Total Laboratory Automation and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Improve Turnaround Times in the Clinical Microbiology Laboratory: a Retrospective Analysis.

PubMed

Theparee, Talent; Das, Sanchita; Thomson, Richard B

2018-01-01

Technological advances have changed the practice of clinical microbiology. We implemented Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and BD Kiestra total laboratory automation (TLA) 4 and 3 years ago, respectively. To assess the impact of these new technologies, we compared turnaround times (TATs) for positive and negative urine cultures before and after implementation. In comparison I, TATs for 61,157 urine cultures were extracted for two periods corresponding to pre-TLA and post-TLA, both using MALDI-TOF MS for organism identification. In comparison II, time to organism identification (ID) and antimicrobial susceptibility (AST) reports were calculated for 5,402 positive culture reports representing four different periods: (i) manual plating and conventional biochemical identification (CONV), (ii) manual plating and MALDI-TOF MS identification (MALDI), (iii) MALDI-TOF MS identification and early phase implementation of TLA (TLA1), and (iv) MALDI-TOF MS identification and late phase implementation of TLA (TLA2). By the comparison I results, median pre- and post-TLA TATs to organism IDs (18.5 to 16.9 h), AST results (41.8 to 40.8 h), and preliminary results for negative cultures (17.7 to 13.6 h), including interquartile ranges for all comparisons, were significantly decreased post-TLA ( P < 0.001). By the comparison II results, MALDI significantly improved TAT to organism ID compared to CONV (21.3 to 18 h). TLA further improved overall TAT to ID (18 to 16.5 h) and AST (42.3 to 40.7 h) results compared to MALDI ( P < 0.001). In summary, TLA significantly improved TAT to organism ID, AST report, and preliminary negative results. MALDI-TOF MS significantly improved TAT for organism ID. Use of MALDI-TOF MS and TLA individually and together results in significant decreases in microbiology report TATs. Copyright © 2017 Theparee et al.

Total Laboratory Automation and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Improve Turnaround Times in the Clinical Microbiology Laboratory: a Retrospective Analysis

PubMed Central

Theparee, Talent; Das, Sanchita

2017-01-01

ABSTRACT Technological advances have changed the practice of clinical microbiology. We implemented Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and BD Kiestra total laboratory automation (TLA) 4 and 3 years ago, respectively. To assess the impact of these new technologies, we compared turnaround times (TATs) for positive and negative urine cultures before and after implementation. In comparison I, TATs for 61,157 urine cultures were extracted for two periods corresponding to pre-TLA and post-TLA, both using MALDI-TOF MS for organism identification. In comparison II, time to organism identification (ID) and antimicrobial susceptibility (AST) reports were calculated for 5,402 positive culture reports representing four different periods: (i) manual plating and conventional biochemical identification (CONV), (ii) manual plating and MALDI-TOF MS identification (MALDI), (iii) MALDI-TOF MS identification and early phase implementation of TLA (TLA1), and (iv) MALDI-TOF MS identification and late phase implementation of TLA (TLA2). By the comparison I results, median pre- and post-TLA TATs to organism IDs (18.5 to 16.9 h), AST results (41.8 to 40.8 h), and preliminary results for negative cultures (17.7 to 13.6 h), including interquartile ranges for all comparisons, were significantly decreased post-TLA (P < 0.001). By the comparison II results, MALDI significantly improved TAT to organism ID compared to CONV (21.3 to 18 h). TLA further improved overall TAT to ID (18 to 16.5 h) and AST (42.3 to 40.7 h) results compared to MALDI (P < 0.001). In summary, TLA significantly improved TAT to organism ID, AST report, and preliminary negative results. MALDI-TOF MS significantly improved TAT for organism ID. Use of MALDI-TOF MS and TLA individually and together results in significant decreases in microbiology report TATs. PMID:29118171

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

PubMed

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 (P<0.05). In addition, we modify the reporting cutoffs for the Biotyper "score" yielding acceptable identification. We found that a score of ≥1.700 can maximize the rate of identification. Of interest, MALDI-TOF MS can correctly identify anaerobes 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 Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

PubMed Central

Barbano, Duane; Diaz, Regina; Zhang, Lin; Sandrin, Todd; Gerken, Henri; Dempster, Thomas

2015-01-01

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures. PMID:26271045

Phenolic antioxidants (hydrolyzable tannins, flavonols, and anthocyanins) identified by LC-ESI-MS and MALDI-QIT-TOF MS from Rosa chinensis flowers.

