)

Trim, Paul J; Djidja, Marie-Claude; Atkinson, Sally J; Oakes, Keith; Cole, Laura M; Anderson, David M G; Hart, Philippa J; Francese, Simona; Clench, Malcolm R

2010-08-01

A commercial hybrid quadrupole time-of-flight mass spectrometer has been modified for high-speed matrix-assisted laser desorption ionisation (MALDI) imaging using a short-pulse optical technology Nd:YVO(4) laser. The laser operating in frequency-tripled mode (lambda = 355 nm) is capable of delivering 1.5-ns pulses of energy at up to 8 microJ at 5-10 kHz and 3 microJ at 20 kHz. Experiments to improve beam homogeneity and reduce laser speckle by mechanical vibration of the fibre-optic laser delivery system are reported along with data from trial and tissue imaging experiments using the modified instrument. The laser appeared to yield best results for MALDI-MS imaging experiments when operating at repetition rates 5-10 kHz. Combining this with raster imaging allowed images of rat brain sections to be recorded in 37 min. Similarly, images of the distribution of peptides in "on-tissue" digest experiments from tumour tissues were recorded in 1 h and 30 min rather than the 8-h acquisition time previously used. A brief investigation of targeted protein analysis/imaging by multiple reaction monitoring experiments "on-tissue" is reported. A total of 26 transitions were recorded over a 3-s cycle time and images of abundant proteins were successfully recorded.

MALDI MS imaging investigation of the host response to visceral leishmaniasis.

Science.gov (United States)

Jaegger, C F; Negrão, F; Assis, D M; Belaz, K R A; Angolini, C F F; Fernandes, A M A P; Santos, V G; Pimentel, A; Abánades, D R; Giorgio, S; Eberlin, M N; Rocha, D F O

2017-09-26

Mass spectrometry imaging (MSI) of animal tissues has become an important tool for in situ molecular analyses and biomarker studies in several clinical areas, but there are few applications in parasitological studies. Leishmaniasis is a neglected tropical disease, and experimental mouse models have been essential to evaluate pathological and immunological processes and to develop diagnostic methods. Herein we have employed MALDI MSI to examine peptides and low molecular weight proteins (2 to 20 kDa) differentially expressed in the liver during visceral leishmaniasis in mice models. We analyzed liver sections of Balb/c mice infected with Leishmania infantum using the SCiLS Lab software for statistical analysis, which facilitated data interpretation and thus highlighted several key proteins and/or peptides. We proposed a decision tree classification for visceral leishmaniasis with distinct phases of the disease, which are named here as healthy, acute infection and chronic infection. Among others, the ion of m/z 4963 was the most important to identify acute infection and was tentatively identified as Thymosin β4. This peptide was previously established as a recovery factor in the human liver and might participate in the response of mice to Leishmania infection. This preliminary investigation shows the potential of MALDI MSI to complement classical compound selective imaging techniques and to explore new features not yet recognized by these approaches.

An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project

DEFF Research Database (Denmark)

Zhang, Xumin; Shi, Liang; Shu, Shaokung

2007-01-01

An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The ...

MALDI FTICR IMS of Intact Proteins: Using Mass Accuracy to Link Protein Images with Proteomics Data

Science.gov (United States)

Spraggins, Jeffrey M.; Rizzo, David G.; Moore, Jessica L.; Rose, Kristie L.; Hammer, Neal D.; Skaar, Eric P.; Caprioli, Richard M.

2015-06-01

MALDI imaging mass spectrometry is a highly sensitive and selective tool used to visualize biomolecules in tissue. However, identification of detected proteins remains a difficult task. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. Here, we demonstrate the capabilities of MALDI FTICR MS for imaging intact proteins. MALDI FTICR IMS provides an unprecedented combination of mass resolving power (~75,000 at m/z 5000) and accuracy (differentiate a series of oxidation products of S100A8 ( m/z 10,164.03, -2.1ppm), a subunit of the heterodimer calprotectin, in kidney tissue from mice infected with Staphylococcus aureus. S100A8 - M37O/C42O3 ( m/z 10228.00, -2.6ppm) was found to co-localize with bacterial microcolonies at the center of infectious foci. The ability of MALDI FTICR IMS to distinguish S100A8 modifications is critical to understanding calprotectin's roll in nutritional immunity.

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.

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

Fundamentals of MALDI-ToF-MS analysis applications in bio-diagnosis, tissue engineering and drug delivery

CERN Document Server

Hosseini, Samira

2017-01-01

This book presents the fundamentals and applications of Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF-MS) technique. It highlights the basic principles, the history of invention as well as the mechanism of ionization and mass determination using this technique. It describes the fundamental principles and methods for MALDI spectra interpretation and determination of exact chemical structures from experimental data. This book guides the reader through the interpretation of MALDI data where complex macromolecular spectra are simplified in order to present the major principles behind data interpretation. In addition, each chapter describes how MALDI-ToF-MS analysis provides necessary understanding of the copolymer systems that have been designed for specialized biomedical applications.

[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.

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

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.

Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation.

Science.gov (United States)

Goodwin, Richard J A; Nilsson, Anna; Borg, Daniel; Langridge-Smith, Pat R R; Harrison, David J; Mackay, C Logan; Iverson, Suzanne L; Andrén, Per E

2012-08-30

Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis. Copyright © 2012 Elsevier B.V. All rights reserved.

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

From whole-body sections down to cellular level, multiscale imaging of phospholipids by MALDI mass spectrometry.

Science.gov (United States)

Chaurand, Pierre; Cornett, Dale S; Angel, Peggi M; Caprioli, Richard M

2011-02-01

Significant progress in instrumentation and sample preparation approaches have recently expanded the potential of MALDI imaging mass spectrometry to the analysis of phospholipids and other endogenous metabolites naturally occurring in tissue specimens. Here we explore some of the requirements necessary for the successful analysis and imaging of phospholipids from thin tissue sections of various dimensions by MALDI time-of-flight mass spectrometry. We address methodology issues relative to the imaging of whole-body sections such as those cut from model laboratory animals, sections of intermediate dimensions typically prepared from individual organs, as well as the requirements for imaging areas of interests from these sections at a cellular scale spatial resolution. We also review existing limitations of MALDI imaging MS technology relative to compound identification. Finally, we conclude with a perspective on important issues relative to data exploitation and management that need to be solved to maximize biological understanding of the tissue specimen investigated.

Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality : Effect of sample preparation on MALDI-MS of synthetic polymers

NARCIS (Netherlands)

Kooijman, Pieter C.; Kok, Sander; Honing, Maarten

2017-01-01

Rationale: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides detailed and in-depth information about the molecular characteristics of synthetic polymers. To obtain the most accurate results the sample preparation parameters should be chosen to suit the sample and the

MALDI MS

Indian Academy of Sciences (India)

Unknown

revealed that there was an upper limit on the size of the molecules that can be .... 5. Sample preparation. The sample preparation for polymer MALDI must ac- ..... copy are in qualitative agreement with those obtained from other conventional ...

Differentiation of Vitis vinifera varieties by MALDI-MS analysis of the grape seed proteins.

Science.gov (United States)

Pesavento, Ivana Chiara; Bertazzo, Antonella; Flamini, Riccardo; Vedova, Antonio Dalla; De Rosso, Mirko; Seraglia, Roberta; Traldi, Pietro

2008-02-01

Until now the study of pathogenic related proteins in grape juice and wine, performed by ESI-MS, LC/ESI-MS, and MALDI/MS, has been proposed for differentiation of varieties. In fact, chitinases and thaumatin-like proteins persist through the vinification process and cause hazes and sediments in bottled wines. An additional instrument, potentially suitable for the grape varieties differentiation, has been developed by MALDI/MS for the grape seed protein analysis. The hydrosoluble protein profiles of seeds extract from three different Vitis vinifera grape (red and white) varieties were analyzed and compared. In order to evaluate the environmental conditions and harvest effects, the seed protein profiles of one grape variety from different locations and harvests were studied. (c) 2008 John Wiley & Sons, Ltd.

Comparison of feature selection and classification for MALDI-MS data

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Yang Mary

2009-07-01

Full Text Available Abstract Introduction In the classification of Mass Spectrometry (MS proteomics data, peak detection, feature selection, and learning classifiers are critical to classification accuracy. To better understand which methods are more accurate when classifying data, some publicly available peak detection algorithms for Matrix assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS data were recently compared; however, the issue of different feature selection methods and different classification models as they relate to classification performance has not been addressed. With the application of intelligent computing, much progress has been made in the development of feature selection methods and learning classifiers for the analysis of high-throughput biological data. The main objective of this paper is to compare the methods of feature selection and different learning classifiers when applied to MALDI-MS data and to provide a subsequent reference for the analysis of MS proteomics data. Results We compared a well-known method of feature selection, Support Vector Machine Recursive Feature Elimination (SVMRFE, and a recently developed method, Gradient based Leave-one-out Gene Selection (GLGS that effectively performs microarray data analysis. We also compared several learning classifiers including K-Nearest Neighbor Classifier (KNNC, Naïve Bayes Classifier (NBC, Nearest Mean Scaled Classifier (NMSC, uncorrelated normal based quadratic Bayes Classifier recorded as UDC, Support Vector Machines, and a distance metric learning for Large Margin Nearest Neighbor classifier (LMNN based on Mahanalobis distance. To compare, we conducted a comprehensive experimental study using three types of MALDI-MS data. Conclusion Regarding feature selection, SVMRFE outperformed GLGS in classification. As for the learning classifiers, when classification models derived from the best training were compared, SVMs performed the best with respect to the expected testing

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...

MALDI-MS analysis and theoretical evaluation of olanzapine as a UV laser desorption ionization (LDI) matrix.

Science.gov (United States)

Musharraf, Syed Ghulam; Ameer, Mariam; Ali, Arslan

2017-01-05

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) being soft ionization technique, has become a method of choice for high-throughput analysis of proteins and peptides. In this study, we have explored the potential of atypical anti-psychotic drug olanzapine (OLZ) as a matrix for MALDI-MS analysis of peptides aided with the theoretical studies. Seven small peptides were employed as target analytes to check performance of olanzapine and compared with conventional MALDI matrix α-cyano-4-hydroxycinnamic acid (HCCA). All peptides were successfully detected when olanzapine was used as a matrix. Moreover, peptides angiotensin Ι and angiotensin ΙΙ were detected with better S/N ratio and resolution with this method as compared to their analysis by HCCA. Computational studies were performed to determine the thermochemical properties of olanzapine in order to further evaluate its similarity to MALDI matrices which were found in good agreement with the data of existing MALDI matrices. Copyright © 2016. Published by Elsevier B.V.

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

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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.

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.

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.

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.

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

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

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.

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.

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.

Analysis of skin derived peptides from the Cuyaba Dwarf Frog Physalaemus nattereri by off-line LC MALDI MS/MS

DEFF Research Database (Denmark)

Castro, Mariana S; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner

2017-01-01

We have investigated the potential for analysis of the complex peptide mixtures secreted from frog skin using off-line LC-MALDI MS/MS. Since only limited information about the sequence of such peptides is available, de novo sequencing followed by Blast search was needed. An automated workflow has...... for future studies of peptides secreted from frogs....

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

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.

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.

MALDI Imaging Mass Spectrometry (MALDI-IMS―Application of Spatial Proteomics for Ovarian Cancer Classification and Diagnosis

Directory of Open Access Journals (Sweden)

Johan O. R. Gustafsson

2011-01-01

Full Text Available MALDI imaging mass spectrometry (MALDI-IMS allows acquisition of mass data for metabolites, lipids, peptides and proteins directly from tissue sections. IMS is typically performed either as a multiple spot profiling experiment to generate tissue specific mass profiles, or a high resolution imaging experiment where relative spatial abundance for potentially hundreds of analytes across virtually any tissue section can be measured. Crucially, imaging can be achieved without prior knowledge of tissue composition and without the use of antibodies. In effect MALDI-IMS allows generation of molecular data which complement and expand upon the information provided by histology including immuno-histochemistry, making its application valuable to both cancer biomarker research and diagnostics. The current state of MALDI-IMS, key biological applications to ovarian cancer research and practical considerations for analysis of peptides and proteins on ovarian tissue are presented in this review.

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.

A Derivatization and Validation Strategy for Determining the Spatial Localization of Endogenous Amine Metabolites in Tissues using MALDI Imaging Mass Spectrometry

Science.gov (United States)

Manier, M. Lisa; Spraggins, Jeffrey M.; Reyzer, Michelle L.; Norris, Jeremy L.; Caprioli, Richard M.

2014-01-01

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid (FA) as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high-mass resolution and MSn imaging mass spectrometry. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high-performance liquid chromatography (HPLC)-MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds. PMID:25044893

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

Detection of Amyloid Beta (Aβ) Oligomeric Composition Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS)

Science.gov (United States)

Wang, Jasmine S.-H.; Whitehead, Shawn N.; Yeung, Ken K.-C.

2018-02-01

The use of MALDI MS as a fast and direct method to detect the Aβ oligomers of different masses is examined in this paper. Experimental results suggest that Aβ oligomers are ionized and detected as singly charged ions, and thus, the resulting mass spectrum directly reports the oligomer size distribution. Validation experiments were performed to verify the MS data against artifacts. Mass spectra collected from modified Aβ peptides with different propensities for aggregation were compared. Generally, the relative intensities of multimers were higher from samples where oligomerization was expected to be more favorable, and vice versa. MALDI MS was also able to detect the differences in oligomeric composition before and after the incubation/oligomerization step. Such differences in sample composition were also independently confirmed with an in vitro Aβ toxicity study on primary rat cortical neurons. An additional validation was accomplished through removal of oligomers from the sample using molecular weight cutoff filters; the resulting MS data correctly reflected the removal at the expected cutoff points. The results collectively validated the ability of MALDI MS to assess the monomeric/multimeric composition of Aβ samples. [Figure not available: see fulltext.

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

DEFF Research Database (Denmark)

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

2010-01-01

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

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

MALDI matrices for low molecular weight compounds: an endless story?

Science.gov (United States)

Calvano, Cosima Damiana; Monopoli, Antonio; Cataldi, Tommaso R I; Palmisano, Francesco

2018-04-23

Since its introduction in the 1980s, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has gained a prominent role in the analysis of high molecular weight biomolecules such as proteins, peptides, oligonucleotides, and polysaccharides. Its application to low molecular weight compounds has remained for long time challenging due to the spectral interferences produced by conventional organic matrices in the low m/z window. To overcome this problem, specific sample preparation such as analyte/matrix derivatization, addition of dopants, or sophisticated deposition technique especially useful for imaging experiments, have been proposed. Alternative approaches based on second generation (rationally designed) organic matrices, ionic liquids, and inorganic matrices, including metallic nanoparticles, have been the object of intense and continuous research efforts. Definite evidences are now provided that MALDI MS represents a powerful and invaluable analytical tool also for small molecules, including their quantification, thus opening new, exciting applications in metabolomics and imaging mass spectrometry. This review is intended to offer a concise critical overview of the most recent achievements about MALDI matrices capable of specifically address the challenging issue of small molecules analysis. Graphical abstract An ideal Book of matrices for MALDI MS of small molecules.

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.

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)

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.

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.

Chemical characterization of Azadirachta indica grafted on Melia azedarach and analyses of azadirachtin by HPLC-MS-MS (SRM) and meliatoxins by MALDI-MS.

Science.gov (United States)

Forim, Moacir Rossi; Cornélio, Vivian Estevam; da Silva, M Fátima das G F; Rodrigues-Filho, Edson; Fernandes, João B; Vieira, Paulo C; Matinez, Sueli Souza; Napolitano, Michael P; Yost, Richard A

2010-01-01

Melia azedarach adapted to cool climates was selected as rootstocks for vegetative propagation of Azadirachta indica. Cleft grafting of A. indica on M. azedarach rootstock showed excellent survival. Little is known about the chemistry of grafting. The roots, stems, leaves and seeds of this graft were examined in order to verify if grafted A. indica would produce limonoids different from those found in non-grafted plants. Intact matured fruits were also studied to verify if they were free of meliatoxins. After successive chromatographic separations the extracts afforded several limonoids. HPLC-MS/MS and MALDI-MS were used to develop sensitive methods for detecting azadirachtin on all aerial parts of this graft and meliatoxins in fruits, respectively. The stem afforded the limonoid salannin, which was previously found in the oil seeds of A. indica. Salannin is also found in the root bark of M. azedarach. Thus, the finding of salannin in this study suggests that it could have been translocated from the M. azedarach rootstock to the A. indica graft. HPLC-MS/MS analyses showed that azadirachtin was present in all parts of the fruits, stem, flowers and root, but absent in the leaves. The results of MALDI-MS analyses confirmed the absence of meliatoxins in graft fruits. This study showed that A. indica grafted onto M. azedarach rootstock produces azadirachtin, and also that its fruits are free of meliatoxins from rootstocks, confirming that this graft forms an excellent basis for breeding vigorous Neem trees in cooler regions.

Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples.

Science.gov (United States)

Zhao, Yaju; Tang, Minmin; Liao, Qiaobo; Li, Zhoumin; Li, Hui; Xi, Kai; Tan, Li; Zhang, Mei; Xu, Danke; Chen, Hong-Yuan

2018-04-27

In this work, we demonstrate, for the first time, the development of a disposable MoS 2 -arrayed matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple sulfonamides (SAs). The disposable MALDI MS chip was designed and fabricated by MoS 2 array formation on a commercial indium tin oxide (ITO) glass slide. A series of SAs were analyzed, and clear deprotonated signals were obtained in negative-ion mode. Compared with MoS 2 -arrayed commercial steel plate, the prepared MALDI MS chip exhibited comparable LDI efficiency, providing a good alternative and disposable substrate for MALDI MS analysis. Furthermore, internal standard (IS) was previously deposited onto the MoS 2 array to simplify the experimental process for MALDI MS quantitation. 96 sample spots could be analyzed within 10 min in one single chip to perform quantitative analysis, recovery studies, and real foodstuff detection. Upon targeted extraction and enrichment by antibody conjugated magnetic beads, five SAs were quantitatively determined by the IS-first method with the linear range of 0.5-10 ng/mL ( R 2 > 0.990). Good recoveries and repeatability were obtained for spiked pork, egg, and milk samples. SAs in several real foodstuffs were successfully identified and quantified. The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.

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

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...

Comparison of MALDI-MSI and LC-MS for pharmacokinetic study of metformin

Czech Academy of Sciences Publication Activity Database

Strnad, Štěpán; Sýkora, D.; Cvačka, Josef; Maletínská, Lenka; Pirník, Z.; Majerčíková, Zuzana; Kuneš, Jaroslav; Vrkoslav, Vladimír

2017-01-01

Roč. 15, č. 1 (2017), s. 35 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : metformin * MALDI-MSI * LC-MS Subject RIV: CB - Analytical Chemistry, Separation

Probing the fungicidal property of CdS quantum dots on Saccharomyces cerevisiae and Candida utilis using MALDI-MS

Energy Technology Data Exchange (ETDEWEB)

Manikandan, Muthu; Wu, Hui-Fen, E-mail: hwu@faculty.nsysu.edu.tw [National Sun Yat-Sen University, Department of Chemistry (China)

2013-07-15

For the first time, we report the successful application of inhouse synthesized CdS quantum dots (QDs) with particle sizes between 1 and 7 nm exhibiting excellent fungicidal activity based on the interactions with Saccharomyces cerevisiae and Candida utilis. The growth curves and the growth rates of both fungi were established in the presence of three varying concentrations of CdS QDs. It was observed that the CdS QDs were highly inhibitory even at the lowest concentration of 10 mg/L used in this study, while the untreated control cells followed a normal growth pattern in the cases of both Saccharomyces and Candida. MALDI-MS was applied to substantiate the observations obtained by direct cell count method. It was observed that the trend observed in the case of Saccharomyces and Candida was well-represented in the MALDI-MS spectra. This study proposes a mechanism for the first time based on MALDI-MS results, that the CdS QDs interact with the extracellular polymeric substances (EPS) and remove small molecules from EPS layer; on the other hand, it was observed that CdS QDs at all concentrations lead to enrichment of protein signals in MALDI-MS. We have substantiated these results by quantifying the EPS in the control and treated cells and also using TEM to further confirm the results.

Probing the fungicidal property of CdS quantum dots on Saccharomyces cerevisiae and Candida utilis using MALDI-MS

International Nuclear Information System (INIS)

Manikandan, Muthu; Wu, Hui-Fen

2013-01-01

For the first time, we report the successful application of inhouse synthesized CdS quantum dots (QDs) with particle sizes between 1 and 7 nm exhibiting excellent fungicidal activity based on the interactions with Saccharomyces cerevisiae and Candida utilis. The growth curves and the growth rates of both fungi were established in the presence of three varying concentrations of CdS QDs. It was observed that the CdS QDs were highly inhibitory even at the lowest concentration of 10 mg/L used in this study, while the untreated control cells followed a normal growth pattern in the cases of both Saccharomyces and Candida. MALDI-MS was applied to substantiate the observations obtained by direct cell count method. It was observed that the trend observed in the case of Saccharomyces and Candida was well-represented in the MALDI-MS spectra. This study proposes a mechanism for the first time based on MALDI-MS results, that the CdS QDs interact with the extracellular polymeric substances (EPS) and remove small molecules from EPS layer; on the other hand, it was observed that CdS QDs at all concentrations lead to enrichment of protein signals in MALDI-MS. We have substantiated these results by quantifying the EPS in the control and treated cells and also using TEM to further confirm the results

Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality: Effect of sample preparation on MALDI-MS of synthetic polymers.

Science.gov (United States)

Kooijman, Pieter C; Kok, Sander; Honing, Maarten

2017-02-28

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides detailed and in-depth information about the molecular characteristics of synthetic polymers. To obtain the most accurate results the sample preparation parameters should be chosen to suit the sample and the aim of the experiment. Because the underlying principles of MALDI are still not fully known, a priori determination of optimal sample preparation protocols is often not possible. Employing an automated sample preparation quality assessment method recently presented by us we quantified the sample preparation quality obtained using various sample preparation protocols. Six conventional matrices with and without added potassium as a cationization agent and six ionic liquid matrices (ILMs) were assessed using poly(ethylene glycol) (PEG), polytetrahydrofuran (PTHF) and poly(methyl methacrylate) (PMMA) as samples. All sample preparation protocols were scored and ranked based on predefined quality parameters and spot-to-spot repeatability. Clearly distinctive preferences were observed in matrix identity and cationization agent for PEG, PTHF and PMMA, as the addition of an excess of potassium cationization agent results in an increased score for PMMA and a contrasting matrix-dependent effect for PTHF and PEG. The addition of excess cationization agent to sample mixtures dissipates any overrepresentation of high molecular weight polymer species. Our results show reduced ionization efficiency and similar sample deposit homogeneity for all tested ILMs, compared with well-performing conventional MALDI matrices. The results published here represent a start in the unsupervised quantification of sample preparation quality for MALDI samples. This method can select the best sample preparation parameters for any synthetic polymer sample and the results can be used to formulate hypotheses on MALDI principles. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

DEFF Research Database (Denmark)

Klenø, Tina Guldberg; Andreasen, Christian Maaløv; Kjeldal, Helle Ørsted

2004-01-01

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increa......-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications....

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

Imaging MALDI mass spectrometry using an oscillating capillary nebulizer matrix coating system and its application to analysis of lipids in brain from a mouse model of Tay-Sachs/Sandhoff disease.

Science.gov (United States)

Chen, Yanfeng; Allegood, Jeremy; Liu, Ying; Wang, Elaine; Cachón-Gonzalez, Begoña; Cox, Timothy M; Merrill, Alfred H; Sullards, M Cameron

2008-04-15

The quality of tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) depends on the effectiveness of the matrix deposition, especially for lipids that may dissolve in the solvent used for the matrix application. This article describes the use of an oscillating capillary nebulizer (OCN) to spray small droplets of matrix aerosol onto the sample surface for improved matrix homogeneity, reduced crystal size, and controlled solvent effects. This system was then applied to the analysis of histological slices of brains from mice with homozygous disruption of the hexb gene (hexb-/-), a model of Tay-Sachs and Sandhoff disease, versus the functionally normal heterozygote (hexb+/-) by imaging MALDI-MS. This allowed profiling and localization of many different lipid species, and of particular interest, ganglioside GM2, asialo-GM2 (GA2), and sulfatides (ST). The presence of these compounds was confirmed by analysis of brain extracts using electrospray ionization in conjunction with tandem mass spectrometry (MS/MS). The major fatty acid of the ceramide backbone of both GM2 and GA2 was identified as stearic acid (18:0) versus nervonic acid (24:1) for ST by both tissue-imaging MS and ESI-MS/MS. GM2 and GA2 were highly elevated in hexb-/- and were both localized in the granular cell region of the cerebellum. ST, however, was localized mainly in myelinated fiber (white matter) region of the cerebellum as well as in the brain stem with a relatively uniform distribution and had similar relative signal intensity for both hexb+/- and hexb-/- brain. It was also observed that there were distinct localizations for numerous other lipid subclasses; hence, imaging MALDI-MS could be used for "lipidomic" studies. These results illustrate the usefulness of tissue-imaging MALDI-MS with matrix deposition by OCN for histologic comparison of lipids in tissues such as brains from this mouse model of Tay-Sachs and Sandhoff disease.

Investigation of the interaction of iron(III) complexes with dAMP by ESI-MS, MALDI-MS and potentiometric titration: insights into synthetic nuclease behavior.

Science.gov (United States)

Fernandes, Christiane; Oliveira Moreira, Rafaela; Lube, Leonardo M; Horn, Adolfo; Szpoganicz, Bruno; Sherrod, Stacy; Russell, David H

2010-06-07

We report herein the characterization by electrospray ionization (ESI) mass spectrometry (MS), matrix assisted laser desorption ionization (MALDI-MS) and potentiometric titration of three iron(III) compounds: [Fe(III)(HPClNOL)Cl2]·NO3 (1), [Cl(HPClNOL)Fe(III)-(μ-O)-Fe(III)(HPClNOL)Cl]·Cl2·H2O (2) and [(SO4)(HPClNOL)Fe(III)-(μ-O)-Fe(III)(HPClNOL)(SO4)]·6H2O (3), where HPClNOL= 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol). Despite the fact that the compounds have distinct structures in solid state and non-buffered solution, all compounds present similar ESI and MALDI mass spectra in a buffered medium (pH 7.0). At this pH, the species [(PClNOL)Fe(III)-(μ-O)-Fe(III)(PClNOL)](2+) (m/z 354) was observed for all the compounds under investigation. Potentiometric titration confirms a similar behavior for all compounds, indicating that the dihydroxo form [(OH)(HPClNOL)Fe(III)-(μ-O)-Fe(III)(HPClNOL)(OH)](2+) is the major species at pH 7.0, for all the compounds. The products of the interaction between compounds (1), (2) and (3) and dAMP (2'-deoxyadenosine-5'-monophosphate) in a buffered medium (pH 7.0) were identified by MALDI-MS/MS. The fragmentation data obtained by MS/MS allow one to identify the nature of the interaction between the iron(III) compounds and dAMP, revealing the direct interaction between the iron center and phosphate groups.

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.

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.

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

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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...

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.

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

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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.

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.

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

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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.

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

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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.

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

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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

Methodological issues in protein and lipidic expressions in brain tissue exposed to Co60 based on DESI/MALDI-MS

International Nuclear Information System (INIS)

Soares, Matheus F.; Campos, Tarcísio P.R.; Augusti, Rodinei; Eberlin, Marcos N.; Vendramini, Pedro H.

2017-01-01

The present paper attempts to present some issues in the methodology of identifying lipid and protein changes in brain tissue induced by radiation. The goal was to address the analysis of the methodology and to investigate the feasibility of the generation of lipid/protein profiles of irradiated brain tissue, in order to identify radioinduced changes. Lipids and proteins are biomolecules with diverse structures and functionalities that participate in important intracellular processes. Changes in the lipid and the tissue protein profiles may indicate a cellular response to an external stimulus as well as the emergence of neoplasms or neurodegenerative diseases such as Alzheimer's. DESI-MS is a convenient method for identifying lipids and their spatial distribution in tissue beyond analytical quantification. DESI-MS allows the creation of an image of several low lipid m/z classes. MALDI-MS has already been a method used in the study of macromolecules as structural, membrane, hormone, neuromediator and immunological peptides. Through a full-scan matrix scan, with a m/z spectrum between 500-1000 for lipids and with a mass spectrum of 1000-15000 Da for proteins, the molecular profile can be analyzed. Generated pixel shape 2D chemical image. The produced image allows to associate the tissue distribution of the lipids and proteins with their chemical profile identified, allowing the verification of the changes radioinduced. Radiation triggers intense oxidative stress by increasing reactive oxygen species (ROS) and free radicals, causing DNA damage with consequent alterations in proteomics and cellular lipid explaining such changes in the lipid and protein expressions. The cellular morphophysiological changes are responsible for both the clonogenic inhibition and the induction of the apoptotic process. The images's production was directly dependent on the rigorous execution of the methodological procedures. Innumerable interferences could impair the image

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.

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

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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

Identification of Apis mellifera gut microbiota with MALDI TOF MS Biotyper

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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.

Preparation of positive blood cultures for direct MALDI-ToF MS identification.

Science.gov (United States)

Robinson, Andrew M; Ussher, James E

2016-08-01

MALDI-ToF MS can be used to identify microorganisms directly from blood cultures. This study compared two methods of sample preparation. Similar levels of genus- (91% vs 90%) and species-level identifications (79% vs 74%) were obtained with differential centrifugation and SDS methods. The SDS method is faster and requires minimal handling. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

MALDI imaging mass spectrometry: discrimination of pathophysiological regions in traumatized skeletal muscle by characteristic peptide signatures.

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Klein, Oliver; Strohschein, Kristin; Nebrich, Grit; Oetjen, Janina; Trede, Dennis; Thiele, Herbert; Alexandrov, Theodore; Giavalisco, Patrick; Duda, Georg N; von Roth, Philipp; Geissler, Sven; Klose, Joachim; Winkler, Tobias

2014-10-01

Due to formation of fibrosis and the loss of contractile muscle tissue, severe muscle injuries often result in insufficient healing marked by a significant reduction of muscle force and motor activity. Our previous studies demonstrated that the local transplantation of mesenchymal stromal cells into an injured skeletal muscle of the rat improves the functional outcome of the healing process. Since, due to the lack of sufficient markers, the accurate discrimination of pathophysiological regions in injured skeletal muscle is inadequate, underlying mechanisms of the beneficial effects of mesenchymal stromal cell transplantation on primary trauma and trauma adjacent muscle area remain elusive. For discrimination of these pathophysiological regions, formalin-fixed injured skeletal muscle tissue was analyzed by MALDI imaging MS. By using two computational evaluation strategies, a supervised approach (ClinProTools) and unsupervised segmentation (SCiLS Lab), characteristic m/z species could be assigned to primary trauma and trauma adjacent muscle regions. Using "bottom-up" MS for protein identification and validation of results by immunohistochemistry, we could identify two proteins, skeletal muscle alpha actin and carbonic anhydrase III, which discriminate between the secondary damage on adjacent tissue and the primary traumatized muscle area. Our results underscore the high potential of MALDI imaging MS to describe the spatial characteristics of pathophysiological changes in muscle. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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.

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

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.

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

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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.

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.

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

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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.

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...

Methodological issues in protein and lipidic expressions in brain tissue exposed to Co{sup 60} based on DESI/MALDI-MS

Energy Technology Data Exchange (ETDEWEB)

Soares, Matheus F.; Campos, Tarcísio P.R.; Augusti, Rodinei, E-mail: matheus.soares@gmail.com, E-mail: tprcampos@pq.cnpq.br, E-mail: augusti.rodinei@gmail.com, E-mail: augusti@ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte (Brazil); Eberlin, Marcos N.; Vendramini, Pedro H., E-mail: eberlin@iqm.unicamp.br, E-mail: ph_vendramini@yahoo.com.br [Universidade de Campinas (UNICAMP), SP (Brazil). Thompson Mass Spectroscopy Laboratory

2017-07-01

The present paper attempts to present some issues in the methodology of identifying lipid and protein changes in brain tissue induced by radiation. The goal was to address the analysis of the methodology and to investigate the feasibility of the generation of lipid/protein profiles of irradiated brain tissue, in order to identify radioinduced changes. Lipids and proteins are biomolecules with diverse structures and functionalities that participate in important intracellular processes. Changes in the lipid and the tissue protein profiles may indicate a cellular response to an external stimulus as well as the emergence of neoplasms or neurodegenerative diseases such as Alzheimer's. DESI-MS is a convenient method for identifying lipids and their spatial distribution in tissue beyond analytical quantification. DESI-MS allows the creation of an image of several low lipid m/z classes. MALDI-MS has already been a method used in the study of macromolecules as structural, membrane, hormone, neuromediator and immunological peptides. Through a full-scan matrix scan, with a m/z spectrum between 500-1000 for lipids and with a mass spectrum of 1000-15000 Da for proteins, the molecular profile can be analyzed. Generated pixel shape 2D chemical image. The produced image allows to associate the tissue distribution of the lipids and proteins with their chemical profile identified, allowing the verification of the changes radioinduced. Radiation triggers intense oxidative stress by increasing reactive oxygen species (ROS) and free radicals, causing DNA damage with consequent alterations in proteomics and cellular lipid explaining such changes in the lipid and protein expressions. The cellular morphophysiological changes are responsible for both the clonogenic inhibition and the induction of the apoptotic process. The images's production was directly dependent on the rigorous execution of the methodological procedures. Innumerable interferences could impair the image

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.

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...

Detection and mapping of illicit drugs and their metabolites in fingermarks by MALDI MS and compatibility with forensic techniques

Science.gov (United States)

Groeneveld, G.; de Puit, M.; Bleay, S.; Bradshaw, R.; Francese, S.

2015-06-01

Despite the proven capabilities of Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) in laboratory settings, research is still needed to integrate this technique into current forensic fingerprinting practice. Optimised protocols enabling the compatible application of MALDI to developed fingermarks will allow additional intelligence to be gathered around a suspect’s lifestyle and activities prior to the deposition of their fingermarks while committing a crime. The detection and mapping of illicit drugs and metabolites in latent fingermarks would provide intelligence that is beneficial for both police investigations and court cases. This study investigated MALDI MS detection and mapping capabilities for a large range of drugs of abuse and their metabolites in fingermarks; the detection and mapping of a mixture of these drugs in marks, with and without prior development with cyanoacrylate fuming or Vacuum Metal Deposition, was also examined. Our findings indicate the versatility of MALDI technology and its ability to retrieve chemical intelligence either by detecting the compounds investigated or by using their ion signals to reconstruct 2D maps of fingermark ridge details.

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

Science.gov (United States)

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

2015-09-01

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

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

Science.gov (United States)

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

2015-09-01

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

Comparison of HPLC/MS and MALDI-MS for characterizing triacylglycerols in insects: Specie-specific composition of lipids in fat bodies of bumblebee males

Czech Academy of Sciences Publication Activity Database

Kofroňová, Edita; Cvačka, Josef; Vrkoslav, Vladimír; Hanus, Robert; Jiroš, Pavel; Kindl, Jiří; Hovorka, Oldřich; Valterová, Irena

2009-01-01

Roč. 877, č. 30 (2009), s. 3878-3884 ISSN 1570-0232 R&D Projects: GA ČR GA203/09/0139; GA ČR GA203/09/1446; GA MŠk 2B06007 Institutional research plan: CEZ:AV0Z40550506 Keywords : Bumblebee * fat body * triacylglycerols * HPLC/APCI-MS * MALDI-MS Subject RIV: CC - Organic Chemistry Impact factor: 2.777, year: 2009

Analysis of hazardous biological material by MALDI mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

KL Wahl; KH Jarman; NB Valentine; MT Kingsley; CE Petersen; ST Cebula; AJ Saenz

2000-03-21

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) has become a valuable tool for analyzing microorganisms. The speed with which data can be obtained from MALDI-MS makes this a potentially important tool for biological health hazard monitoring and forensic applications. The excitement in the mass spectrometry community in this potential field of application is evident by the expanding list of research laboratories pursuing development of MALDI-MS for bacterial identification. Numerous research groups have demonstrated the ability to obtain unique MALDI-MS spectra from intact bacterial cells and bacterial cell extracts. The ability to differentiate strains of the same species has been investigated. Reproducibility of MALDI-MS spectra from bacterial species under carefully controlled experimental conditions has also been demonstrated. Wang et al. have reported on interlaboratory reproducibility of the MALDI-MS analysis of several bacterial species. However, there are still issues that need to be addressed, including the careful control of experimental parameters for reproducible spectra and selection of optimal experimental parameters such as solvent and matrix.

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.

Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

Science.gov (United States)

Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter

1997-12-01

In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.

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

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.

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.

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.

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

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

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

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

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.

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.

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

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

Murine cutaneous leishmaniasis investigated by MALDI mass spectrometry imaging.

Science.gov (United States)

Negrão, Fernanda; de O Rocha, Daniele F; Jaeeger, Caroline F; Rocha, Francisca J S; Eberlin, Marcos N; Giorgio, Selma

2017-09-26

Imaging mass spectrometry (IMS) is recognized as a powerful tool to investigate the spatial distribution of untargeted or targeted molecules of a wide variety of samples including tissue sections. Leishmania is a protozoan parasite that causes different clinical manifestations in mammalian hosts. Leishmaniasis is a major public health risk in different continents and represents one of the most important neglected diseases. Cutaneous lesions from mice experimentally infected with Leishmania spp. were investigated by matrix-assisted laser desorption ionization MS using the SCiLS Lab software for statistical analysis. Being applied to cutaneous leishmaniasis (CL) for the first time, MALDI-IMS was used to search for peptides and low molecular weight proteins (2-10 kDa) as candidates for potential biomarkers. Footpad sections of Balb/c mice infected with (i) Leishmania amazonensis or (ii) Leishmania major were imaged. The comparison between healthy and infected skin highlighted a set of twelve possible biomarker proteins for L. amazonenis and four proteins for L. major. Further characterization of these proteins could reveal how these proteins act in pathology progression and confirm their values as biomarkers.

Improved Spectra for MALDI MSI of Peptides Using Ammonium Phosphate Monobasic in MALDI Matrix.

Science.gov (United States)

Ucal, Yasemin; Ozpinar, Aysel

2018-05-10

MALDI mass spectrometry imaging (MSI) enables analysis of peptides along with histology. However, there are several critical steps in MALDI MSI of peptides, one of which is spectral quality. Suppression of MALDI matrix clusters by the aid of ammonium salts in MALDI experiments is well-known. It is asserted that addition of ammonium salts dissociates potential matrix adducts and thereafter decreases matrix cluster formation. Consequently, MALDI MS sensitivity and mass accuracy increases. Up to our knowledge, a limited number of MALDI MSI studies used ammonium salts as matrix additives to suppress matrix clusters and enhance peptide signals. In this work, we investigated the effect of ammonium phosphate monobasic (AmP) as alpha-cyano-4-hydroxycinnamic acid (α-CHCA) matrix additive in MALDI MSI of peptides. Prior to MALDI MSI, the effect of varying concentrations of AmP in α-CHCA were assessed in bovine serum albumin (BSA) tryptic digests and compared with the control (α-CHCA without AmP). Based on our data, the addition of AmP as matrix additive decreased matrix cluster formation regardless of its concentration and, specifically 8 mM AmP and 10 mM AmP increased BSA peptide signal intensities. In MALDI MSI of peptides, both 8 mM, and 10 mM AmP in α-CHCA improved peptide signals especially in the mass range of m/z 2000 to 3000. In particular, 9 peptide signals were found to have differential intensities within the tissues deposited with AmP in α-CHCA (AUC>0.60). To the best of our knowledge, this is the first MALDI MSI of peptides work investigating different concentrations of AmP as α-CHCA matrix additive in order to enhance peptide signals in formalin fixed paraffin embedded (FFPE) tissues. Further, AmP as part of α-CHCA matrix could enhance protein identifications and support MALDI MSI based proteomic approaches. This article is protected by copyright. All rights reserved.

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.

Utility of imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) on an ion trap mass spectrometer in the analysis of drugs and metabolites in biological tissues.

Science.gov (United States)

Drexler, Dieter M; Garrett, Timothy J; Cantone, Joseph L; Diters, Richard W; Mitroka, James G; Prieto Conaway, Maria C; Adams, Stephen P; Yost, Richard A; Sanders, Mark

2007-01-01

The properties and potential liabilities of drug candidate are investigated in detailed ADME assays and in toxicity studies, where findings are placed in context of exposure to dosed drug and metabolites. The complex nature of biological samples may necessitate work-up procedures prior to high performance liquid chromatography-mass spectrometric (HPLC-MS) analysis of endogenous or xenobiotic compounds. This concept can readily be applied to biological fluids such as blood or urine, but in localized samples such as organs and tissues potentially important spatial, thus anatomical, information is lost during sample preparation as the result of homogenization and extraction procedures. However, the localization of test article or spatial identification of metabolites may be critical to the understanding of the mechanism of target-organ toxicity and its relevance to clinical safety. Tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) and ion trap mass spectrometry (MS) with higher order mass spectrometric scanning functions was utilized for localization of dosed drug or metabolite in tissue. Laser capture microscopy (LCM) was used to obtain related samples from tissue for analyses by standard MALDI-MS and HPLC-MS. In a toxicology study, rats were administered with a high dosage of a prodrug for 2 weeks. Birefringent microcrystalline material (10-25 microm) was observed in histopathologic formalin-fixed tissue samples. Direct analysis by IMS provided the identity of material in the microcrystals as circulating active drug while maintaining spatial orientation. Complementary data from visual cross-polarized light microscopy as well as standard MALDI-MS and HPLC-MS experiments on LCM samples validated the qualitative results obtained by IMS. Furthermore, the HPLC-MS analysis on the LCM samples afforded a semi-quantitative assessment of the crystalline material in the tissue samples. IMS by MALDI ion trap MS proved sensitive

On the Importance of Mathematical Methods for Analysis of MALDI-Imaging Mass Spectrometry Data

Directory of Open Access Journals (Sweden)

Trede Dennis

2012-03-01

Full Text Available In the last decade, matrix-assisted laser desorption/ionization (MALDI imaging mass spectrometry (IMS, also called as MALDI-imaging, has proven its potential in proteomics and was successfully applied to various types of biomedical problems, in particular to histopathological label-free analysis of tissue sections. In histopathology, MALDI-imaging is used as a general analytic tool revealing the functional proteomic structure of tissue sections, and as a discovery tool for detecting new biomarkers discriminating a region annotated by an experienced histologist, in particular, for cancer studies.