PubMed

Cai, Yi-Zhong; Xing, Jie; Sun, Mei; Zhan, Zhao-Qi; Corke, Harold

2005-12-28

Rosa chinensis (Yuejihua) is a well-known ornamental plant, and its flowers are commonly used in traditional Chinese medicine. Methanolic crude extracts of dried R. chinensis flowers were used for simultaneous determination of phenolic constituents by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS). A total of 36 known and unknown phenolics were identified as hydrolyzable tannins, flavonols, and anthocyanins, mainly including gallotannins (mono-, di-, or trigalloylglucopyranosides), ellagitannins, quercetin, quercetin/kaempferol mono- and diglycosides, and cyanidin/pelargonidin diglycosides. MALDI-QIT-TOF MS was applied not only to verify most phenolics isolated and identified by LC-MS but also to tentatively identify two ellagitannins (rugosins B and C) not isolated and unidentified by LC-MS. This study is the first to demonstrate the rapid and successful use of MALDI-QIT-TOF MS and LC-MS to directly and simultaneously identify phenolics in the crude extracts of R. chinensis flowers without any purification. The antioxidant activity of the crude extracts from R. chinensis flowers was also measured with three assay methods. The results showed that the phenolic antioxidants from R. chinensis flowers exhibited very strong radical scavenging effect and antioxidant power. High levels of flavonols and hydrolyzable tannins might be important bioactive principles in the dried R. chinensis flowers.

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

PubMed

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) <2.00, the on-plate extraction method is characterized by high-performance. For samples for which Biotyper produces more than one reliable result, MALDI-TOF MS must be used in combination with genotypic techniques to achieve unambiguous results. 16S-ARDRA is simple, repetitive method with high power of discrimination, whose sole limitation is its inability to discriminate between species with very high 16S rDNA sequence homology, such as L. casei and L. zeae. The assays can be used for discrimination of Lactobacillus bacteria from different habitats.

[Application of mass spectrometry in mycology].

PubMed

Quiles Melero, Inmaculada; Peláez, Teresa; Rezusta López, Antonio; Garcia-Rodríguez, Julio

2016-06-01

MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) is becoming an essential tool in most microbiology laboratories. At present, by using a characteristic fungal profile obtained from whole cells or through simple extraction protocols, MALDI-TOF MS allows the identification of pathogenic fungi with a high performance potential. This methodology decreases the laboratory turnaround time, optimizing the detection of mycoses. This article describes the state-of-the-art of the use of MALDI-TOF MS for the detection of human clinical fungal pathogens in the laboratory and discusses the future applications of this technology, which will further improve routine mycological diagnosis. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

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

PubMed

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

2017-01-18

Patient mortality is significantly reduced by rapid identification of bacteria from sterile sites. MALDI-TOF can identify bacteria directly from positive blood cultures and multiple sample preparation methods are available. We evaluated three sample preparation methods and two MALDI-TOF score cut-off values. Positive blood culture bottles with organisms present in Gram stains were prospectively analysed by MALDI-TOF. Three lysis reagents (Saponin, SDS, and SepsiTyper lysis bufer) were applied to each positive culture followed by centrifugation, washing and protein extraction steps. Methods were compared using the McNemar test and 16S rDNA sequencing was used to assess discordant results. In 144 monomicrobial cultures, using ≥2.000 as the cut-off value, species level identifications were obtained from 69/144 (48%) samples using Saponin, 86/144 (60%) using SDS, and 91/144 (63%) using SepsiTyper. The difference between SDS and SepsiTyper was not statistically significant (P = 0.228). Differences between Saponin and the other two reagents were significant (P < 0.01). Using ≥1.700 plus top three results matching as the cut-off value, species level identifications were obtained from 100/144 (69%) samples using Saponin, 103/144 (72%) using SDS, and 106/144 (74%) using SepsiTyper and there was no statistical difference between the methods. No true discordances between culture and direct MALDI-TOF identification were observed in monomicrobial cultures. In 32 polymicrobial cultures, MALDI-TOF identified one organism in 34-75% of samples depending on the method. This study demonstrates two inexpensive in-house detergent lysis methods are non-inferior to a commercial kit for analysis of positive blood cultures by direct MALDI-TOF in a clinical diagnostic microbiology laboratory.

Evaluation of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Mycobacterium species, Nocardia species, and Other Aerobic Actinomycetes

PubMed Central

Buckwalter, S. P.; Olson, S. L.; Connelly, B. J.; Lucas, B. C.; Rodning, A. A.; Walchak, R. C.; Deml, S. M.; Wohlfiel, S. L.

2015-01-01

The value of matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer's supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed. PMID:26637381

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

PubMed

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

2007-03-26

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. 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. 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.