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.

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.

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.

Visualizing fungal metabolites during mycoparasitic interaction by MALDI mass spectrometry imaging

Science.gov (United States)

Holzlechner, Matthias; Reitschmidt, Sonja; Gruber, Sabine; Zeilinger, Susanne

2016-01-01

Studying microbial interactions by MALDI mass spectrometry imaging (MSI) directly from growing media is a difficult task if high sensitivity is demanded. We present a quick and robust sample preparation strategy for growing fungi (Trichoderma atroviride, Rhizoctonia solani) on glass slides to establish a miniaturized confrontation assay. By this we were able to visualize metabolite distributions by MALDI MSI after matrix deposition with a home‐built sublimation device and thorough recrystallization. We present for the first time MALDI MSI data for secondary metabolite release during active mycoparasitism. PMID:26959280

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.

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.

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

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

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...

Identifying modifications in RNA by MALDI mass spectrometry

DEFF Research Database (Denmark)

Douthwaite, Stephen; Kirpekar, Finn

2007-01-01

as RNA modifications added in cell-free in vitro systems. MALDI-MS is particularly useful in cases in which other techniques such as those involving primer extension or chromatographic analyses are not practicable. To date, MALDI-MS has been used to localize rRNA modifications that are involved......Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass...... spectra produced by MALDI are relatively straightforward to interpret, because they are dominated by singly charged ions, making it possible to analyze complex mixtures of RNA oligonucleotides ranging from trinucleotides up to 20-mers. Analysis of modifications within much longer RNAs, such as ribosomal...

Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS.

Science.gov (United States)

Zhou, Hui; Warren, Peter G; Froehlich, John W; Lee, Richard S

2014-07-01

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.

A new classification method for MALDI imaging mass spectrometry data acquired on formalin-fixed paraffin-embedded tissue samples.

Science.gov (United States)

Boskamp, Tobias; Lachmund, Delf; Oetjen, Janina; Cordero Hernandez, Yovany; Trede, Dennis; Maass, Peter; Casadonte, Rita; Kriegsmann, Jörg; Warth, Arne; Dienemann, Hendrik; Weichert, Wilko; Kriegsmann, Mark

2017-07-01

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) shows a high potential for applications in histopathological diagnosis, and in particular for supporting tumor typing and subtyping. The development of such applications requires the extraction of spectral fingerprints that are relevant for the given tissue and the identification of biomarkers associated with these spectral patterns. We propose a novel data analysis method based on the extraction of characteristic spectral patterns (CSPs) that allow automated generation of classification models for spectral data. Formalin-fixed paraffin embedded (FFPE) tissue samples from N=445 patients assembled on 12 tissue microarrays were analyzed. The method was applied to discriminate primary lung and pancreatic cancer, as well as adenocarcinoma and squamous cell carcinoma of the lung. A classification accuracy of 100% and 82.8%, resp., could be achieved on core level, assessed by cross-validation. The method outperformed the more conventional classification method based on the extraction of individual m/z values in the first application, while achieving a comparable accuracy in the second. LC-MS/MS peptide identification demonstrated that the spectral features present in selected CSPs correspond to peptides relevant for the respective classification. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

[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.

Structural study of synthetic polymers by MALDI-TOFMS; MALDI-TOFMS ni yoru gosei kobunshi no kozo kaiseki

Energy Technology Data Exchange (ETDEWEB)

Noguchi, K.; Hirayama, K. [Ajinomoto Co. Inc., Tokyo (Japan)

1998-08-01

As observation results on the time-dependent change in the ring-opening reaction of novolac epoxy resin with acetic acid by MALDI-TOFMS, the epoxy ring was opened with reaction time, the hydroxy group formed by the ring-opening reaction was acetylated, those components were measured. In the case of the FABMS/MS observation of materials and the reaction products, the estimation structure could be confirmed from the measured results of MALDI-TOFMS. In the polymerization of bisphenol A epoxy resin with N, N`-dimethylethylenediamine, it was observed by MALDI-TOFMS that many kinds of polymers with high molecular weight were formed with an increase of reaction time. In this case, the LSIMS/MS observation of materials and the reaction products was carried out, the estimation structure could be confirmed from the measured results of MALDI-TOFMS. 19 refs., 8 figs.

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.

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.

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

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.

Surface-MALDI mass spectrometry in biomaterials research

DEFF Research Database (Denmark)

Griesser, H.J.; Kingshott, P.; McArthur, S.L.

2004-01-01

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new...... surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological...... solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established...

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.

Thermoresponsive Arrays Patterned via Photoclick Chemistry: Smart MALDI Plate for Protein Digest Enrichment, Desalting, and Direct MS Analysis.

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Meng, Xiao; Hu, Junjie; Chao, Zhicong; Liu, Ying; Ju, Huangxian; Cheng, Quan

2018-01-10

Sample desalting and concentration are crucial steps before matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis. Current sample pretreatment approaches require tedious fabrication and operation procedures, which are unamenable to high-throughput analysis and also result in sample loss. Here, we report the development of a smart MALDI substrate for on-plate desalting, enrichment, and direct MS analysis of protein digests based on thermoresponsive, hydrophilic/hydrophobic transition of surface-grafted poly(N-isopropylacrylamide) (PNIPAM) microarrays. Superhydrophilic 1-thioglycerol microwells are first constructed on alkyne-silane-functionalized rough indium tin oxide substrates based on two sequential thiol-yne photoclick reactions, whereas the surrounding regions are modified with hydrophobic 1H,1H,2H,2H-perfluorodecanethiol. Surface-initiated atom-transfer radical polymerization is then triggered in microwells to form PNIPAM arrays, which facilitate sample loading and enrichment of protein digests by concentrating large-volume samples into small dots and achieving on-plate desalting through PNIPAM configuration change at elevated temperature. The smart MALDI plate shows high performance for mass spectrometric analysis of cytochrome c and neurotensin in the presence of 1 M urea and 100 mM NaHCO 3 , as well as improved detection sensitivity and high sequence coverage for α-casein and cytochrome c digests in femtomole range. The work presents a versatile sample pretreatment platform with great potential for proteomic research.

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

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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.

MALDI-TOF-mass spectrometry applications in clinical microbiology.

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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.

Major phytopathogens and strains from cocoa (Theobroma cacao L.) are differentiated by MALDI-MS lipid and/or peptide/protein profiles.

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Dos Santos, Fábio Neves; Tata, Alessandra; Belaz, Kátia Roberta Anacleto; Magalhães, Dilze Maria Argôlo; Luz, Edna Dora Martins Newman; Eberlin, Marcos Nogueira

2017-03-01

Phytopathogens are the main disease agents that promote attack of cocoa plantations in all tropical countries. The similarity of the symptoms caused by different phytopathogens makes the reliable identification of the diverse species a challenge. Correct identification is important in the monitoring and management of these pests. Here we show that matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis is able to rapidly and reliably differentiate cocoa phytopathogens, namely Moniliophthora perniciosa, Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C. paradoxa, and C. fimbriata. MALDI-MS reveals unique peptide/protein and lipid profiles which differentiate these phytopathogens at the level of genus, species, and single strain coming from different hosts or cocoa tissues collected in several plantations/places. This fast methodology based on molecular biomarkers is also shown to be sufficiently reproducible and selective and therefore seems to offer a suitable tool to guide the correct application of sanitary defense approaches for infected cocoa plantations. International trading of cocoa plants and products could also be efficiently monitored by MALDI-MS. It could, for instance, prevent the entry of new phytopathogens into a country, e.g., as in the case of Moniliophthora roreri fungus that is present in all cocoa plantations of countries bordering Brazil, but that has not yet attacked Brazilian plantations. Graphical Abstract Secure identification of phytopathogens attacking cocoa plantations has been demonstrated via typical chemical profiles provided by mass spectrometric screening.

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.

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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

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

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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.

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

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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.

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

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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.

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

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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.

DIFFERENTIATION OF AEROMONAS ISOLATES OBTAINED FROM DRINKING WATER DISTRIBUTION SYSTEM USING MATRIX-ASSISTED LASER DESCRIPTION/IONIZATION-MASS SPECTROMETRY (MALDI-MS)

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The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to analyze the...

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

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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.

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

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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.

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

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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.

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

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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.

An update on MALDI mass spectrometry based technology for the analysis of fingermarks - stepping into operational deployment.

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Francese, S; Bradshaw, R; Denison, N

2017-07-10

Since 2009, when Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) was firstly reported for the molecular mapping of latent fingermarks, the range of information and operational capabilities have steadily increased. Pioneering work from our Fingermark Research Group exploited different modalities, including Profiling (MALDI MSP), tandem mass spectrometry (MS/MS) and Ion Mobility MS/MS; a number of methodologies were also developed to conquer a main challenge, namely profiling the suspect and their actions prior to or whilst committing the crime. Suspect profiling here is no longer based on behavioural science but complements this discipline and the investigations by detecting and visualising the molecular make-up of fingermarks onto the identifying ridges. This forensic opportunity provides the link between the biometric information (ridge detail) and the corpus delicti or intelligence on the circumstances of the crime. In 2013, a review was published covering the research work and developments of four years supported by the Home Office, UK, and the local regional Police with some insights (and comparison) into similar research being reported employing other mass spectrometric techniques. The present review is an extensive update on the MALDI MS based methods' achievements, limitations and work in progress in fingermark analysis; it also offers an outlook on further necessary research into this subject. The main highlights are the increased number of possible information retrievable around a suspect and the more extended compatibility of this technology. The latter has allowed MALDI MS based methods to integrate well with current forensic fingerprinting, leading to the investigation of real police casework.

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

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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

Microorganism Identification Based On MALDI-TOF-MS Fingerprints

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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.

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.

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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.

The Effect of Collimating Lens Focusing on Laser Beam Shape in Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS).

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O'Rourke, Matthew B; Raymond, Benjamin B A; Djordjevic, Steven P; Padula, Matthew P

2018-03-01

Tissue imaging using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established technique that, in recent years, has seen wider adoption and novel application. Applications such imaging mass spectrometry (IMS) and biotyping are beginning to gain greater exposure and use; however, with limitations in optimization methods, producing the best result often relies on the ability to customize the physical characteristics of the instrumentation, a task that is challenging for most mass spectrometry laboratories. With this in mind, we have described the effect of making simple adjustments to the laser optics at the final collimating lens area, to adjust the laser beam size and shape in order to allow greater customization of the instrument for improving techniques such as IMS. We have therefore been able to demonstrate that improvements can be made without requiring the help of an electrical engineer or external funding in a way that only costs a small amount of time. Graphical Abstract ᅟ.

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

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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.

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

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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.

On-target digestion of collected bacteria for MALDI mass spectrometry.

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Dugas, Alton J; Murray, Kermit K

2008-10-03

An on-target protein digestion system was developed for the identification of microorganisms in collected bioaerosols using off-line matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Bacteria analysis techniques based on MALDI-MS were adapted for use with an orthogonal MALDI quadrupole-time-of-flight mass spectrometer. Bioaerosols were generated using a pneumatic nebulizer and infused into a chamber for sampling. An Andersen N6 single-stage impactor was used to collect the bioaerosols on a MALDI target. On-target digestion was carried out inside temporary mini-wells placed over the impacted samples. The wells served as miniature reactors for proteolysis. Collected test aerosol particles containing the protein cytochrome c and E. coli bacteria were proteolyzed in situ using trypsin or cyanogen bromide. A total of 19 unique proteins were identified for E. coli. Using the TOF-MS spectra of the digested samples, peptide mass mapping was performed using the MASCOT search engine and an iterative search technique.

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

Scores for standardization of on-tissue digestion of formalin-fixed paraffin-embedded tissue in MALDI-MS imaging.

Science.gov (United States)

Erich, Katrin; Sammour, Denis A; Marx, Alexander; Hopf, Carsten

2017-07-01

On-slide digestion of formalin-fixed and paraffin-embedded human biopsy tissue followed by mass spectrometry imaging of resulting peptides may have the potential to become an additional analytical modality in future ePathology. Multiple workflows have been described for dewaxing, antigen retrieval, digestion and imaging in the past decade. However, little is known about suitable statistical scores for method comparison and systematic workflow standardization required for development of processes that would be robust enough to be compatible with clinical routine. To define scores for homogeneity of tissue processing and imaging as well as inter-day repeatability for five different processing methods, we used human liver and gastrointestinal stromal tumor tissue, both judged by an expert pathologist to be >98% histologically homogeneous. For mean spectra-based as well as pixel-wise data analysis, we propose the coefficient of determination R 2 , the natural fold-change (natFC) value and the digest efficiency DE% as readily accessible scores. Moreover, we introduce two scores derived from principal component analysis, the variance of the mean absolute deviation, MAD, and the interclass overlap, J overlap , as computational scores that may help to avoid user bias during future workflow development. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

RAPID AND AUTOMATED PROCESSING OF MALDI-FTICR/MS DATA FOR N-METABOLIC LABELING IN A SHOTGUN PROTEOMICS ANALYSIS.

Science.gov (United States)

Jing, Li; Amster, I Jonathan

2009-10-15

Offline high performance liquid chromatography combined with matrix assisted laser desorption and Fourier transform ion cyclotron resonance mass spectrometry (HPLC-MALDI-FTICR/MS) provides the means to rapidly analyze complex mixtures of peptides, such as those produced by proteolytic digestion of a proteome. This method is particularly useful for making quantitative measurements of changes in protein expression by using (15)N-metabolic labeling. Proteolytic digestion of combined labeled and unlabeled proteomes produces complex mixtures that with many mass overlaps when analyzed by HPLC-MALDI-FTICR/MS. A significant challenge to data analysis is the matching of pairs of peaks which represent an unlabeled peptide and its labeled counterpart. We have developed an algorithm and incorporated it into a compute program which significantly accelerates the interpretation of (15)N metabolic labeling data by automating the process of identifying unlabeled/labeled peak pairs. The algorithm takes advantage of the high resolution and mass accuracy of FTICR mass spectrometry. The algorithm is shown to be able to successfully identify the (15)N/(14)N peptide pairs and calculate peptide relative abundance ratios in highly complex mixtures from the proteolytic digest of a whole organism protein extract.

Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS

DEFF Research Database (Denmark)

Boyd, A. R.; Burke, G. A.; Duffy, H.

2011-01-01

Protein adsorption onto calcium phosphate (Ca–P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation...... to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role...

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.

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.

Detection of high molecular weight proteins by MALDI imaging mass spectrometry.

Science.gov (United States)

Mainini, Veronica; Bovo, Giorgio; Chinello, Clizia; Gianazza, Erica; Grasso, Marco; Cattoretti, Giorgio; Magni, Fulvio

2013-06-01

MALDI imaging mass spectrometry (IMS) is a unique technology to explore the spatial distribution of biomolecules directly on tissues. It allows the in situ investigation of a large number of small proteins and peptides. Detection of high molecular weight proteins through MALDI IMS still represents an important challenge, as it would allow the direct investigation of the distribution of more proteins involved in biological processes, such as cytokines, enzymes, neuropeptide precursors and receptors. In this work we compare the traditional method performed with sinapinic acid with a comparable protocol using ferulic acid as the matrix. Data show a remarkable increase of signal acquisition in the mass range of 20k to 150k Th. Moreover, we report molecular images of biomolecules above 70k Th, demonstrating the possibility of expanding the application of this technology both in clinical investigations and basic science.

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues.

Science.gov (United States)

Drake, R R; Powers, T W; Jones, E E; Bruner, E; Mehta, A S; Angel, P M

2017-01-01

Glycosylated proteins account for a majority of the posttranslation modifications of cell surface, secreted, and circulating proteins. Within the tumor microenvironment, the presence of immune cells, extracellular matrix proteins, cell surface receptors, and interactions between stroma and tumor cells are all processes mediated by glycan binding and recognition reactions. Changes in glycosylation during tumorigenesis are well documented to occur and affect all of these associated adhesion and regulatory functions. A MALDI imaging mass spectrometry (MALDI-IMS) workflow for profiling N-linked glycan distributions in fresh/frozen tissues and formalin-fixed paraffin-embedded tissues has recently been developed. The key to the approach is the application of a molecular coating of peptide-N-glycosidase to tissues, an enzyme that cleaves asparagine-linked glycans from their protein carrier. The released N-linked glycans can then be analyzed by MALDI-IMS directly on tissue. Generally 40 or more individual glycan structures are routinely detected, and when combined with histopathology localizations, tumor-specific glycans are readily grouped relative to nontumor regions and other structural features. This technique is a recent development and new approach in glycobiology and mass spectrometry imaging research methodology; thus, potential uses such as tumor-specific glycan biomarker panels and other applications are discussed. © 2017 Elsevier Inc. All rights reserved.

Correlation between Sweet Spots of Glycopeptides and Polymorphism of the Matrix Crystal in MALDI Samples.

Science.gov (United States)

Nishikaze, Takashi; Okumura, Hisako; Jinmei, Hiroshi; Amano, Junko

2012-01-01

A standard dried-droplet preparation using 2,5-dihydroxybenzoic acid (2,5-DHBA) as the matrix results in a large variation in signal intensity and poor shot-to-shot reproducibility in matrix-assisted laser desorption/ionization (MALDI). We expected that the differences can be attributed to the nature of the crystal structures in the region of the "sweet spot" within the MALDI samples. 2,5-DHBA crystals with and without analytes on a target plate obtained by means of a dried-droplet preparation contain two polymorphs, which can be distinguished by Raman spectra. In comparing the Raman image with the MS image, a clear correlation between the signal distribution of glycopeptides and hydrophilic peptides and the specific crystal form of 2,5-DHBA could be made. The ionization of hydrophobic peptides appears to proceed in both types of polymorphic crystals. In addition, the derivatization of glycopeptides with a pyrene group enabled us to detect glycopeptides regardless the crystal form. As the result, the number of sweet spots increased and MS spectra with a high signal intensity were obtained. The results suggest that the introduction of a hydrophobic/aromatic moiety to glycopeptides results in a more successful MALDI analysis due to the effective incorporation of the analyte into matrix crystals.

MALDI Imaging of Neutral Cuticular Lipids in Insects and Plants

Czech Academy of Sciences Publication Activity Database

Vrkoslav, Vladimír; Muck, A.; Cvačka, Josef; Svatoš, A.

2010-01-01

Roč. 21, č. 2 (2010), s. 220-231 ISSN 1044-0305 R&D Projects: GA ČR GA203/09/0139 Institutional research plan: CEZ:AV0Z40550506 Keywords : MALDI imaging * epicuticular waxes * neutral lipids Subject RIV: CC - Organic Chemistry Impact factor: 3.830, year: 2010

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

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.

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET.

Science.gov (United States)

Beeman, Katrin; Baumgärtner, Jens; Laubenheimer, Manuel; Hergesell, Karlheinz; Hoffmann, Martin; Pehl, Ulrich; Fischer, Frank; Pieck, Jan-Carsten

2017-12-01

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated "in-line reader" for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

Offline coupling of low-pressure anion-exchange chromatography with MALDI-MS to determine the elution order of human milk oligosaccharides.

Science.gov (United States)

Finke, B; Mank, M; Daniel, H; Stahl, B

2000-09-10

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution "fingerprint" of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before. Copyright 2000 Academic Press.

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.

Nanoparticle-assisted laser desorption/ionization mass spectrometry: Novel sample preparation methods and nanoparticle screening for plant metabolite imaging

Energy Technology Data Exchange (ETDEWEB)

Yagnik, Gargey B. [Iowa State Univ., Ames, IA (United States)

2016-02-19

The main goal of the presented research is development of nanoparticle based matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). This dissertation includes the application of previously developed data acquisition methods, development of novel sample preparation methods, application and comparison of novel nanoparticle matrices, and comparison of two nanoparticle matrix application methods for MALDI-MS and MALDI-MS imaging.

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.

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 .

Quantification of low molecular weight compounds by MALDI imaging mass spectrometry - A tutorial review.

Science.gov (United States)

Rzagalinski, Ignacy; Volmer, Dietrich A

2017-07-01

Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) permits label-free in situ analysis of chemical compounds directly from the surface of two-dimensional biological tissue slices. It links qualitative molecular information of compounds to their spatial coordinates and distribution within the investigated tissue. MALDI-MSI can also provide the quantitative amounts of target compounds in the tissue, if proper calibration techniques are performed. Obviously, as the target molecules are embedded within the biological tissue environment and analysis must be performed at their precise locations, there is no possibility for extensive sample clean-up routines or chromatographic separations as usually performed with homogenized biological materials; ion suppression phenomena therefore become a critical side effect of MALDI-MSI. Absolute quantification by MALDI-MSI should provide an accurate value of the concentration/amount of the compound of interest in relatively small, well-defined region of interest of the examined tissue, ideally in a single pixel. This goal is extremely challenging and will not only depend on the technical possibilities and limitations of the MSI instrument hardware, but equally on the chosen calibration/standardization strategy. These strategies are the main focus of this article and are discussed and contrasted in detail in this tutorial review. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

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.

Cyclodextrin-supported organic matrix for application of MALDI-MS for forensics. Soft-ionization to obtain protonated molecules of low molecular weight compounds

International Nuclear Information System (INIS)

Yonezawa, Tetsu; Asano, Takashi; Fujino, Tatsuya; Nishihara, Hiroshi

2013-01-01

Highlights: ► MALDI-MS applications for drug identification in forensic science is investigated. ► Cyclodextrin-supported organic matrices strongly suppress the obstacle peaks of organic matrix compounds. ► Cyclodextrin-supported organic matrices also suppress the alkali adducted molecule peaks. ► Sugar units of cyclodextrins work for this specific features. - Abstract: A mass measurement technique for detecting low-molecular-weight drugs with a cyclodextrin-supported organic matrix was investigated. By using cyclodextrin-supported 2,4,6-trihydroxyacetophenone (THAP), the matrix-related peaks of drugs were suppressed. The peaks of protonated molecules of the sample and THAP were mainly observed, and small fragments were detected in a few cases. Despite the Na + and K + peaks were observed in the spectrum, Na + or K + adduct sample molecules were undetected, owing to the sugar units of cyclodextrin. The advantages of MALDI-MS with cyclodextrin-supported matrices as an analytical tool for forensic samples are discussed. The suppression of alkali adducted molecules and desorption process are also discussed

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.

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

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

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.

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.

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.

3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution.

Science.gov (United States)

Körsgen, Martin; Pelster, Andreas; Dreisewerd, Klaus; Arlinghaus, Heinrich F

2016-02-01

The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-μm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution. Graphical Abstract ᅟ.

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...

Multimodal MSI in Conjunction with Broad Coverage Spatially Resolved MS² Increases Confidence in Both Molecular Identification and Localization

Energy Technology Data Exchange (ETDEWEB)

Veličković, Dušan; Chu, Rosalie K.; Carrell, Alyssa A. [Biosciences; Thomas, Mathew; Paša-Tolić, Ljiljana; Weston, David J. [Biosciences; Anderton, Christopher R.

2017-12-18

One critical aspect of mass spectrometry imaging (MSI) is the need to confidently identify detected analytes. While orthogonal tandem MS (e.g., LC-MS²) experiments from sample extracts can assist in annotating ions, the spatial information about these molecules is lost. Accordingly, this could cause mislead conclusions, especially in cases where isobaric species exhibit different distributions within a sample. In this Technical Note, we employed a multimodal imaging approach, using matrix assisted laser desorption/ionization (MALDI)-MSI and liquid extraction surface analysis (LESA)-MS²I, to confidently annotate and One critical aspect of mass spectrometry imaging (MSI) is the need to confidently identify detected analytes. While orthogonal tandem MS (e.g., LC-MS2) experiments from sample extracts can assist in annotating ions, the spatial information about these molecules is lost. Accordingly, this could cause mislead conclusions, especially in cases where isobaric species exhibit different distributions within a sample. In this Technical Note, we employed a multimodal imaging approach, using matrix assisted laser desorption/ionization (MALDI)-MSI and liquid extraction surface analysis (LESA)-MS²I, to confidently annotate and localize a broad range of metabolites involved in a tripartite symbiosis system of moss, cyanobacteria, and fungus. We found that the combination of these two imaging modalities generated very congruent ion images, providing the link between highly accurate structural information onfered by LESA and high spatial resolution attainable by MALDI. These results demonstrate how this combined methodology could be very useful in differentiating metabolite routes in complex systems.

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.

MALDI imaging facilitates new topical drug development process by determining quantitative skin distribution profiles.

Science.gov (United States)

Bonnel, David; Legouffe, Raphaël; Eriksson, André H; Mortensen, Rasmus W; Pamelard, Fabien; Stauber, Jonathan; Nielsen, Kim T

2018-04-01

Generation of skin distribution profiles and reliable determination of drug molecule concentration in the target region are crucial during the development process of topical products for treatment of skin diseases like psoriasis and atopic dermatitis. Imaging techniques like mass spectrometric imaging (MSI) offer sufficient spatial resolution to generate meaningful distribution profiles of a drug molecule across a skin section. In this study, we use matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to generate quantitative skin distribution profiles based on tissue extinction coefficient (TEC) determinations of four different molecules in cross sections of human skin explants after topical administration. The four drug molecules: roflumilast, tofacitinib, ruxolitinib, and LEO 29102 have different physicochemical properties. In addition, tofacitinib was administrated in two different formulations. The study reveals that with MALDI-MSI, we were able to observe differences in penetration profiles for both the four drug molecules and the two formulations and thereby demonstrate its applicability as a screening tool when developing a topical drug product. Furthermore, the study reveals that the sensitivity of the MALDI-MSI techniques appears to be inversely correlated to the drug molecules' ability to bind to the surrounding tissues, which can be estimated by their Log D values. Graphical abstract.

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.

Development of matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) for plant metabolite analysis

Energy Technology Data Exchange (ETDEWEB)

Korte, Andrew R [Iowa State Univ., Ames, IA (United States)

2014-12-01

This thesis presents efforts to improve the methodology of matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) as a method for analysis of metabolites from plant tissue samples. The first chapter consists of a general introduction to the technique of MALDI-MSI, and the sixth and final chapter provides a brief summary and an outlook on future work.

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.

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.

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.

Cyclodextrin-supported organic matrix for application of MALDI-MS for forensics. Soft-ionization to obtain protonated molecules of low molecular weight compounds

Energy Technology Data Exchange (ETDEWEB)

Yonezawa, Tetsu, E-mail: tetsu@eng.hokudai.ac.jp [Division of Materials Science and Engineering, Faculty of Engineering, Hokkaido University, Kita 13, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-8628 (Japan); Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Asano, Takashi [Division of Materials Science and Engineering, Faculty of Engineering, Hokkaido University, Kita 13, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-8628 (Japan); Criminal Investigation Laboratory, Metropolitan Police Department, 2-1-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8929 (Japan); Fujino, Tatsuya [Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo 192-0397 (Japan); Nishihara, Hiroshi [Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

2013-06-20

Highlights: ► MALDI-MS applications for drug identification in forensic science is investigated. ► Cyclodextrin-supported organic matrices strongly suppress the obstacle peaks of organic matrix compounds. ► Cyclodextrin-supported organic matrices also suppress the alkali adducted molecule peaks. ► Sugar units of cyclodextrins work for this specific features. - Abstract: A mass measurement technique for detecting low-molecular-weight drugs with a cyclodextrin-supported organic matrix was investigated. By using cyclodextrin-supported 2,4,6-trihydroxyacetophenone (THAP), the matrix-related peaks of drugs were suppressed. The peaks of protonated molecules of the sample and THAP were mainly observed, and small fragments were detected in a few cases. Despite the Na{sup +} and K{sup +} peaks were observed in the spectrum, Na{sup +} or K{sup +} adduct sample molecules were undetected, owing to the sugar units of cyclodextrin. The advantages of MALDI-MS with cyclodextrin-supported matrices as an analytical tool for forensic samples are discussed. The suppression of alkali adducted molecules and desorption process are also discussed.

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

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.

Recent advances in matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) for in situ analysis of endogenous molecules in plants.

Science.gov (United States)

Qin, Liang; Zhang, Yawen; Liu, Yaqin; He, Huixin; Han, Manman; Li, Yanyan; Zeng, Maomao; Wang, Xiaodong

2018-04-17

Mass spectrometry imaging (MSI) as a label-free and powerful imaging technique enables in situ evaluation of a tissue metabolome and/or proteome, becoming increasingly popular in the detection of plant endogenous molecules. The characterization of structure and spatial information of endogenous molecules in plants are both very important aspects to better understand the physiological mechanism of plant organism. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a commonly-used tissue imaging technique, which requires matrix to assist in situ detection of a variety of molecules on the surface of a tissue section. In previous studies, MALDI-MSI was mostly used for the detection of molecules from animal tissue sections, compared to plant samples due to cell structural limitations, such as plant cuticles, epicuticular waxes, and cell walls. Despite the enormous progress that has been made in tissue imaging, there is still a challenge for MALDI-MSI suitable for the imaging of endogenous compounds in plants. This review summarises the recent advances in MALDI-MSI, focusing on the application of in situ detection of endogenous molecules in different plant organs, i.e. root, stem, leaf, flower, fruit, and seed. Further improvements on instrumentation sensitivity, matrix selection, image processing and sample preparation will expand the application of MALDI-MSI in plant research. Copyright © 2018 John Wiley & Sons, Ltd.

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

The quest for improved reproducibility in MALDI mass spectrometry.

Science.gov (United States)

O'Rourke, Matthew B; Djordjevic, Steven P; Padula, Matthew P

2018-03-01

Reproducibility has been one of the biggest hurdles faced when attempting to develop quantitative protocols for MALDI mass spectrometry. The heterogeneous nature of sample recrystallization has made automated sample acquisition somewhat "hit and miss" with manual intervention needed to ensure that all sample spots have been analyzed. In this review, we explore the last 30 years of literature and anecdotal evidence that has attempted to address and improve reproducibility in MALDI MS. Though many methods have been attempted, we have discovered a significant publication history surrounding the use of nitrocellulose as a substrate to improve homogeneity of crystal formation and therefore reproducibility. We therefore propose that this is the most promising avenue of research for developing a comprehensive and universal preparation protocol for quantitative MALDI MS analysis. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:217-228, 2018. © 2016 Wiley Periodicals, Inc.

MALDI (matrix assisted laser desorption ionization) Imaging Mass Spectrometry (IMS) of skin: Aspects of sample preparation.

Science.gov (United States)

de Macedo, Cristiana Santos; Anderson, David M; Schey, Kevin L

2017-11-01

MALDI (matrix assisted laser desorption ionization) Imaging Mass Spectrometry (IMS) allows molecular analysis of biological materials making possible the identification and localization of molecules in tissues, and has been applied to address many questions on skin pathophysiology, as well as on studies about drug absorption and metabolism. Sample preparation for MALDI IMS is the most important part of the workflow, comprising specimen collection and preservation, tissue embedding, cryosectioning, washing, and matrix application. These steps must be carefully optimized for specific analytes of interest (lipids, proteins, drugs, etc.), representing a challenge for skin analysis. In this review, critical parameters for MALDI IMS sample preparation of skin samples will be described. In addition, specific applications of MALDI IMS of skin samples will be presented including wound healing, neoplasia, and infection. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

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.

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...

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.

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

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

Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality: A novel statistical approach for quality scoring.

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Kooijman, Pieter C; Kok, Sander J; Weusten, Jos J A M; Honing, Maarten

2016-05-05

Preparation of samples according to an optimized method is crucial for accurate determination of polymer sample characteristics by Matrix-Assisted Laser Desorption Ionization (MALDI) analysis. Sample preparation conditions such as matrix choice, cationization agent, deposition technique or even the deposition volume should be chosen to suit the sample of interest. Many sample preparation protocols have been developed and employed, yet finding the optimal sample preparation protocol remains a challenge. Because an objective comparison between the results of diverse protocols is not possible, "gut-feeling" or "good enough" is often decisive in the search for an optimum. This implies that sub-optimal protocols are used, leading to a loss of mass spectral information quality. To address this problem a novel analytical strategy based on MALDI imaging and statistical data processing was developed in which eight parameters were formulated to objectively quantify the quality of sample deposition and optimal MALDI matrix composition and finally sum up to an overall quality score of the sample deposition. These parameters can be established in a fully automated way using commercially available mass spectrometry imaging instruments without any hardware adjustments. With the newly developed analytical strategy the highest quality MALDI spots were selected, resulting in more reproducible and more valuable spectra for PEG in a variety of matrices. Moreover, our method enables an objective comparison of sample preparation protocols for any analyte and opens up new fields of investigation by presenting MALDI performance data in a clear and concise way. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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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.

On the origin of increased sensitivity and mass resolution using silicon masks in MALDI.

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Diologent, Laurent; Franck, Julien; Wisztorski, Maxence; Treizebre, Anthony; Focsa, Cristian; Fournier, Isabelle; Ziskind, Michael

2014-02-04

Since its development, MALDI has proved its performance in the analysis of intact biomolecules up to high molecular weights, regardless of their polarity. Sensitivity of MALDI instruments is a key point for breaking the limits of observing biomolecules of lower abundances. Instrumentation is one way to improve sensitivity by increasing ion transmission and using more sensitive detection systems. On the other side, improving MALDI ion production yields would have important outcomes. MALDI ion production is still not well-controlled and, indeed, the amount of ions produced per laser shot with respect to the total volume of desorbed material is very low. This has particular implications for certain applications, such as MALDI MS imaging where laser beam focusing as fine as possible (5-10 μm) is searched in order to reach higher spatial resolution images. However, various studies point out an intrinsic decrease in signal intensity for strong focusing. We have therefore been interested in developing silicon mask systems to decrease an irradiated area by cutting rather than focusing the laser beam and to study the parameters affecting sensitivity using such systems. For this, we systematically examined variation with laser fluence of intensity and spectral resolution in MALDI of standard peptides when using silicon-etched masks of various aperture sizes. These studies demonstrate a simultaneous increase in spectral resolution and signal intensity. Origin of this effect is discussed in the frame of the two-step ionization model. Experimental data in the low fluence range are fitted with an increase of the primary ionization through matrix-silicon edge contact provided by the masks. On the other hand, behavior at higher fluence could be explained by an effect on the secondary ionization via changes in the plume dynamics.

Optimizing UV laser focus profiles for improved MALDI performance.

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Holle, Armin; Haase, Andreas; Kayser, Markus; Höhndorf, Jens

2006-06-01

Matrix assisted laser desorption/ionization (MALDI) applications, such as proteomics, genomics, clinical profiling and MALDI imaging, have created a growing demand for faster instrumentation. Since the commonly used nitrogen lasers have throughput and life span limitations, diode-pumped solid-state lasers are an alternative. Unfortunately this type of laser shows clear performance limitations in MALDI in terms of sensitivity, resolution and ease of use, for applications such as thin-layer sample preparations, acceptance of various matrices (e.g. DHB for glycopeptides) and MALDI imaging. While it is obvious that the MALDI process has some dependence on the characteristics of the laser used, it is unclear which features are the most critical in determining laser performance for MALDI. In this paper we show, for the first time, that a spatially structured laser beam profile in lieu of a Gaussian profile is of striking importance. This result enabled us to design diode-pumped Nd : YAG lasers that on various critical applications perform as well for MALDI as the nitrogen lasers and in some respects even better. The modulation of the beam profile appears to be a new parameter for optimizing the MALDI process. In addition, the results trigger new questions directing us to a better understanding of the MALDI process. Copyright (c) 2006 John Wiley & Sons, Ltd.

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

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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.

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

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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.

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.

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.

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

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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…

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

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

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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

Matching the laser wavelength to the absorption properties of matrices increases the ion yield in UV-MALDI mass spectrometry.

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Wiegelmann, Marcel; Soltwisch, Jens; Jaskolla, Thorsten W; Dreisewerd, Klaus

2013-09-01

A high analytical sensitivity in ultraviolet matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is only achieved if the laser wavelength corresponds to a high optical absorption of the matrix. Laser fluence and the physicochemical properties of the compounds, e.g., the proton affinity, also influence analytical sensitivity significantly. In combination, these parameters determine the amount of material ejected per laser pulse and the ion yield, i.e., the fraction of ionized biomolecules. Here, we recorded peptide ion signal intensities as a function of these parameters. Three cinnamic acid matrices were investigated: α-cyano-4-hydroxycinnamic acid, α-cyano-4-chlorocinnamic acid, and α-cyano-2,4-difluorocinnamic acid. In addition, 2,5-dihydroxybenzoic acid was used in comparison experiments. Ion signal intensities "per laser shot" and integrated ion signal intensities were acquired over 900 consecutive laser pulses applied on distinct positions on the dried-droplet sample preparations. With respect to laser wavelength, the two standard MALDI wavelengths of 337/355 nm were investigated. Also, 305 or 320 nm was selected to account for the blue-shifted absorption profiles of the halogenated derivatives. Maximal peptide ion intensities were obtained if the laser wavelength fell within the peak of the absorption profile of the compound and for fluences two to three times the corresponding ion detection threshold. The results indicate ways for improving the analytical sensitivity in MALDI-MS, and in particular for MALDI-MS imaging applications where a limited amount of material is available per irradiated pixel.

Characterization of biodegradation intermediates of nonionic surfactants by MALDI-MS. 2. Oxidative biodegradation profiles of uniform octylphenol polyethoxylate in 18O-labeled water.

Science.gov (United States)

Sato, Hiroaki; Shibata, Atsushi; Wang, Yang; Yoshikawa, Hiromichi; Tamura, Hiroto

2003-01-01

This paper reports the characterization of the biodegradation intermediates of octylphenol octaethoxylate (OP(8)EO) by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The biodegradation test study was carried out in a pure culture (Pseudomonas putida S-5) under aerobic conditions using OP(8)EO as the sole carbon source and (18)O-labeled water as an incubation medium. In the MALDI-MS spectra of biodegraded samples, a series of OP(n)EO molecules with n = 2-8 EO units and their corresponding carboxylic acid products (OP(n)EC) were observed. The use of purified OP(8)EO enabled one to distinguish the shortened OPEO molecules as biodegradation intermediates. Furthermore, the formation of OP(8)EC (the oxidized product of OP(8)EO) supported the notion that terminal oxidation is a step in the biodegradation process. When biodegradation study was carried out in (18)O-labeled water, incorporation of (18)O atoms into the carboxyl group was observed for OPEC, while no incorporation was observed for the shortened OPEO products. These results could provide some rationale to the biodegradation mechanism of alkylphenol polyethoxylates.

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

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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.

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

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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.

Atmospheric pressure MALDI for the noninvasive characterization of carbonaceous ink from Renaissance documents.

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Grasso, Giuseppe; Calcagno, Marzia; Rapisarda, Alessandro; D'Agata, Roberta; Spoto, Giuseppe

2017-06-01

The analytical methods that are usually applied to determine the compositions of inks from ancient manuscripts usually focus on inorganic components, as in the case of iron gall ink. In this work, we describe the use of atmospheric pressure/matrix-assisted laser desorption ionization-mass spectrometry (AP/MALDI-MS) as a spatially resolved analytical technique for the study of the organic carbonaceous components of inks used in handwritten parts of ancient books for the first time. Large polycyclic aromatic hydrocarbons (L-PAH) were identified in situ in the ink of XVII century handwritten documents. We prove that it is possible to apply MALDI-MS as a suitable microdestructive diagnostic tool for analyzing samples in air at atmospheric pressure, thus simplifying investigations of the organic components of artistic and archaeological objects. The interpretation of the experimental MS results was supported by independent Raman spectroscopic investigations. Graphical abstract Atmospheric pressure/MALDI mass spectrometry detects in situ polycyclic aromatic hydrocarbons in the carbonaceous ink of XVII century manuscripts.

MALDI Mass Spectral Imaging of Bile Acids Observed as Deprotonated Molecules and Proton-Bound Dimers from Mouse Liver Sections

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Rzagalinski, Ignacy; Hainz, Nadine; Meier, Carola; Tschernig, Thomas; Volmer, Dietrich A.

2018-02-01

Bile acids (BAs) play two vital roles in living organisms, as they are involved in (1) the secretion of cholesterol from liver, and (2) the lipid digestion/absorption in the intestine. Abnormal bile acid synthesis or secretion can lead to severe liver disorders. Even though there is extensive literature on the mass spectrometric determination of BAs in biofluids and tissue homogenates, there are no reports on the spatial distribution in the biliary network of the liver. Here, we demonstrate the application of high mass resolution/mass accuracy matrix-assisted laser desorption/ionization (MALDI)-Fourier-transform ion cyclotron resonance (FTICR) to MS imaging (MSI) of BAs at high spatial resolutions (pixel size, 25 μm). The results show chemical heterogeneity of the mouse liver sections with a number of branching biliary and blood ducts. In addition to ion signals from deprotonation of the BA molecules, MALDI-MSI generated several further intense signals at larger m/z for the BAs. These signals were spatially co-localized with the deprotonated molecules and easily misinterpreted as additional products of BA biotransformations. In-depth analysis of accurate mass shifts and additional electrospray ionization and MALDI-FTICR experiments, however, confirmed them as proton-bound dimers. Interestingly, dimers of bile acids, but also unusual mixed dimers of different taurine-conjugated bile acids and free taurine, were identified. Since formation of these complexes will negatively influence signal intensities of the desired [M - H]- ions and significantly complicate mass spectral interpretations, two simple broadband techniques were proposed for non-selective dissociation of dimers that lead to increased signals for the deprotonated BAs. [Figure not available: see fulltext.

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...

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.