Application of pressure probe and UV-MALDI-TOF MS for direct analysis of plant underivatized carbohydrates in subpicoliter single-cell cytoplasm extract.

PubMed

Gholipour, Yousef; Nonami, Hiroshi; Erra-Balsells, Rosa

2008-12-01

Single-cell cytoplasm sap (1-10 pL) was extracted by using a pressure probe glass microcapillary tip from tulip leaf and bulb and analyzed by UV-MALDI-TOF MS for free underivatized carbohydrate content. Three matrices including 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone (THAP), and carbon nanotubes (CNTs) in positive ion mode were selected for analysis because of acceptable carbohydrate-related signal reproducibility. Disaccharide and oligosaccharide (up to 15 Hex when THAP was used, 11 Hex with DHB, and 7 Hex with CNTs) were detected in tulip bulb cell cytoplasm sample. When DHB was used as matrix, neutral carbohydrates were more abundantly detected as sodiated cations; the sugar-related signals, however, appeared as dominant potassiated cations when THAP and CNTs were used. Small amount of monosaccharide was also detected in bulb cell cytoplasm with CNTs as matrix. UV-MALDI-TOF MS of leaf cell extract resulted in high-resolution detection of hexose and disaccharide with DHB, THAP, and CNTs.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification.

PubMed

Bizzini, A; Greub, G

2010-11-01

Until recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for the identification of microorganisms remained confined to research laboratories. In the last 2 years, the availability of relatively simple to use MALDI-TOF MS devices, which can be utilized in clinical microbiology laboratories, has changed the laboratory workflows for the identification of pathogens. Recently, the first prospective studies regarding the performance in routine bacterial identification showed that MALDI-TOF MS is a fast, reliable and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification for most bacterial strains isolated in clinical microbiology laboratories. For routine bacterial isolates, correct identification by MALDI-TOF MS at the species level was obtained in 84.1-93.6% of instances. In one of these studies, a protein extraction step clearly improved the overall valid identification yield, from 70.3% to 93.2%. This review focuses on the current state of use of MALDI-TOF MS for the identification of routine bacterial isolates and on the main difficulties that may lead to erroneous or doubtful identifications. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

Direct bacterial identification in positive blood cultures by use of two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.

PubMed

Chen, Jonathan H K; Ho, Pak-Leung; Kwan, Grace S W; She, Kevin K K; Siu, Gilman K H; Cheng, Vincent C C; Yuen, Kwok-Yung; Yam, Wing-Cheong

2013-06-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and fungi was recently introduced in microbiology laboratories. This technology could greatly improve the clinical management of patients and guidance for chemotherapy. In this study, we used a commercial MALDI Sepsityper extraction method to evaluate the performance of two commercial MALDI-TOF MS systems, the Vitek MS IVD (bioMérieux) and the Microflex LT Biotyper (Bruker Daltonics) for direct bacterial identification in positive blood cultures. In 181 monomicrobial cultures, both systems generated genus to species level identifications for >90% of the specimens (Biotyper, 177/181 [97.8%]; Vitek MS IVD, 167/181 [92.3%]). Overall, the Biotyper system generated significantly more accurate identifications than the Vitek MS IVD system (P = 0.016; 177 versus 167 out of 181 specimens). The Biotyper system identified the minority species among polymicrobial blood cultures. We also compared the performance of an in-house extraction method with that of the Sepsityper on both MALDI-TOF MS systems. The in-house method generated more correct identifications at the genus level than the Sepsityper (96.7% versus 93.5%) on the Biotyper system, whereas the two methods exhibited the same performance level (88.0% versus 88.0%) on the Vitek MS IVD system. Our study confirmed the practical advantages of MALDI-TOF MS, and our in-house extraction method reduced the reagent cost to $1 per specimen, with a shorter turnaround time of 3 h, which is highly cost-effective for a diagnostic microbiology service.

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

PubMed

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.

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

PubMed

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.

Identification and Characterization of Lipopeptides from Bacillus subtilis B1 Against Sapstain Fungus of Rubberwood Through MALDI-TOF-MS and RT-PCR.