Laser desorption/ionization mass spectrometry for direct profiling and imaging of small molecules from raw biological materials

Energy Technology Data Exchange (ETDEWEB)

Cha, Sangwon [Iowa State Univ., Ames, IA (United States)

2008-01-01

Matrix-assisted laser desorption/ionization(MALDI) mass spectrometry(MS) has been widely used for analysis of biological molecules, especially macromolecules such as proteins. However, MALDI MS has a problem in small molecule (less than 1 kDa) analysis because of the signal saturation by organic matrixes in the low mass region. In imaging MS (IMS), inhomogeneous surface formation due to the co-crystallization process by organic MALDI matrixes limits the spatial resolution of the mass spectral image. Therefore, to make laser desorption/ionization (LDI) MS more suitable for mass spectral profiling and imaging of small molecules directly from raw biological tissues, LDI MS protocols with various alternative assisting materials were developed and applied to many biological systems of interest. Colloidal graphite was used as a matrix for IMS of small molecules for the first time and methodologies for analyses of small metabolites in rat brain tissues, fruits, and plant tissues were developed. With rat brain tissues, the signal enhancement for cerebroside species by colloidal graphite was observed and images of cerebrosides were successfully generated by IMS. In addition, separation of isobaric lipid ions was performed by imaging tandem MS. Directly from Arabidopsis flowers, flavonoids were successfully profiled and heterogeneous distribution of flavonoids in petals was observed for the first time by graphite-assisted LDI(GALDI) IMS.

MALDI-imaging guided microproteomics workflow for biomarker discovery of intra-tumor heterogeneity

OpenAIRE

Alberts, Deborah; Longuespée, Rémi; Smargiasso, Nicolas; Mazzucchelli, Gabriel; Baiwir, Dominique; DELVENNE, Philippe; Pamelard, Fabien; Picard de Meller, Gael; De Pauw, Edwin

2016-01-01

Introduction A single tumoral tissue can bear phenotypically different cell populations. This phenomenon called intra-tumor heterogeneity can lead to differential behaviors regarding metastasis seeding and therapy resistance [Zardavas et al., Nature Rev. Clin. Onc. 2015]. MALDI imaging has proven its efficiency for revealing hidden molecular features offering an insight into distinct cellular regions based on their molecular content. Further, proteomics applied to these regions could allo...

Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry for the Investigation of Proteins and Peptides

Science.gov (United States)

Burnum, Kristin E.; Frappier, Sara L.; Caprioli, Richard M.

2008-07-01

Mass spectrometry (MS) is an excellent technology for molecular imaging because of its high data dimensionality. MS can monitor thousands of individual molecular data channels measured as mass-to-charge (m/z). We describe the use of matrix-assisted laser desorption/ionization (MALDI) MS for the image analysis of proteins, peptides, lipids, drugs, and metabolites in tissues. We discuss the basic instrumentation and sample preparation methods needed to produce high-resolution images and high image reproducibility. Matrix-addition protocols are briefly discussed along with normal operating procedures, and selected biological and medical applications of MALDI imaging MS are described. We give examples of both two- and three-dimensional imaging, including normal mouse embryo implantation, sperm maturation in mouse epididymis, protein distributions in brain sections, protein alterations as a result of drug administration, and protein changes in brain due to neurodegeneration and tumor formation. Advantages of this technology and future challenges for its improvement are discussed.

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.

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

Directory of Open Access Journals (Sweden)

Marko Jovanović

2014-04-01

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

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...

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

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

Mevalonate-derived quinonemethide triterpenoid from in vitro roots of Peritassa laevigata and their localization in root tissue by MALDI imaging

Science.gov (United States)

Pina, Edieidia S.; Silva, Denise B.; Teixeira, Simone P.; Coppede, Juliana S.; Furlan, Maysa; França, Suzelei C.; Lopes, Norberto P.; Pereira, Ana Maria S.; Lopes, Adriana A.

2016-03-01

Biosynthetic investigation of quinonemethide triterpenoid 22β-hydroxy-maytenin (2) from in vitro root cultures of Peritassa laevigata (Celastraceae) was conducted using 13C-precursor. The mevalonate pathway in P. laevigata is responsible for the synthesis of the quinonemethide triterpenoid scaffold. Moreover, anatomical analysis of P. laevigata roots cultured in vitro and in situ showed the presence of 22β-hydroxy-maytenin (2) and maytenin (1) in the tissues from transverse or longitudinal sections with an intense orange color. MALDI-MS imaging confirmed the distribution of (2) and (1) in the more distal portions of the root cap, the outer cell layers, and near the vascular cylinder of P. laevigata in vitro roots suggesting a role in plant defense against infection by microorganisms as well as in the root exudation processes.

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

Influence of the Laser Spot Size, Focal Beam Profile, and Tissue Type on the Lipid Signals Obtained by MALDI-MS Imaging in Oversampling Mode.

Science.gov (United States)

Wiegelmann, Marcel; Dreisewerd, Klaus; Soltwisch, Jens

2016-12-01

To improve the lateral resolution in matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) beyond the dimensions of the focal laser spot oversampling techniques are employed. However, few data are available on the effect of the laser spot size and its focal beam profile on the ion signals recorded in oversampling mode. To investigate these dependencies, we produced 2 times six spots with dimensions between ~30 and 200 μm. By optional use of a fundamental beam shaper, square flat-top and Gaussian beam profiles were compared. MALDI-MSI data were collected using a fixed pixel size of 20 μm and both pixel-by-pixel and continuous raster oversampling modes on a QSTAR mass spectrometer. Coronal mouse brain sections coated with 2,5-dihydroxybenzoic acid matrix were used as primary test systems. Sizably higher phospholipid ion signals were produced with laser spots exceeding a dimension of ~100 μm, although the same amount of material was essentially ablated from the 20 μm-wide oversampling pixel at all spot size settings. Only on white matter areas of the brain these effects were less apparent to absent. Scanning electron microscopy images showed that these findings can presumably be attributed to different matrix morphologies depending on tissue type. We propose that a transition in the material ejection mechanisms from a molecular desorption at large to ablation at smaller spot sizes and a concomitant reduction in ion yields may be responsible for the observed spot size effects. The combined results indicate a complex interplay between tissue type, matrix crystallization, and laser-derived desorption/ablation and finally analyte ionization. Graphical Abstract ᅟ.

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.

Direct Visualization of Neurotransmitters in Rat Brain Slices by Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI - MS)

Science.gov (United States)

Fernandes, Anna Maria A. P.; Vendramini, Pedro H.; Galaverna, Renan; Schwab, Nicolas V.; Alberici, Luciane C.; Augusti, Rodinei; Castilho, Roger F.; Eberlin, Marcos N.

2016-12-01

Mass spectrometry imaging (MSI) of neurotransmitters has so far been mainly performed by matrix-assisted laser desorption/ionization (MALDI) where derivatization reagents, deuterated matrix and/or high resolution, or tandem MS have been applied to circumvent problems with interfering ion peaks from matrix and from isobaric species. We herein describe the application of desorption electrospray ionization mass spectrometry imaging (DESI)-MSI in rat brain coronal and sagittal slices for direct spatial monitoring of neurotransmitters and choline with no need of derivatization reagents and/or deuterated materials. The amino acids γ-aminobutyric (GABA), glutamate, aspartate, serine, as well as acetylcholine, dopamine, and choline were successfully imaged using a commercial DESI source coupled to a hybrid quadrupole-Orbitrap mass spectrometer. The spatial distribution of the analyzed compounds in different brain regions was determined. We conclude that the ambient matrix-free DESI-MSI is suitable for neurotransmitter imaging and could be applied in studies that involve evaluation of imbalances in neurotransmitters levels.

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...

Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil

Czech Academy of Sciences Publication Activity Database

Uhlík, Ondřej; Strejček, M.; Junková, P.; Šanda, Miloslav; Hroudová, Miluše; Vlček, Čestmír; Macková, Martina; Macek, Tomáš

2011-01-01

Roč. 77, č. 19 (2011), s. 6858-6866 ISSN 0099-2240 Grant - others:GA MŠk(CZ) ME09024; GA ČR(CZ) GA525/09/1058; GA MŠk(CZ) 2B06156 Program:GA; 2B Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : MALDI-TOF MS * bioremediation * MALDI Biotyper * bacterial identification Subject RIV: CC - Organic Chemistry Impact factor: 3.829, year: 2011

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.

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

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.

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

Protein biomarkers on tissue as imaged via MALDI mass spectrometry: A systematic approach to study the limits of detection.

Science.gov (United States)

van de Ven, Stephanie M W Y; Bemis, Kyle D; Lau, Kenneth; Adusumilli, Ravali; Kota, Uma; Stolowitz, Mark; Vitek, Olga; Mallick, Parag; Gambhir, Sanjiv S

2016-06-01

MALDI mass spectrometry imaging (MSI) is emerging as a tool for protein and peptide imaging across tissue sections. Despite extensive study, there does not yet exist a baseline study evaluating the potential capabilities for this technique to detect diverse proteins in tissue sections. In this study, we developed a systematic approach for characterizing MALDI-MSI workflows in terms of limits of detection, coefficients of variation, spatial resolution, and the identification of endogenous tissue proteins. Our goal was to quantify these figures of merit for a number of different proteins and peptides, in order to gain more insight in the feasibility of protein biomarker discovery efforts using this technique. Control proteins and peptides were deposited in serial dilutions on thinly sectioned mouse xenograft tissue. Using our experimental setup, coefficients of variation were biomarkers and a new benchmarking strategy that can be used for comparing diverse MALDI-MSI workflows. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

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.

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.

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.

Utilizing a Robotic Sprayer for High Lateral and Mass Resolution MALDI FT-ICR MSI of Microbial Cultures

Energy Technology Data Exchange (ETDEWEB)

Anderton, Christopher R.; Chu, Rosalie K.; Tolic, Nikola; Creissen, Alain V.; Pasa-Tolic, Ljiljana

2016-01-07

The ability to visualize biochemical interactions between microbial communities using MALDI MSI has provided tremendous insights into a variety of biological fields. Matrix application using a sieve proved to be incredibly useful, but it had many limitations that include uneven matrix coverage and limitation in the types of matrices one could employ in their studies. Recently, there has been a concerted effort to improve matrix application for studying agar plated microbial cultures, many of which utilized automated matrix sprayers. Here, we describe the usefulness of using a robotic sprayer for matrix application. The robotic sprayer has two-dimensional control over where matrix is applied and a heated capillary that allows for rapid drying of the applied matrix. This method provided a significant increase in MALDI sensitivity over the sieve method, as demonstrated by FT-ICR MS analysis, facilitating the ability to gain higher lateral resolution MS images of Bacillus Subtilis than previously reported. This method also allowed for the use of different matrices to be applied to the culture surfaces.

MALDI-ISD Mass Spectrometry Analysis of Hemoglobin Variants: a Top-Down Approach to the Characterization of Hemoglobinopathies

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Théberge, Roger; Dikler, Sergei; Heckendorf, Christian; Chui, David H. K.; Costello, Catherine E.; McComb, Mark E.

2015-08-01

Hemoglobinopathies are the most common inherited disorders in humans and are thus the target of screening programs worldwide. Over the past decade, mass spectrometry (MS) has gained a more important role as a clinical means to diagnose variants, and a number of approaches have been proposed for characterization. Here we investigate the use of matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) with sequencing using in-source decay (MALDI-ISD) for the characterization of Hb variants. We explored the effect of matrix selection using super DHB or 1,5-diaminonaphthalene on ISD fragment ion yield and distribution. MALDI-ISD MS of whole blood using super DHB simultaneously provided molecular weights for the alpha and beta chains, as well as extensive fragmentation in the form of sequence defining c-, (z + 2)-, and y-ion series. We observed sequence coverage on the first 70 amino acids positions from the N- and C-termini of the alpha and beta chains in a single experiment. An abundant beta chain N-terminal fragment ion corresponding to βc34 was determined to be a diagnostic marker ion for Hb S (β6 Glu→Val, sickle cell), Hb C (β6 Glu→Lys), and potentially for Hb E (β26 Glu→Lys). The MALDI-ISD analysis of Hb S and HbSC yielded mass shifts corresponding to the variants, demonstrating the potential for high-throughput screening. Characterization of an alpha chain variant, Hb Westmead (α122 His→Gln), generated fragments that established the location of the variant. This study is the first clinical application of MALDI-ISD MS for the determination and characterization of hemoglobin variants.

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

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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

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

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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.

Biomedical application of MALDI mass spectrometry for small-molecule analysis.

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van Kampen, Jeroen J A; Burgers, Peter C; de Groot, Ronald; Gruters, Rob A; Luider, Theo M

2011-01-01

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is an emerging analytical tool for the analysis of molecules with molar masses below 1,000 Da; that is, small molecules. This technique offers rapid analysis, high sensitivity, low sample consumption, a relative high tolerance towards salts and buffers, and the possibility to store sample on the target plate. The successful application of the technique is, however, hampered by low molecular weight (LMW) matrix-derived interference signals and by poor reproducibility of signal intensities during quantitative analyses. In this review, we focus on the biomedical application of MALDI-MS for the analysis of small molecules and discuss its favorable properties and its challenges as well as strategies to improve the performance of the technique. Furthermore, practical aspects and applications are presented. © 2010 Wiley Periodicals, Inc.

Matrix-assisted laser-desorption/ionization mass spectrometric imaging of olanzapine in a single hair using esculetin as a matrix.

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Wang, Hang; Wang, Ying; Wang, Ge; Hong, Lizhi

2017-07-15

Matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for the analysis of intact hair is a powerful tool for monitoring changes in drug consumption. The embedding of a low drug concentration in the hydrophobic hair matrix makes it difficult to extract and detect, and requires an improved method to increase detection sensitivity. In this study, an MSI method using MALDI-Fourier transform ion cyclotron resonance was developed for direct identification and imaging of olanzapine in hair samples using the positive ion mode. Following decontamination, scalp hair samples from an olanzapine user were scraped from the proximal to the distal end three times, and 5mm hair sections were fixed onto an Indium-Tin-Oxide (ITO)-coated microscopic glass slide. Esculetin (6,7-dihydroxy-2H-chromen-2-one) was used as a new hydrophobic matrix to increase the affinity, extraction and ionization efficiency of olanzapine in the hair samples. The spatial distribution of olanzapine was observed using five single hairs from the same drug user. This matrix improves the affinity of olanzapine in hair for molecular imaging with mass spectrometry. This method may provide a detection power for olanzapine to the nanogram level per 5mm hair. Time course changes in the MSI results were also compared with quantitative HPLC-MS/MS for each 5mm segment of single hair shafts selected from the MALDI target. MALDI imaging intensities in single hairs showed good semi-quantitative correlation with the results from conventional HPLC-MS/MS. MALDI-MSI is suitable for monitoring drug intake with a high time resolution. Copyright © 2017 Elsevier B.V. All rights reserved.

Mass Spectrometry Imaging of Biological Tissue: An Approach for Multicenter Studies

Energy Technology Data Exchange (ETDEWEB)

Rompp, Andreas; Both, Jean-Pierre; Brunelle, Alain; Heeren, Ronald M.; Laprevote, Olivier; Prideaux, Brendan; Seyer, Alexandre; Spengler, Bernhard; Stoeckli, Markus; Smith, Donald F.

2015-03-01

Mass spectrometry imaging has become a popular tool for probing the chemical complexity of biological surfaces. This led to the development of a wide range of instrumentation and preparation protocols. It is thus desirable to evaluate and compare the data output from different methodologies and mass spectrometers. Here, we present an approach for the comparison of mass spectrometry imaging data from different laboratories (often referred to as multicenter studies). This is exemplified by the analysis of mouse brain sections in five laboratories in Europe and the USA. The instrumentation includes matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF), MALDI-QTOF, MALDIFourier transform ion cyclotron resonance (FTICR), atmospheric-pressure (AP)-MALDI-Orbitrap, and cluster TOF-secondary ion mass spectrometry (SIMS). Experimental parameters such as measurement speed, imaging bin width, and mass spectrometric parameters are discussed. All datasets were converted to the standard data format imzML and displayed in a common open-source software with identical parameters for visualization, which facilitates direct comparison of MS images. The imzML conversion also allowed exchange of fully functional MS imaging datasets between the different laboratories. The experiments ranged from overview measurements of the full mouse brain to detailed analysis of smaller features (depending on spatial resolution settings), but common histological features such as the corpus callosum were visible in all measurements. High spatial resolution measurements of AP-MALDI-Orbitrap and TOF-SIMS showed comparable structures in the low-micrometer range. We discuss general considerations for planning and performing multicenter studies in mass spectrometry imaging. This includes details on the selection, distribution, and preparation of tissue samples as well as on data handling. Such multicenter studies in combination with ongoing activities for reporting guidelines, a common

[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.

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

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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.

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.

Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

International Nuclear Information System (INIS)

McDonald, Chris; Li Liang

2005-01-01

Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC

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.

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.

MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

Science.gov (United States)

Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R

2014-01-01

A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

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

Directory of Open Access Journals (Sweden)

Mariela S Zárate

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

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

MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue

DEFF Research Database (Denmark)

Hanrieder, Jørg; Wicher, Grzegorz; Bergquist, Jonas

2011-01-01

. Complementary proteomic experiments revealed the identity of these signature proteins that were predominantly expressed in the different glial cell types, including histone H4 for oligodendrocytes and S100-A10 for astrocytes. MALDI imaging MS was performed, and signature masses were employed as molecular...... tracers for prediction of oligodendroglial and astroglial localization in brain tissue. The different cell type specific protein distributions in tissue were validated using immunohistochemistry. ICMS of intact neuroglia is a simple and straightforward approach for characterization and discrimination...

Automated MALDI Matrix Coating System for Multiple Tissue Samples for Imaging Mass Spectrometry

Science.gov (United States)

Mounfield, William P.; Garrett, Timothy J.

2012-03-01

Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions, including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality and more reproducible than from tissue specimens prepared by the inkjet deposition method.

Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms.

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Amantonico, Andrea; Urban, Pawel L; Fagerer, Stephan R; Balabin, Roman M; Zenobi, Renato

2010-09-01

Heterogeneity is a characteristic feature of all populations of living organisms. Here we make an attempt to validate a single-cell mass spectrometric method for detection of changes in metabolite levels occurring in populations of unicellular organisms. Selected metabolites involved in central metabolism (ADP, ATP, GTP, and UDP-Glucose) could readily be detected in single cells of Closterium acerosum by means of negative-mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The analytical capabilities of this approach were characterized using standard compounds. The method was then used to study populations of individual cells with different levels of the chosen metabolites. With principal component analysis and support vector machine algorithms, it was possible to achieve a clear separation of individual C. acerosum cells in different metabolic states. This study demonstrates the suitability of mass spectrometric analysis of metabolites in single cells to measure cell-population heterogeneity.

Application of the MALDI Biotyper to clinical microbiology: progress and potential.

Science.gov (United States)

Kostrzewa, Markus

2018-03-01

The introduction of the MALDI Biotyper in laboratories substantially changed microbiology practice, this has been called a revolution. The system accelerated diagnostic while costs were reduced and accuracy was increased. In just a few years MALDI-TOF MS became the first-line identification tool for microorganisms. Ten years after its introduction, more than 2000 MALDI Biotyper systems are installed in laboratories which are performing routine diagnostic, and the number is still increasing. Areas covered: This article summarises changes in clinical microbiology introduced by the MALDI Biotyper and its effects, as it has been published in peer reviewed articles found in PubMed. Further, the potential of novel developments to increase the value of the system is described. Expert commentary: The MALDI Biotyper has significantly improved clinical microbiology in the area of microorganism identification. Now new developments and applications, e.g. for typing and resistance testing, might further increase its value in clinical microbiology. The systems might get the central diagnostic analyser which is getting integrated into the widely automated microbiology laboratories of the future.

Comprehensive characterization of natural organic matter by MALDI- and ESI-Fourier transform ion cyclotron resonance mass spectrometry

International Nuclear Information System (INIS)

Cao, Dong; Huang, Huogao; Hu, Ming; Cui, Lin; Geng, Fanglan; Rao, Ziyu; Niu, Hongyun; Cai, Yaqi; Kang, Yuehui

2015-01-01

Highlights: • MALDI-FT-ICR-MS was firstly employed for molecular characterization of NOM. • 1,8-Bis(dimethyl-amino)-naphthalene (DMAN) was used as matrix. • Mass spectra of NOM generated by MALDI and ESI methods were compared. • Complementary molecular information of NOM was provided by MALDI. - Abstract: Natural organic matter (NOM) is a complex and non-uniform mixture of organic compounds which plays an important role in environmental processes. Due to the complexity, it is challenging to obtain fully detailed structural information about NOM. Although Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has been demonstrated to be a powerful tool for providing molecular information about NOM, multiple ionization methods are needed for comprehensive characterization of NOM at the molecular level considering the ionizing selectivity of different ionization methods. This paper reports the first use of matrix assisted laser desorption/ionization (MALDI) method coupled with FT-ICR-MS for molecular characterization of NOM within a mass range of 200–800 Da. The mass spectral data obtained by MALDI were systematically compared with data generated by electrospray ionization (ESI). It showed that complementary molecular information about NOM which could not be detected by ESI, were provided by MALDI. More unsaturated and aromatic constituents of NOM with lower O/C ratio (O/C ratio < 0.5) were preferentially ionized in MALDI negative mode, whereas more polar constituents of NOM with higher O/C ratio were preferentially ionized in ESI negative mode. Molecular anions of NOM appearing at even m/z in MALDI negative ion mode were detected. The results show that NOM molecules with aromatic structures, moderate O/C ratio (0.7 > O/C ratio > 0.25) and lower H/C ratio were liable to form molecular anions at even m/z, whereas those with higher H/C ratio are more likely to form deprotonated ions at odd m/z. It is speculated that almost half of the NOM

MALDI imaging of enzymatic degradation of glycerides by lipase on textile surface

DEFF Research Database (Denmark)

Hall-Andersen, Jonatan; Kaasgaard, Svend G; Janfelt, Christian

2018-01-01

Most modern laundry detergents contain enzymes such as proteases, amylases, and lipases for more efficient removal of stains containing proteins, carbohydrates, and lipids during wash at low temperature. The function of the lipases is to hydrolyse the hydrophobic triglycerides from fats and oils...... stain and simulating washing cycles using well-defined detergents with lipase concentrations ranging between 0 and 0.5ppm. After washing, the textile swatches as well as cryo-sections of the swatches were imaged using MALDI imaging in positive ion mode at pixel sizes of 15-75μm. Similar samples were...... an inhomogeneous presence of diglycerides after lipase treatment both in planar images of the textile surface as well as in cross-sections suggesting a non-uniform enzyme effect or extraction of the lipase reaction products from the textile....

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

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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.

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...

The effect of temperature on the stability of compounds used as UV-MALDI-MS matrix: 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, alpha-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, nor-harmane and harmane.

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Tarzi, Olga I; Nonami, Hiroshi; Erra-Balsells, Rosa

2009-02-01

The thermal stability of several commonly used crystalline matrix-assisted ultraviolet laser desorption/ionization mass spectrometry (UV-MALDI-MS) matrices, 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THA), alpha-cyano-4-hydroxycinnamic acid (CHC), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid; SA), 9H-pirido[3,4-b]indole (nor-harmane; nor-Ho), 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho), perchlorate of nor-harmanonium ([nor-Ho+H]+) and perchlorate of harmanonium ([Ho+H]+) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI-MS), ultraviolet laserdesorption/ionization-time-of-flight-mass spectrometry (UV-LDI-TOF-MS) and electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV-absorption, fluorescence emission and excitation spectroscopy) and 1H nuclear magnetic resonance spectroscopy (1H-NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO2, yielding the trans-/cis-4-hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and 1H-NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well-known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV-MALDI-MS. Commercial SA (SA 98%; trans-SA/cis-SA 5:1) showed mainly cis- to-trans thermal isomerization and, with very poor yield, loss of CO2, yielding (3',5'-dimethoxy-4'-hydroxyphenyl)-1-ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV-MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted. Copyright (c) 2008 John Wiley & Sons, Ltd.

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.

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...

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.

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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.

Inter- and intra-organ spatial distributions of sea star saponins by MALDI imaging.

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Demeyer, Marie; Wisztorski, Maxence; Decroo, Corentin; De Winter, Julien; Caulier, Guillaume; Hennebert, Elise; Eeckhaut, Igor; Fournier, Isabelle; Flammang, Patrick; Gerbaux, Pascal

2015-11-01

Saponins are secondary metabolites that are abundant and diversified in echinoderms. Mass spectrometry is increasingly used not only to identify saponin congeners within animal extracts but also to decipher the structure/biological activity relationships of these molecules by determining their inter-organ and inter-individual variability. The usual method requires extensive purification procedures to prepare saponin extracts compatible with mass spectrometry analysis. Here, we selected the sea star Asterias rubens as a model animal to prove that direct analysis of saponins can be performed on tissue sections. We also demonstrated that carboxymethyl cellulose can be used as an embedding medium to facilitate the cryosectioning procedure. Matrix-assisted laser desorption/ionization (MALDI) imaging was also revealed to afford interesting data on the distribution of saponin molecules within the tissues. We indeed highlight that saponins are located not only inside the body wall of the animals but also within the mucus layer that probably protects the animal against external aggressions. Graphical Abstract Saponins are the most abundant secondary metabolites in sea stars. They should therefore participate in important biological activities. Here, MALDI imaging is presented as a powerful method to determine the spatial distribution of saponins within the animal tissues. The inhomogeneity of the intra-organ saponin distribution is highlighted, paving the way for future elegant structure/activity relationship investigations.

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

Electrospray and MALDI mass spectrometry in the identification of spermicides in criminal investigations.

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Hollenbeck, T P; Siuzdak, G; Blackledge, R D

1999-07-01

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been used to examine evidence in a sexual assault investigation. Because condoms are being used increasingly by sexual assailants and some condom brands include the spermicide nonoxynol-9 (nonylphenoxy polyethoxyethanol) in the lubricant formulation, the recovery, and identification of nonoxynol-9 from evidence items may assist in proving corpus delicti. A method was developed for the recovery of nonoxynol-9 from internal vaginal swabs and for its identification by reverse phase liquid chromatography/electrospray ionization mass spectrometry (LC ESI-MS), nanoelectrospray ionization (nanoESI) mass spectrometry, and high resolution MALDI Fourier transform mass spectrometry (MALDI-FTMS). The method was tested on extracts from precoitus, immediate postcoitus, and four-hours postcoitus vaginal swabs provided by a volunteer whose partner does not normally use condoms, but for this trial used a condom having a water-soluble gel-type lubricant that includes 5% nonoxynol-9 in its formulation. Subsequently, LC ESI-MS was used to identify traces of nonoxynol-9 from the internal vaginal swab of a victim of a sexual assault.

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

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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.

Mass spectrometry of rhenium complexes: a comparative study by using LDI-MS, MALDI-MS, PESI-MS and ESI-MS.

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Petroselli, Gabriela; Mandal, Mridul Kanti; Chen, Lee Chuin; Ruiz, Gustavo T; Wolcan, Ezequiel; Hiraoka, Kenzo; Nonami, Hiroshi; Erra-Balsells, Rosa

2012-03-01

A group of rhenium (I) complexes including in their structure ligands such as CF(3)SO(3)-, CH(3)CO(2)-, CO, 2,2'-bipyridine, dipyridil[3,2-a:2'3'-c]phenazine, naphthalene-2-carboxylate, anthracene-9-carboxylate, pyrene-1-carboxylate and 1,10-phenanthroline have been studied for the first time by mass spectrometry. The probe electrospray ionization (PESI) is a technique based on electrospray ionization (ESI) that generates electrospray from the tip of a solid metal needle. In this work, mass spectra for organometallic complexes obtained by PESI were compared with those obtained by classical ESI and high flow rate electrospray ionization assisted by corona discharge (HF-ESI-CD), an ideal method to avoid decomposition of the complexes and to induce their oxidation to yield intact molecular cation radicals in gas state [M](+·) and to produce their reduction yielding the gas species [M](-·). It was found that both techniques showed in general the intact molecular ions of the organometallics studied and provided additional structure characteristic diagnostic fragments. As the rhenium complexes studied in the present work showed strong absorption in the UV-visible region, particularly at 355 nm, laser desorption ionization (LDI) mass spectrometry experiments could be conducted. Although intact molecular ions could be detected in a few cases, LDI mass spectra showed diagnostic fragments for characterization of the complexes structure. Furthermore, matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained. Nor-harmane, a compound with basic character, was used as matrix, and the intact molecular ions were detected in two examples, in negative ion mode as the [M](-·) species. Results obtained with 2-[(2E)-3-(4-tert-buthylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) as matrix are also described. LDI experiments provided more information about the rhenium complex structures than did the MALDI ones. Copyright © 2012 John Wiley & Sons, Ltd.

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.

MALDI Mass Spectrometry Imaging for Visualizing In Situ Metabolism of Endogenous Metabolites and Dietary Phytochemicals

Science.gov (United States)

Fujimura, Yoshinori; Miura, Daisuke

2014-01-01

Understanding the spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmaceutical roles. Mass spectrometry imaging (MSI) enables determination of the distribution of ionizable molecules present in tissue sections of whole-body or single heterogeneous organ samples by direct ionization and detection. This emerging technique is now widely used for in situ label-free molecular imaging of endogenous or exogenous small molecules. MSI allows the simultaneous visualization of many types of molecules including a parent molecule and its metabolites. Thus, MSI has received much attention as a potential tool for pathological analysis, understanding pharmaceutical mechanisms, and biomarker discovery. On the other hand, several issues regarding the technical limitations of MSI are as of yet still unresolved. In this review, we describe the capabilities of the latest matrix-assisted laser desorption/ionization (MALDI)-MSI technology for visualizing in situ metabolism of endogenous metabolites or dietary phytochemicals (food factors), and also discuss the technical problems and new challenges, including MALDI matrix selection and metabolite identification, that need to be addressed for effective and widespread application of MSI in the diverse fields of biological, biomedical, and nutraceutical (food functionality) research. PMID:24957029

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

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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.

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.

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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.

Laser-induced hydrogen radical removal in UV MALDI-MS allows for the differentiation of flavonoid monoglycoside isomers.

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Yamagaki, Tohru; Watanabe, Takehiro; Tanaka, Masaki; Sugahara, Kohtaro

2014-01-01

Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M - H](-)) and the sugar-unit lost fragment ions ([M - Sugar - H](-)). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M - H* - H](-), [M - Sugar - H* - H](-), and [M - Sugar - 2H* - H](-). It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser. The glycosyl positions depend on the extent of the hydrogen radical removals and that of the homolytic cleavage of the glycosidic bonds. Flavonoid mono-glycoside isomers were distinguished according to their TOF MS and tandem mass spectra.

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.

MALDI Mass Spectrometry Imaging for Evaluation of Therapeutics in Colorectal Tumor Organoids

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Liu, Xin; Flinders, Colin; Mumenthaler, Shannon M.; Hummon, Amanda B.

2018-03-01

Patient-derived colorectal tumor organoids (CTOs) closely recapitulate the complex morphological, phenotypic, and genetic features observed in in vivo tumors. Therefore, evaluation of drug distribution and metabolism in this model system can provide valuable information to predict the clinical outcome of a therapeutic response in individual patients. In this report, we applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine the spatial distribution of the drug irinotecan and its metabolites in CTOs from two patients. Irinotecan is a prodrug and is often prescribed as part of therapeutic regimes for patients with advanced colorectal cancer. Irinotecan shows a time-dependent and concentration-dependent permeability and metabolism in the CTOs. More interestingly, the active metabolite SN-38 does not co-localize well with the parent drug irinotecan and the inactive metabolite SN-38G. The phenotypic effect of irinotecan metabolism was also confirmed by a viability study showing significantly reduced proliferation in the drug treated CTOs. MALDI-MSI can be used to investigate various pharmaceutical compounds in CTOs derived from different patients. By analyzing multiple CTOs from a patient, this method could be used to predict patient-specific drug responses and help to improve personalized dosing regimens. [Figure not available: see fulltext.

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.

Analytical investigations of toxic p-phenylenediamine (PPD) levels in clinical urine samples with special focus on MALDI-MS/MS.

Science.gov (United States)

Hooff, Gero P; van Huizen, Nick A; Meesters, Roland J W; Zijlstra, Eduard E; Abdelraheem, Mohamed; Abdelraheem, Waleed; Hamdouk, Mohamed; Lindemans, Jan; Luider, Theo M

2011-01-01

Para-phenylenediamine (PPD) is a common chromophoric ingredient in oxidative hair-dyes. In some African countries like Sudan, Egypt and Morocco but also in India this chemical is used alone or in combination with colouring extracts like Henna for dyeing of the hair or the skin. Excessive dermal exposure to PPD mainly leads to the N-mono- and N,N'-diacetylated products (MAPPD, DAPPD) by N-acetyltransferase 1 and 2 (NAT1 and 2) catalyzed reactions. Metabolites and PPD are mainly excreted via renal clearance. Despite a low risk of intoxication when used in due form, there are numerous cases of acute intoxication in those countries every year. At the ENT Hospital - Khartoum (Sudan) alone more than 300 cases are reported every year (~10% fatal), mostly caused by either an accidental or intended (suicidal) high systemic exposure to pure PPD. Intoxication leads to a severe clinical syndrome including laryngeal edema, rhabdomyolysis and subsequent renal failure, neurotoxicity and acute toxic hepatitis. To date, there is no defined clinical treatment or antidote available and treatment is largely supportive. Herein, we show the development of a quick on-site identification assay to facilitate differential diagnosis in the clinic and, more importantly, the implementation of an advanced analytical platform for future in-depth investigations of PPD intoxication and metabolism is described. The current work shows a sensitive (~25 µM) wet chemistry assay, a validated MALDI-MS/MS and HPLC-UV assay for the determination of PPD and its metabolites in human urine. We show the feasibility of the methods for measuring PPD over a range of 50-1000 µM. The validation criteria included linearity, lower limit of quantification (LLOQ), accuracy and precision, recovery and stability. Finally, PPD concentrations were determined in clinical urine samples of cases of acute intoxication and the applied technique was expanded to identify MAPPD and DAPPD in the identical samples.

In situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization MS analysis of polypeptide mixtures

DEFF Research Database (Denmark)

Kjellström, Sven; Jensen, Ole Nørregaard

2003-01-01

A novel liquid-liquid extraction (LLE) procedure was investigated for preparation of peptide and protein samples for matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). LLE using ethyl acetate as the water-immiscible organic solvent enabled segregation of hydrophobic...... matrix to the organic solvent enhanced the efficiency of the LLE-MALDI MS method for analysis of hydrophobic peptides and proteins. LLE-MALDI MS enabled the detection of the hydrophobic membrane protein bacteriorhodopsin as a component in a simple protein mixture. Peptide mixtures containing...... phosphorylated, glycosylated, or acylated peptides were successfully separated and analyzed by the in situ LLE-MALDI MS technique and demonstrate the potential of this method for enhanced separation and structural analysis of posttranslationally modified peptides in proteomics research....

Matrix-assisted laser desorption/ionization mass spectrometric imaging for the rapid segmental analysis of methamphetamine in a single hair using umbelliferone as a matrix.

Science.gov (United States)

Wang, Hang; Wang, Ying

2017-07-04

Segmental hair analysis offers a longer period for retrospective drug detection than blood or urine. Hair is a keratinous fiber and is strongly hydrophobic. The embedding of drugs in hydrophobic hair at low concentrations makes it difficult for extraction and detection with matrix-assisted laser desorption/ionization (MALDI) coupled with mass spectrometric imaging (MSI). In this study, a single scalp hair was longitudinally cut with a cryostat section to a length of 4 mm and fixed onto a stainless steel MALDI plate. Umbelliferone was used as a new hydrophobic matrix to enrich and assist the ionization efficiency of methamphetamine in the hair sample. MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS profiling and imaging were performed for direct detection and mapping of methamphetamine on the longitudinal sections of the single hair sample in positive ion mode. Using MALDI-MSI, the distribution of methamphetamine was observed throughout five longitudinally sectioned hair samples from a drug abuser. The changes of methamphetamine were also semi-quantified by comparing the ratios of methamphetamine/internal standard (I.S). This method improves the detection sensitivity of target drugs embedded in a hair matrix for imaging with mass spectrometry. The method could provide a detection level of methamphetamine down to a nanogram per milligram incorporated into hair. The results were also compared with the conventional high performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS) method. Changes in the imaging results over time by the MSI method showed good semi-quantitative correlation to the results from the HPLC-MS/MS method. This study provides a powerful tool for drug abuse control and forensic medicine analysis in a narrow time frame, and a reduction in the sample amount required. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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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.

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

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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.

Semiconductor Nanomaterials-Based Fluorescence Spectroscopic and Matrix-Assisted Laser Desorption/Ionization (MALDI Mass Spectrometric Approaches to Proteome Analysis

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Suresh Kumar Kailasa

2013-12-01

Full Text Available Semiconductor quantum dots (QDs or nanoparticles (NPs exhibit very unusual physico-chemcial and optical properties. This review article introduces the applications of semiconductor nanomaterials (NMs in fluorescence spectroscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS for biomolecule analysis. Due to their unique physico-chemical and optical properties, semiconductors NMs have created many new platforms for investigating biomolecular structures and information in modern biology. These semiconductor NMs served as effective fluorescent probes for sensing proteins and cells and acted as affinity or concentrating probes for enriching peptides, proteins and bacteria proteins prior to MALDI-MS analysis.

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

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Carolina Firacative

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

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.

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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

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

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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.

Z-sinapinic acid: the change of the stereochemistry of cinnamic acids as rational synthesis of a new matrix for carbohydrate MALDI-MS analysis.

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Salum, María L; Itovich, Lucia M; Erra-Balsells, Rosa

2013-11-01

Successful application of matrix-assisted laser desorption/ionization (MALDI) MS started with the introduction of efficient matrices such as cinnamic acid derivatives (i.e. 3,5-dimethoxy-4-hydroxycinnamic acid, SA; α-cyano-4-hydroxycinnamic acid). Since the empirical founding of these matrices, other commercial available cinnamic acids with different nature and location of substituents at benzene ring were attempted. Rational design and synthesis of new cinnamic acids have been recently described too. Because the presence of a rigid double bond in its molecule structure, cinnamic acids can exist as two different geometric isomers, the E-form and Z-form. Commercial available cinnamic acids currently used as matrices are the geometric isomers trans or E (E-cinnamic and trans-cinnamic acids). As a new rational design of MALDI matrices, Z-cinnamic acids were synthesized, and their properties as matrices were studied. Their performance was compared with that of the corresponding E-isomer and classical crystalline matrices (3,5-dihydroxybenzoic acid; norharmane) in the analysis of neutral/sulfated carbohydrates. Herein, we demonstrate the outstanding performance for Z-SA. Sulfated oligosaccharides were detected in negative ion mode, and the dissociation of sulfate groups was almost suppressed. Additionally, to better understand the quite different performance of each geometric isomer as matrix, the physical and morphological properties as well as the photochemical stability in solid state were studied. The influence of the E/Z photoisomerization of the matrix during MALDI was evaluated. Finally, molecular modeling (density functional theory study) of the optimized geometry and stereochemistry of E-cinnamic and Z-cinnamic acids revealed some factors governing the analyte-matrix interaction. Copyright © 2013 John Wiley & Sons, Ltd.

Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. botulinum Neurotoxin Detection

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Michael J. Perry

2017-03-01

Full Text Available Currently, the gold standard method for active botulinum neurotoxin (BoNT detection is the mouse bioassay (MBA. A Centers for Disease Control and Prevention-developed mass spectrometry (MS-based assay that detects active BoNT was successfully validated and implemented in a public health laboratory in clinical matrices using the Bruker MALDI-TOF MS (Matrix-assisted laser desorption ionization–time of flight mass spectrometry Biotyper. For the first time, a direct comparison with the MBA was performed to determine MS-based assay sensitivity using the Bruker MALDI Biotyper. Mice were injected with BoNT/A, /B, /E, and /F at concentrations surrounding the established MS assay limit of detection (LOD and analyzed simultaneously. For BoNT/B, /E, and /F, MS assay sensitivity was equivalent or better than the MBA at 25, 0.3, and 8.8 mLD50, respectively. BoNT/A was detected by the MBA between 1.8 and 18 mLD50, somewhat more sensitive than the MS method of 18 mLD50. Studies were performed to compare assay performance in clinical specimens. For all tested specimens, the MS method rapidly detected BoNT activity and serotype in agreement with, or in the absence of, results from the MBA. We demonstrate that the MS assay can generate reliable, rapid results while eliminating the need for animal testing.

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.

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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.

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.

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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.

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.

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

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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.

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

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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/.

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

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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.

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

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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.

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

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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.

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

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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.

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

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...

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

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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.

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

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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

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

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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.

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

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).

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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.

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

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

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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.

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.

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...

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.

ToF-SIMS Parallel Imaging MS/MS of Lipid Species in Thin Tissue Sections.

Science.gov (United States)

Bruinen, Anne Lisa; Fisher, Gregory L; Heeren, Ron M A

2017-01-01

Unambiguous identification of detected species is essential in complex biomedical samples. To date, there are not many mass spectrometry imaging techniques that can provide both high spatial resolution and identification capabilities. A new and patented imaging tandem mass spectrometer, exploiting the unique characteristics of the nanoTOF II (Physical Electronics, USA) TOF-SIMS TRIFT instrument, was developed to address this.Tandem mass spectrometry is based on the selection of precursor ions from the full secondary ion spectrum (MS 1 ), followed by energetic activation and fragmentation, and collection of the fragment ions to obtain a tandem MS spectrum (MS 2 ). The PHI NanoTOF II mass spectrometer is equipped with a high-energy collision induced dissociation (CID) fragmentation cell as well as a second time-of-flight analyzer developed for simultaneous ToF-SIMS and tandem MS imaging experiments.We describe here the results of a ToF-SIMS imaging experiment on a thin tissue section of an infected zebrafish as a model organism for tuberculosis. The focus is on the obtained ion distribution plot of a fatty acid as well as its identification by tandem mass spectrometry.

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

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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

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

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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.

Ionic solution and nanoparticle assisted MALDI-MS as bacterial biosensors for rapid analysis of yogurt.

Science.gov (United States)

Lee, Chia-Hsun; Gopal, Judy; Wu, Hui-Fen

2012-01-15

Bacterial analysis from food samples is a highly challenging task because food samples contain intensive interferences from proteins and carbohydrates. Three different conditions of yogurt were analyzed: (1) the fresh yogurt immediately after purchasing, (2) the yogurt after expiry date stored in the refrigerator and (3) the yogurt left outside, without refrigeration. The shelf lives of both these yogurt was compared in terms of the decrease in bacterial signals. AB which initially contained 10(9) cells/mL drastically reduced to 10(7) cells/mL. However, Lin (Feng-Yin) yogurt which initially (fresh) had 10(8) cells/mL, even after two weeks beyond the expiry period showed no marked drop in bacterial count. Conventional MALDI-MS analysis showed limited sensitivity for analysis of yogurt bacteria amidst the complex milk proteins present in yogurt. A cost effective ionic solution, CrO(4)(2-) solution was used to enable the successful detection of bacterial signals (40-fold increased in sensitivity) selectively without the interference of the milk proteins. 0.035 mg of Ag nanoparticles (NPs) were also found to improve the detection of bacteria 2-6 times in yogurt samples. The current approach can be further applied as a rapid, sensitive and effective platform for bacterial analysis from food. Copyright © 2011 Elsevier B.V. All rights reserved.

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...

A novel tantalum-based sol-gel packed microextraction syringe for highly specific enrichment of phosphopeptides in MALDI-MS applications.

Science.gov (United States)

Çelikbıçak, Ömür; Atakay, Mehmet; Güler, Ülkü; Salih, Bekir

2013-08-07

A new tantalum-based sol-gel material was synthesized using a unique sol-gel synthesis pathway by PEG incorporation into the sol-gel structure without performing a calcination step. This improved its chemical and physical properties for the high capacity and selective enrichment of phosphopeptides from protein digests in complex biological media. The specificity of the tantalum-based sol-gel material for phosphopeptides was evaluated and compared with tantalum(V) oxide (Ta2O5) in different phosphopeptide enrichment applications. The tantalum-based sol-gel and tantalum(V) oxide were characterized in detail using FT-IR spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM), and also using a surface area and pore size analyzer. In the characterization studies, the surface morphology, pore volume, crystallinity of the materials and PEG incorporation into the sol-gel structure to produce a more hydrophilic material were successfully demonstrated. The X-ray diffractograms of the two different materials were compared and it was noted that the broad signals of the tantalum-based sol-gel clearly represented the amorphous structure of the sol-gel material, which was more likely to create enough surface area and to provide more accessible tantalum atoms for phosphopeptides to be easily adsorbed when compared with the neat and more crystalline structure of Ta2O5. Therefore, the phosphopeptide enrichment performance of the tantalum-based sol-gels was found to be remarkably higher than the more crystalline Ta2O5 in our studies. Phosphopeptides at femtomole levels could be selectively enriched using the tantalum-based sol-gel and detected with a higher signal-to-noise ratio by matrix-assisted laser desorption/ionization-mass spectrometer (MALDI-MS). Moreover, phosphopeptides in a tryptic digest of non-fat bovine milk as a complex real-world biological sample were retained with higher yield using a tantalum-based sol-gel. Additionally, the sol-gel material

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.

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

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.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling

Science.gov (United States)

Comi, Troy J.; Neumann, Elizabeth K.; Do, Thanh D.; Sweedler, Jonathan V.

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. [Figure not available: see fulltext.

Structural Characterization of Neutral Saccharides by Negative Ion MALDI Mass Spectrometry Using a Superbasic Proton Sponge as Deprotonating Matrix

Science.gov (United States)

Calvano, Cosima Damiana; Cataldi, Tommaso R. I.; Kögel, Julius F.; Monopoli, Antonio; Palmisano, Francesco; Sundermeyer, Jorge

2017-08-01

The superbasic proton sponge 1,8-bis(tripyrrolidinylphosphazenyl)naphthalene (TPPN) has been successfully employed for the structural characterization of neutral saccharides, cyclodextrins, and saccharide alditols by matrix assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). Owing to its inherently high basicity, TPPN is capable of deprotonating neutral carbohydrates (M) providing an efficient and simple way to produce gas-phase [M - H]- ions. Highly informative negative ions MS/MS spectra showing several diagnostic fragment ions were obtained, mainly A-type cross-ring and C-type glycosidic cleavages. Indeed, cross-ring cleavages of monosaccharides with formation of 0,2A, 0,3A, 2,4A, 2,5A, 3,5A, and 0,3X product ions dominate the MS/MS spectra. A significant difference between reducing (e.g., lactose, maltose) and non-reducing disaccharides (e.g., sucrose, trehalose) was observed. Though disaccharides with the anomeric positions blocked give rise to deprotonated molecules, [M - H]-, at m/ z 341.1, reducing ones exhibited a peak at m/ z 340.1, most likely as radical anion, [M - H•- H]-•. The superiority of TPPN was clearly demonstrated by comparison with well recognized matrices, such as 2,5-dihydroxybenzoic acid and 2',4',6'-trihydroxyacetophenone (positive ion mode) and nor-harman (negative ion mode). MALDI MS/MS experiments on isotopically labeled sugars have greatly supported the interpretation of plausible fragmentation pathways.

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

Multi-imaging of Cytokinin and Abscisic Acid on the Roots of Rice (Oryza sativa) Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Science.gov (United States)

Shiono, Katsuhiro; Hashizaki, Riho; Nakanishi, Toyofumi; Sakai, Tatsuko; Yamamoto, Takushi; Ogata, Koretsugu; Harada, Ken-Ichi; Ohtani, Hajime; Katano, Hajime; Taira, Shu

2017-09-06

Plant hormones act as important signaling molecules that regulate responses to abiotic stress as well as plant growth and development. Because their concentrations of hormones control the physiological responses in the target tissue, it is important to know the distributions and concentrations in the tissues. However, it is difficult to determine the hormone concentration on the plant tissue as a result of the limitations of conventional methods. Here, we report the first multi-imaging of two plant hormones, one of cytokinin [i.e., trans-zeatin (tZ)] and abscisic acid (ABA) using a new technology, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) imaging. Protonated signals of tZ (m/z 220.1) and ABA (m/z 265.3) were chosen on longitudinal sections of rice roots for MS imaging. tZ was broadly distributed about 40 mm behind the root apex but was barely detectable at the apex, whereas ABA was mainly detected at the root apex. Multi-imaging using MALDI-TOF-MS enabled the visualization of the localization and quantification of plant hormones. Thus, this tool is applicable to a wide range of plant species growing under various environmental conditions.

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.

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.

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

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.

Hydrazinonicotinic acid derivatization for selective ionization and improved glycan structure characterization by MALDI-MS.

Science.gov (United States)

Jiao, Jing; Yang, Lijun; Zhang, Ying; Lu, Haojie

2015-08-21

The analysis of glycan is important for understanding cell biology and disease processes because the glycans play a key role in many important biological behaviors, such as cell division, cellular localization, tumor immunology and inflammation. Nevertheless, it is still hard work to analyze glycans by MALDI-MS, which generally stems from the inherent low abundance and the low ionization efficiency of glycans. Moreover, the difficulty in generating informative fragmentations further hinders glycans structure characterization. In this work, hydrazinonicotinic acid (HYNIC) was used as a novel derivatized reagent for improved and selective detection of glycans. Through tagging the reducing terminus of glycans with the diazanyl group of HYNIC, significant enhancement of the ionization efficiency of glycans was achieved. After derivatization, the signal to noise ratio (S/N) of the maltoheptaose was improved by more than one order of magnitude in positive mode. HYNIC derivatization also allowed the sensitive detection of sialylated glycan in negative mode, with a 15 fold enhancement of S/N. Interestingly, it is noteworthy that the HYNIC reagent not only effectively labeled the reducing end of glycans in the presence of tryptic peptides, but also suppressed the ionization of peptides, enabling the direct detection of glycans from glycoprotein without separation. Therefore, analysis of glycans became easier due to the omission of a pre-separation step. Importantly, by using different acid reagents as the catalyst, derivatized product signals corresponding to [M + Na](+) or [M + H](+) were obtained respectively, which yield complementary fragmentation patterns for the structure elucidation of glycans. Finally, more than 40 N-glycans were successfully detected in 10 μL human serum using this method.

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.

Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry.

OpenAIRE

Kirpekar, F; Douthwaite, S; Roepstorff, P

2000-01-01

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of post...

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

Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

Science.gov (United States)

Zampieri, Dávila; Santos, Vanessa G; Braga, Patrícia A C; Ferreira, Christina R; Ballottin, Daniela; Tasic, Ljubica; Basso, Andréa C; Sanches, Bruno V; Pontes, José H F; da Silva, Bárbara Pereira; Garboggini, Fabiana Fantinatti; Eberlin, Marcos N; Tata, Alessandra

2013-09-01

Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples. Copyright © 2013 Elsevier Inc. All rights reserved.

Imaging MS in Toxicology: An Investigation of Juvenile Rat Nephrotoxicity Associated with Dabrafenib Administration

Science.gov (United States)

Groseclose, M. Reid; Laffan, Susan B.; Frazier, Kendall S.; Hughes-Earle, Angela; Castellino, Stephen

2015-06-01

As part of an investigative nephrotoxicity study, kidney tissues from juvenile rats orally administered dabrafenib at different age intervals between postnatal day (PND) 7 to 35 were investigated by MALDI and LDI imaging mass spectrometry (IMS) to determine the chemical composition of tubular deposits. In the youngest age group (PND 7-13), MALDI IMS demonstrated that a dabrafenib carboxylic acid metabolite was diffusely localized to the regions of tubular deposits (medulla and corticomedullary junction); however, no dabrafenib-related material was detected directly from the deposits. Rather, the LDI IMS analysis determined that the deposits were composed primarily of calcium phosphate. Based on these data, the dabrafenib associated nephrotoxicity, including the formation of tubular deposits, was determined to be age dependent. Furthermore, immature renal function was hypothesized to be responsible for the susceptibility of the youngest pups.

Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry.

Science.gov (United States)

Zemski Berry, Karin A; Gordon, William C; Murphy, Robert C; Bazan, Nicolas G

2014-03-01

MALDI imaging mass spectrometry (IMS) was used to characterize lipid species within sections of human eyes. Common phospholipids that are abundant in most tissues were not highly localized and observed throughout the accessory tissue, optic nerve, and retina. Triacylglycerols were highly localized in accessory tissue, whereas sulfatide and plasmalogen glycerophosphoethanolamine (PE) lipids with a monounsaturated fatty acid were found enriched in the optic nerve. Additionally, several lipids were associated solely with the inner retina, photoreceptors, or retinal pigment epithelium (RPE); a plasmalogen PE lipid containing DHA (22:6), PE(P-18:0/22:6), was present exclusively in the inner retina, and DHA-containing glycerophosphatidylcholine (PC) and PE lipids were found solely in photoreceptors. PC lipids containing very long chain (VLC)-PUFAs were detected in photoreceptors despite their low abundance in the retina. Ceramide lipids and the bis-retinoid, N-retinylidene-N-retinylethanolamine, was tentatively identified and found only in the RPE. This MALDI IMS study readily revealed the location of many lipids that have been associated with degenerative retinal diseases. Complex lipid localization within retinal tissue provides a global view of lipid organization and initial evidence for specific functions in localized regions, offering opportunities to assess their significance in retinal diseases, such as macular degeneration, where lipids have been implicated in the disease process.

Analysis of antioxidants in insulation cladding of copper wire: a comparison of different mass spectrometric techniques (ESI-IT, MALDI-RTOF and RTOF-SIMS).

Science.gov (United States)

Schnöller, Johannes; Pittenauer, Ernst; Hutter, Herbert; Allmaier, Günter

2009-12-01

Commercial copper wire and its polymer insulation cladding was investigated for the presence of three synthetic antioxidants (ADK STAB AO412S, Irganox 1010 and Irganox MD 1024) by three different mass spectrometric techniques including electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS), matrix-assisted laser desorption/ionization reflectron time-of-flight (TOF) mass spectrometry (MALDI-RTOF-MS) and reflectron TOF secondary ion mass spectrometry (RTOF-SIMS). The samples were analyzed either directly without any treatment (RTOF-SIMS) or after a simple liquid/liquid extraction step (ESI-IT-MS, MALDI-RTOF-MS and RTOF-SIMS). Direct analysis of the copper wire itself or of the insulation cladding by RTOF-SIMS allowed the detection of at least two of the three antioxidants but at rather low sensitivity as molecular radical cations and with fairly strong fragmentation (due to the highly energetic ion beam of the primary ion gun). ESI-IT- and MALDI-RTOF-MS-generated abundant protonated and/or cationized molecules (ammoniated or sodiated) from the liquid/liquid extract. Only ESI-IT-MS allowed simultaneous detection of all three analytes in the extract of insulation claddings. The latter two so-called 'soft' desorption/ionization techniques exhibited intense fragmentation only by applying low-energy collision-induced dissociation (CID) tandem MS on a multistage ion trap-instrument and high-energy CID on a tandem TOF-instrument (TOF/RTOF), respectively. Strong differences in the fragmentation behavior of the three analytes could be observed between the different CID spectra obtained from either the IT-instrument (collision energy in the very low eV range) or the TOF/RTOF-instrument (collision energy 20 keV), but both delivered important structural information. Copyright 2009 John Wiley & Sons, Ltd.

Structural characterization of native high-methoxylated pectin using nuclear magnetic resonance spectroscopy and ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comparative use of 2,5-dihydroxybenzoic acid and nor-harmane as UV-MALDI matrices.

Science.gov (United States)

Monge, María Eugenia; Negri, R Martín; Kolender, Adriana A; Erra-Balsells, Rosa

2007-01-01

The successful analysis by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS) of native and hydrolyzed high-methoxylated pectin samples is described. In order to find the optimal conditions for UV-MALDI-TOF MS analysis several experimental variables were studied such as: different UV-MALDI matrices (nor-harmane, 2,5-dihydroxybenzoic acid), sample preparation methods (mixture, sandwich), inorganic salt addition (doping salts, NaCl, KCl, NH(4)Cl), ion mode (positive, negative), linear and reflectron mode, etc. nor-Harmane has never been used as a UV-MALDI matrix for the analysis of pectins but its use avoids pre-treatment of the sample, such as an enzymatic digestion or an acid hydrolysis, and there is no need to add salts, making the analysis easier and faster. This study suggested an alternative way of analyzing native high-methoxylated pectins, with UV-MALDI-TOF MS, by using nor-harmane as the matrix in negative ion mode. The analysis by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy of the native and hydrolyzed pectin is also briefly described. Copyright (c) 2007 John Wiley & Sons, Ltd.

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.

The combination of simple MALDI matrices for the improvement of intact glycoproteins and glycans analysis

Czech Academy of Sciences Publication Activity Database

Laštovičková, Markéta; Chmelík, Josef; Bobálová, Janette

2009-01-01

Roč. 281, 1-2 (2009), s. 82-88 ISSN 1387-3806 R&D Projects: GA AV ČR IAA600040701; GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : glycoproteins * binary matrices * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.117, year: 2009

The Development of Novel Nanodiamond Based MALDI Matrices for the Analysis of Small Organic Pharmaceuticals

Science.gov (United States)

Chitanda, Jackson M.; Zhang, Haixia; Pahl, Erica; Purves, Randy W.; El-Aneed, Anas

2016-10-01

The utility of novel functionalized nanodiamonds (NDs) as matrices for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) is described herein. MALDI-MS analysis of small organic compounds (<1000 Da) is typically complex because of interferences from numerous cluster ions formed when using conventional matrices. To expand the use of MALDI for the analysis of small molecules, novel matrices were designed by covalently linking conventional matrices (or a lysine moiety) to detonated NDs. Four new functionalized NDs were evaluated for their ionization capabilities using five pharmaceuticals with varying molecular structures. Two ND matrices were able to ionize all tested pharmaceuticals in the negative ion mode, producing the deprotonated ions [M - H]-. Ion intensity for target analytes was generally strong with enhanced signal-to-noise ratios compared with conventional matrices. The negative ion mode is of great importance for biological samples as interference from endogenous compounds is inherently minimized in the negative ion mode. Since the molecular structures of the tested pharmaceuticals did not suggest that negative ion mode would be preferable, this result magnifies the importance of these findings. On the other hand, conventional matrices primarily facilitated the ionization as expected in the positive ion mode, producing either the protonated molecules [M + H]+ or cationic adducts (typically producing complex spectra with numerous adduct peaks). The data presented in this study suggests that these matrices may offer advantages for the analysis of low molecular weight pharmaceuticals/metabolites.

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

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...

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

Molecules and elements for quantitative bioanalysis: The allure of using electrospray, MALDI, and ICP mass spectrometry side-by-side.

Science.gov (United States)

Linscheid, Michael W

2018-03-30

slightly adapted workflows, already in use for quantification in bioanalysis. Imaging mass spectrometry (MSI) with MALDI and laser ablation ICP-MS complemented the range of application in recent years. © 2018 Wiley Periodicals, Inc.

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

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).

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

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.

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.

Rapid Quantification of 25-Hydroxyvitamin D3 in Human Serum by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

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Qi, Yulin; Müller, Miriam; Stokes, Caroline S.; Volmer, Dietrich A.

2018-04-01

LC-MS/MS is widely utilized today for quantification of vitamin D in biological fluids. Mass spectrometric assays for vitamin D require very careful method optimization for precise and interference-free, accurate analyses however. Here, we explore chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) as a rapid alternative for quantitative measurement of 25-hydroxyvitamin D3 in human serum, and compare it to results from LC-MS/MS. The method implemented an automated imaging step of each MALDI spot, to locate areas of high intensity, avoid sweet spot phenomena, and thus improve precision. There was no statistically significant difference in vitamin D quantification between the MALDI-MS/MS and LC-MS/MS: mean ± standard deviation for MALDI-MS—29.4 ± 10.3 ng/mL—versus LC-MS/MS—30.3 ± 11.2 ng/mL (P = 0.128)—for the sum of the 25-hydroxyvitamin D epimers. The MALDI-based assay avoided time-consuming chromatographic separation steps and was thus much faster than the LC-MS/MS assay. It also consumed less sample, required no organic solvents, and was readily automated. In this proof-of-concept study, MALDI-MS readily demonstrated its potential for mass spectrometric quantification of vitamin D compounds in biological fluids.

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

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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.

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

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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.

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

Distribution of erlotinib in rash and normal skin in cancer patients receiving erlotinib visualized by matrix assisted laser desorption/ionization mass spectrometry imaging.

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Nishimura, Meiko; Hayashi, Mitsuhiro; Mizutani, Yu; Takenaka, Kei; Imamura, Yoshinori; Chayahara, Naoko; Toyoda, Masanori; Kiyota, Naomi; Mukohara, Toru; Aikawa, Hiroaki; Fujiwara, Yasuhiro; Hamada, Akinobu; Minami, Hironobu

2018-04-06

The development of skin rashes is the most common adverse event observed in cancer patients treated with epidermal growth factor receptor-tyrosine kinase inhibitors such as erlotinib. However, the pharmacological evidence has not been fully revealed. Erlotinib distribution in the rashes was more heterogeneous than that in the normal skin, and the rashes contained statistically higher concentrations of erlotinib than adjacent normal skin in the superficial skin layer (229 ± 192 vs. 120 ± 103 ions/mm 2 ; P = 0.009 in paired t -test). LC-MS/MS confirmed that the concentration of erlotinib in the skin rashes was higher than that in normal skin in the superficial skin layer (1946 ± 1258 vs. 1174 ± 662 ng/cm 3 ; P = 0.028 in paired t -test). The results of MALDI-MSI and LC-MS/MS were well correlated (coefficient of correlation 0.879, P distribution of erlotinib in the skin tissue was visualized using non-labeled MALDI-MSI. Erlotinib concentration in the superficial layer of the skin rashes was higher than that in the adjacent normal skin. We examined patients with advanced pancreatic cancer who developed skin rashes after treatment with erlotinib and gemcitabine. We biopsied both the rash and adjacent normal skin tissues, and visualized and compared the distribution of erlotinib within the skin using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The tissue concentration of erlotinib was also measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with laser microdissection.

Different target surfaces for the analysis of peptides, peptide mixtures and peptide mass fingerprints by AP-MALDI ion trap-mass spectrometry.

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Pittenauer, Ernst; Kassler, Alexander; Haubner, Roland; Allmaier, Günter

2011-06-10

The desorption/ionization behavior of individual peptides, an equimolare peptide mixture and a tryptic digest was investigated by AP-MALDI-IT-MS using four different target materials (gold-covered stainless steel (SS), titanium nitride-covered SS, hand-polished SS, and microdiamond-covered hardmetal) under identical conditions. Gold-covered as well as polished SS targets yielded comparable mass spectra for peptides and peptide mixture in the low pMol-range. The first target exhibited superior data down to the 10fMol-range. In contrast, titanium nitride-covered SS and microdiamond-covered hardmetal AP-MALDI-targets yielded poor sensitivity. These observations could be correlated with the surface roughness of the targets determined by 3D-confocal-white-light-microscopy. The roughest surfaces were found for titanium nitride-covered SS and microdiamond-covered hardmetal material showing both poor MS sensitivity. A less rough surface could be determined for the hand-polished SS target and the smoothest surface was found for the gold-covered target yielding the best sensitivity of all surfaces. These differences in the roughness having a strong impact on the ultimate sensitivity obtainable for peptide samples could be corroborated by electron microscopy. A peptide mixture covering a wide range of molecular weights and a tryptic protein digest (from 2-DE) exhibit the same behavior. This clearly indicates that the smooth gold-covered SS target is the surface of choice in AP-MALDI MS proteomics. Copyright © 2010. Published by Elsevier B.V.

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

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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.

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

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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.

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

The Characterization of Laser Ablation Patterns and a New Definition of Resolution in Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS).

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O'Rourke, Matthew B; Raymond, Benjamin B A; Padula, Matthew P

2017-05-01

Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) is a technique that has seen a sharp rise in both use and development. Despite this rapid adoption, there have been few thorough investigations into the actual physical mechanisms that underlie the acquisition of IMS images. We therefore set out to characterize the effect of IMS laser ablation patterns on the surface of a sample. We also concluded that the governing factors that control spatial resolution have not been correctly defined and therefore propose a new definition of resolution. Graphical Abstract ᅟ.

Ion yields in UV-MALDI mass spectrometry as a function of excitation laser wavelength and optical and physico-chemical properties of classical and halogen-substituted MALDI matrixes.

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Soltwisch, Jens; Jaskolla, Thorsten W; Hillenkamp, Franz; Karas, Michael; Dreisewerd, Klaus

2012-08-07

The laser wavelength constitutes a key parameter in ultraviolet-matrix-assisted laser desorption ionization-mass spectrometry (UV-MALDI-MS). Optimal analytical results are only achieved at laser wavelengths that correspond to a high optical absorption of the matrix. In the presented work, the wavelength dependence and the contribution of matrix proton affinity to the MALDI process were investigated. A tunable dye laser was used to examine the wavelength range between 280 and 355 nm. The peptide and matrix ion signals recorded as a function of these irradiation parameters are displayed in the form of heat maps, a data representation that furnishes multidimensional data interpretation. Matrixes with a range of proton affinities from 809 to 866 kJ/mol were investigated. Among those selected are the standard matrixes 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA) as well as five halogen-substituted cinnamic acid derivatives, including the recently introduced 4-chloro-α-cyanocinnamic acid (ClCCA) and α-cyano-2,4-difluorocinnamic acid (DiFCCA) matrixes. With the exception of DHB, the highest analyte ion signals were obtained toward the red side of the peak optical absorption in the solid state. A stronger decline of the molecular analyte ion signals generated from the matrixes was consistently observed at the low wavelength side of the peak absorption. This effect is mainly the result of increased fragmentation of both analyte and matrix ions. Optimal use of multiply halogenated matrixes requires adjustment of the excitation wavelength to values below that of the standard MALDI lasers emitting at 355 (Nd:YAG) or 337 nm (N(2) laser). The combined data provide new insights into the UV-MALDI desorption/ionization processes and indicate ways to improve the analytical sensitivity.

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.

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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.

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).

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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.

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

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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.

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

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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.

Proteomics approaches for identification of tumor relevant protein targets in pulmonary squamous cell carcinoma by 2D-DIGE-MS.

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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.

Exploring surface photoreaction dynamics using pixel imaging mass spectrometry (PImMS)

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Kershis, Matthew D.; Wilson, Daniel P.; White, Michael G.; John, Jaya John; Nomerotski, Andrei; Brouard, Mark; Lee, Jason W. L.; Vallance, Claire; Turchetta, Renato

2013-08-01

A new technique for studying surface photochemistry has been developed using an ion imaging time-of-flight mass spectrometer in conjunction with a fast camera capable of multimass imaging. This technique, called pixel imaging mass spectrometry (PImMS), has been applied to the study of butanone photooxidation on TiO2(110). In agreement with previous studies of this system, it was observed that the main photooxidation pathway for butanone involves ejection of an ethyl radical into vacuum which, as confirmed by our imaging experiment, undergoes fragmentation after ionization in the mass spectrometer. This proof-of-principle experiment illustrates the usefulness and applicability of PImMS technology to problems of interest within the surface science community.

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

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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.

Molecular imaging of myocardial infarction with Gadofluorine P – A combined magnetic resonance and mass spectrometry imaging approach

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Fabian Lohöfer

2018-04-01

Full Text Available Background: Molecular MRI is becoming increasingly important for preclinical research. Validation of targeted gadolinium probes in tissue however has been cumbersome up to now. Novel methodology to assess gadolinium distribution in tissue after in vivo application is therefore needed. Purpose: To establish combined Magnetic Resonance Imaging (MRI and Mass Spectrometry Imaging (MSI for improved detection and quantification of Gadofluorine P deposition in scar formation and myocardial remodeling. Materials and methods: Animal studies were performed according to institutionally approved protocols. Myocardial infarction was induced by permanent ligation of the left ascending artery (LAD in C57BL/6J mice. MRI was performed at 7T at 1 week and 6 weeks after myocardial infarction. Gadofluorine P was used for dynamic T1 mapping of extracellular matrix synthesis during myocardial healing and compared to Gd-DTPA. After in vivo imaging contrast agent concentration as well as distribution in tissue were validated and quantified by spatially resolved Matrix-Assisted Laser Desorption Ionization (MALDI MSI and Laser Ablation – Inductively Coupled Plasma – Mass Spectrometry (LA-ICP-MS imaging. Results: Both Gadofluorine P enhancement as well as local tissue content in the myocardial scar were highest at 15 minutes post injection. R1 values increased from 1 to 6 weeks after MI (1.62 s−1 vs 2.68 s−1, p = 0.059 paralleled by an increase in Gadofluorine P concentration in the infarct from 0.019 mM at 1 week to 0.028 mM at 6 weeks (p = 0.048, whereas Gd-DTPA enhancement showed no differences (3.95 s−1 vs 3.47 s−1, p = 0.701. MALDI-MSI results were corroborated by elemental LA-ICP-MS of Gadolinium in healthy and infarcted myocardium. Histology confirmed increased extracellular matrix synthesis at 6 weeks compared to 1 week. Conclusion: Adding quantitative MSI to MR imaging enables a quantitative validation of Gadofluorine P distribution in the heart

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.

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.

A major allergen in rainbow trout (Oncorhynchus mykiss): complete sequences of parvalbumin by MALDI tandem mass spectrometry.

Science.gov (United States)

Aiello, Donatella; Materazzi, Stefano; Risoluti, Roberta; Thangavel, Hariprasad; Di Donna, Leonardo; Mazzotti, Fabio; Casadonte, Francesca; Siciliano, Carlo; Sindona, Giovanni; Napoli, Anna

2015-08-01

Fish parvalbumin (PRVB) is an abundant and stable protein in fish meat. The variation in cross-reactivity among individuals is well known and explained by a broad repertoire of molecular forms and differences between IgE-binding epitopes in fish species. PVRB has "sequential" epitopes, which retain their IgE-binding capacity and allergenicity also after heating and digestion using proteolytic enzymes. From the allergonomics perspective, PRVB is still a challenging target due to its multiple isoforms present at different degrees of distribution. Little information is available in the databases about PVRBs from Oncorhynchus mykiss. At present, only two validated, incomplete isoforms of this species are included in the protein databases: parvalbumin beta 1 (P86431) and parvalbumin beta 2 (P86432). A simple and rapid protocol has been developed for selective solubilization of PRVB from the muscle of farmed rainbow trout (Oncorhynchus mykiss), followed by calcium depletion, proteolytic digestion, MALDI MS, and MS/MS analysis. With this strategy thermal allergen release was assessed and PRVB1 (P86431), PRVB1.1, PRVB2 (P86432) and PRVB2.1 variants from the rainbow trout were sequenced. The correct ordering of peptide sequences was aided by mapping the overlapping enzymatic digests. The deduced peptide sequences were arranged and the theoretical molecular masses (Mr) of the resulting sequences were calculated. Experimental masses (Mr) of each PRVB variant were measured by linear MALDI-TOF.

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

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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%.

Utilizing mass spectrometry imaging to map the thyroid hormones triiodothyronine and thyroxine in Xenopus tropicalis tadpoles.

Science.gov (United States)

Goto-Inoue, Naoko; Sato, Tomohiko; Morisasa, Mizuki; Kashiwagi, Akihiko; Kashiwagi, Keiko; Sugiura, Yuki; Sugiyama, Eiji; Suematsu, Makoto; Mori, Tsukasa

2018-02-01

Thyroid hormones are not only responsible for thermogenesis and energy metabolism in animals, but also have an important role in cell differentiation and development. Amphibian metamorphosis provides an excellent model for studying the remodeling of the body. This metamorphic organ remodeling is induced by thyroid hormones, and a larval body is thus converted into an adult one. The matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) imaging technology is expected to be a suitable tool for investigating small bioreactive molecules. The present study describes the distribution of the thyroid hormones, i.e., triiodothyronine (T3) and thyroxine (T4) and their inactive form reverse T3 (rT3) in Xenopus tropicalis tadpoles using two different types of imaging techniques, MS/MS and Fourier transform (FT)-MS imaging. As a result of MS/MS imaging, we demonstrated that T3 was mainly distributed in the gills. T4 was faintly localized in the eyes, inner gills, and intestine during metamorphosis. The intensity of T3 in the gills and the intensity of T4 in the body fluids were increased during metamorphosis. Moreover, the localization of the inactive form rT3 was demonstrated to be separate from T3, namely in the intestine and muscles. In addition, FT-MS imaging could utilize simultaneous imaging including thyroid hormone. This is the first report to demonstrate the molecular distribution of thyroid hormones themselves and to discriminate T3, T4, and rT3 in animal tissues.

The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer.

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Takayuki Goto

Full Text Available High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS is an emerging application for the comprehensive and detailed analysis of the spatial distribution of ionized molecules in situ on tissue slides. HR-MALDI-IMS in negative mode in a mass range of m/z 500-1000 was performed on optimal cutting temperature (OCT compound-embedded human prostate tissue samples obtained from patients with prostate cancer at the time of radical prostatectomy. HR-MALDI-IMS analysis of the 14 samples in the discovery set identified 26 molecules as highly expressed in the prostate. Tandem mass spectrometry (MS/MS showed that these molecules included 14 phosphatidylinositols (PIs, 3 phosphatidylethanolamines (PEs and 3 phosphatidic acids (PAs. Among the PIs, the expression of PI(18:0/18:1, PI(18:0/20:3 and PI(18:0/20:2 were significantly higher in cancer tissue than in benign epithelium. A biomarker algorithm for prostate cancer was formulated by analyzing the expression profiles of PIs in cancer tissue and benign epithelium of the discovery set using orthogonal partial least squares discriminant analysis (OPLS-DA. The sensitivity and specificity of this algorithm for prostate cancer diagnosis in the 24 validation set samples were 87.5 and 91.7%, respectively. In conclusion, HR-MALDI-IMS identified several PIs as being more highly expressed in prostate cancer than benign prostate epithelium. These differences in PI expression profiles may serve as a novel diagnostic tool for prostate cancer.

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

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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

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

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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.

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

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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.

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.

Imaging mass spectrometry in papillary thyroid carcinoma for the identification and validation of biomarker proteins.

Science.gov (United States)

Min, Kyueng-Whan; Bang, Joo-Young; Kim, Kwang Pyo; Kim, Wan-Seop; Lee, Sang Hwa; Shanta, Selina Rahman; Lee, Jeong Hwa; Hong, Ji Hye; Lim, So Dug; Yoo, Young-Bum; Na, Chan-Hyun

2014-07-01

Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-µm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2.

Quantification and localization of hesperidin and rutin in Citrus sinensis grafted on C. limonia after Xylella fastidiosa infection by HPLC-UV and MALDI imaging mass spectrometry.

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Soares, Márcio Santos; da Silva, Danielle Fernandes; Forim, Moacir Rossi; da Silva, Maria Fátima das Graças Fernandes; Fernandes, João Batista; Vieira, Paulo Cezar; Silva, Denise Brentan; Lopes, Norberto Peporine; de Carvalho, Sérgio Alves; de Souza, Alessandra Alves; Machado, Marcos Antônio

2015-07-01

A high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed for quantifying hesperidin and rutin levels in leaves and stems of Citrus limonia, with a good linearity over a range of 1.0-80.0 and 1.0-50.0 μg mL(-1) respectively, with r(2)>0.999 for all curves. The limits of detection (LOD) for both flavonoids were 0.6 and 0.5 μg mL(-1), respectively, with quantification (LOQ) being 2.0 and 1.0 μg mL(-1), respectively. The quantification method was applied to Citrus sinensis grafted onto C. limonia with and without CVC (citrus variegated chlorosis) symptoms after Xylella fastidiosa infection. The total content of rutin was low and practically constant in all analyses in comparison with hesperidin, which showed a significant increase in its amount in symptomatic leaves. Scanning electron microscopy studies on leaves with CVC symptoms showed vessel occlusion by biofilm, and a crystallized material was noted. Considering the difficulty in isolating these crystals for analysis, tissue sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to confirm the presence of hesperidin at the site of infection. The images constructed from MS/MS data with a specific diagnostic fragment ion (m/z 483) also showed higher ion intensities for it in infected plants than in healthy ones, mainly in the vessel regions. These data suggest that hesperidin plays a role in the plant-pathogen interaction, probably as a phytoanticipin. This method was also applied to C. sinensis and C. limonia seedlings, and comparison with the graft results showed that the rootstock had an increased hesperidin content ∼3.6 fold greater in the graft stem than in the stem of C. sinensis seedlings. Increase in hesperidin content by rootstock can be related to induced internal defense mechanisms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

Science.gov (United States)

Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

2011-02-01

In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

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

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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.

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

Comparison of public peak detection algorithms for MALDI mass spectrometry data analysis.

Science.gov (United States)

Yang, Chao; He, Zengyou; Yu, Weichuan

2009-01-06

In mass spectrometry (MS) based proteomic data analysis, peak detection is an essential step for subsequent analysis. Recently, there has been significant progress in the development of various peak detection algorithms. However, neither a comprehensive survey nor an experimental comparison of these algorithms is yet available. The main objective of this paper is to provide such a survey and to compare the performance of single spectrum based peak detection methods. In general, we can decompose a peak detection procedure into three consequent parts: smoothing, baseline correction and peak finding. We first categorize existing peak detection algorithms according to the techniques used in different phases. Such a categorization reveals the differences and similarities among existing peak detection algorithms. Then, we choose five typical peak detection algorithms to conduct a comprehensive experimental study using both simulation data and real MALDI MS data. The results of comparison show that the continuous wavelet-based algorithm provides the best average performance.

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.

Observation of Intravascular Changes of Superabsorbent Polymer Microsphere (SAP-MS) with Monochromatic X-Ray Imaging

International Nuclear Information System (INIS)

Tanimoto, Daigo; Ito, Katsuyoshi; Yamamoto, Akira; Sone, Teruki; Kobatake, Makito; Tamada, Tsutomu; Umetani, Keiji

2010-01-01

This study was designed to evaluate the intravascular transformation behavior of superabsorbent polymer microsphere (SAP-MS) in vivo macroscopically by using monochromatic X-ray imaging and to quantitatively compare the expansion rate of SAP-MS among different kinds of mixtures. Fifteen rabbits were used for our study and transcatheter arterial embolization (TAE) was performed for their auricular arteries using monochromatic X-ray imaging. We used three kinds of SAP-MS (particle diameter 100-150 μm) mixture as embolic spherical particles: SAP-MS(H) absorbed with sodium meglumine ioxaglate (Hexabrix 320), SAP-MS(V) absorbed with isosmolar contrast medium (Visipaque 270), and SAP-MS(S) absorbed with 0.9% sodium saline. The initial volume of SAP-MS particles just after TAE and its final volume 10 minutes after TAE in the vessel were measured to calculate the expansion rate (ER) (n = 30). Intravascular behavior of SAP-MS particles was clearly observed in real time at monochromatic X-ray imaging. Averaged initial volumes of SAP-MS (H) (1.24 x 10 7 μm 3 ) were significantly smaller (p 7 μm 3 ) and SAP-MS (S) (5.85 x 10 7 μm 3 ). Averaged final volumes of SAP-MS (H) were significantly larger than averaged initial volumes (4.41 x 10 7 μm 3 vs. 1.24 x 10 7 μm 3 ; p < 0.0001, ER = 3.55). There were no significant difference between averaged final volumes and averaged initial volumes of SAP-MS (V) and SAP-MS (S). SAP-MS (H), which first travels distally, reaches to small arteries, and then expands to adapt to the vessel lumen, is an effective particle as an embolic agent, causing effective embolization.

[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

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

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...

BioSunMS: a plug-in-based software for the management of patients information and the analysis of peptide profiles from mass spectrometry

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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/.

A comprehensive high-resolution mass spectrometry approach for characterization of metabolites by combination of ambient ionization, chromatography and imaging methods.

Science.gov (United States)

Berisha, Arton; Dold, Sebastian; Guenther, Sabine; Desbenoit, Nicolas; Takats, Zoltan; Spengler, Bernhard; Römpp, Andreas

2014-08-30

An ideal method for bioanalytical applications would deliver spatially resolved quantitative information in real time and without sample preparation. In reality these requirements can typically not be met by a single analytical technique. Therefore, we combine different mass spectrometry approaches: chromatographic separation, ambient ionization and imaging techniques, in order to obtain comprehensive information about metabolites in complex biological samples. Samples were analyzed by laser desorption followed by electrospray ionization (LD-ESI) as an ambient ionization technique, by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging for spatial distribution analysis and by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) for quantitation and validation of compound identification. All MS data were acquired with high mass resolution and accurate mass (using orbital trapping and ion cyclotron resonance mass spectrometers). Grape berries were analyzed and evaluated in detail, whereas wheat seeds and mouse brain tissue were analyzed in proof-of-concept experiments. In situ measurements by LD-ESI without any sample preparation allowed for fast screening of plant metabolites on the grape surface. MALDI imaging of grape cross sections at 20 µm pixel size revealed the detailed distribution of metabolites which were in accordance with their biological function. HPLC/ESI-MS was used to quantify 13 anthocyanin species as well as to separate and identify isomeric compounds. A total of 41 metabolites (amino acids, carbohydrates, anthocyanins) were identified with all three approaches. Mass accuracy for all MS measurements was better than 2 ppm (root mean square error). The combined approach provides fast screening capabilities, spatial distribution information and the possibility to quantify metabolites. Accurate mass measurements proved to be critical in order to reliably combine data from different MS

The challenge of on-tissue digestion for MALDI MSI- a comparison of different protocols to improve imaging experiments.

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Diehl, Hanna C; Beine, Birte; Elm, Julian; Trede, Dennis; Ahrens, Maike; Eisenacher, Martin; Marcus, Katrin; Meyer, Helmut E; Henkel, Corinna

2015-03-01

Mass spectrometry imaging (MSI) has become a powerful and successful tool in the context of biomarker detection especially in recent years. This emerging technique is based on the combination of histological information of a tissue and its corresponding spatial resolved mass spectrometric information. The identification of differentially expressed protein peaks between samples is still the method's bottleneck. Therefore, peptide MSI compared to protein MSI is closer to the final goal of identification since peptides are easier to measure than proteins. Nevertheless, the processing of peptide imaging samples is challenging due to experimental complexity. To address this issue, a method development study for peptide MSI using cryoconserved and formalin-fixed paraffin-embedded (FFPE) rat brain tissue is provided. Different digestion times, matrices, and proteases were tested to define an optimal workflow for peptide MSI. All practical experiments were done in triplicates and analyzed by the SCiLS Lab software, using structures derived from myelin basic protein (MBP) peaks, principal component analysis (PCA) and probabilistic latent semantic analysis (pLSA) to rate the experiments' quality. Blinded experimental evaluation in case of defining countable structures in the datasets was performed by three individuals. Such an extensive method development for peptide matrix-assisted laser desorption/ionization (MALDI) imaging experiments has not been performed so far, and the resulting problems and consequences were analyzed and discussed.

Proteomics with Mass Spectrometry Imaging: Beyond Amyloid Typing.

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Lavatelli, Francesca; Merlini, Giampaolo

2018-04-01

Detection and typing of amyloid deposits in tissues are two crucial steps in the management of systemic amyloidoses. The presence of amyloid deposits is routinely evaluated through Congo red staining, whereas proteomics is now a mainstay in the identification of the deposited proteins. In article number 1700236, Winter et al. [Proteomics 2017, 17, Issue 22] describe a novel method based on MALDI-MS imaging coupled to ion mobility separation and peptide filtering, to detect the presence of amyloid in histology samples and to identify its composition, while preserving the spatial distribution of proteins in tissues. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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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.

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.

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