PubMed

Sajitha, K L; Dev, Suma Arun; Maria Florence, E J

2016-07-01

Bacillus subtilis is a potent biocontrol agent producing a wide array of antifungal lipopeptides for the inhibition of fungal growth. B. subtilis B1 isolated from market-available compost provided an efficient control of rubberwood sapstain fungus, Lasiodiplodia theobromae. The current study is aimed to identify and characterize the lipopeptides responsible for the biocontrol of rubberwood sapstain fungus by Bacillus subtilis B1. The bacterial whole-cell surface extract from the dual culture of B. subtilis B1 and sapstain fungus (L. theobromae) was analysed using MALDI-TOF-MS. The protonated as well as sodium, potassium adducts of homologues of iturin C, surfactin, bacillomycin D and fengycin A and B were identified and expression of the lipopeptide biosynthetic genes could be confirmed through RT-PCR. This is the first report of mycobacillin and trimethylsilyl derivative of bacilysin during antagonism through MALDI-TOF-MS. MALDI-TOF-MS with RT-PCR offered easy platforms to characterize the antifungal lipopeptides. The identification of antifungal lipopeptides can lead to the formulation of prospective biocontrol by-products which have wide-scale utility.

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

PubMed

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.

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Panda, A; Kurapati, S; Samantaray, J C; Myneedu, V P; Verma, A; Srinivasan, A; Ahmad, H; Behera, D; Singh, U B

2013-01-01

The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

Validation of MALDI-TOF MS for rapid classification and identification of lactic acid bacteria, with a focus on isolates from traditional fermented foods in Northern Vietnam.

PubMed

Doan, N T L; Van Hoorde, K; Cnockaert, M; De Brandt, E; Aerts, M; Le Thanh, B; Vandamme, P

2012-10-01

To evaluate the potential use of MALDI-TOF MS for fast and reliable classification and identification of lactic acid bacteria (LAB) from traditional fermented foods. A total of 119 strains of LAB from fermented meat (nem chua) were analysed with both (GTG)(5)-PCR fingerprinting and MALDI-TOF MS. Cluster analysis of the profiles revealed five species represented by a single isolate both in (GTG)(5)-PCR and in MALDI-TOF MS; five species grouped alike for (GTG)(5)-PCR and for MALDI-TOF MS; however, differences in minimal similarity between the delineated (GTG)(5)-PCR and MALDI-TOF MS clusters could be observed; three species showed more heterogeneity in their MALDI-TOF MS profiles compared to their (GTG)(5)-PCR profiles; two species, each represented by a single MALDI-TOF cluster, were subdivided in the corresponding (GTG)(5)-PCR dendrogram. As proof of the identification potential of MALDI-TOF MS, LAB diversity from one fermented mustard sample was analysed using MALDI-TOF MS. PheS gene sequencing was used for validation. MALDI-TOF MS is a powerful, fast, reliable and cost-effective technique for the identification of LAB associated with the production of fermented foods. Food LAB can be identified using MALDI-TOF MS, and its application could possibly be extended to other food matrices and/or other food-derived micro-organisms. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

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

PubMed Central

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 MSMS spectra to obtain structural information was focused only on discriminative ions in order to minimize analysis time. Conclusion Contrary to other classical approaches with direct online coupling of chromatographic separation and on the flight MS and/or MSMS data acquisition for all detected analytes, our dual decoupling strategy allowed us to focus on the most discriminative analytes, giving us more time to acquire more replicates of the same urine samples thus increasing detection sensitivity and mass precision. PMID:19014585

MALDI-TOF MS for the identification of veterinary non-C. neoformans-C. gattii Cryptococcus spp. isolates from Italy.

PubMed

Danesi, Patrizia; Drigo, Ilenia; Iatta, Roberta; Firacative, Carolina; Capelli, Gioia; Cafarchia, Claudia; Meyer, Wieland

2014-08-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) offers an effective alternative to phenotypic and molecular methods for the rapid identification of microorganisms. Our aim in this study was to create an in-house library for a set of strains of nine uncommonly reported human and animal cryptococcal species, including Cryptococcus adeliensis, C. albidosimilis, C. albidus, C. aureus, C. carnescens, C. laurentii, C. magnus, C. victoriae and C. uniguttulatus, and to use this library to make timely and correct identifications using MALDI-TOF MS for use in routine laboratory diagnostics. Protein extracts obtained via the formic acid extraction method of 62 veterinary non-C. neoformans-C. gattii cryptococcal isolates were studied. The obtained mass spectra correctly grouped all 62 studied isolates according to species identification previously obtained by internal transcribe spacer sequence analysis. The in-house database was than exported and successfully uploaded to the Microflex LT (Maldi Biotyper; Bruker Daltonics) instrument at a different diagnostic laboratory in Italy. Scores >2.7 obtained from isolates reanalyzed in the latter laboratory supported the high reproducibility of the method. The possibility of creating and transferring an in-house library adds to the usefulness MALDI-TOF MS an important tool for the rapid and inexpensive identification of pathogenic and saprophytic fungi as required for differential diagnosis of human and animal mycoses. © The Author 2014. 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.

MALDI-TOF to compare polysaccharide profiles from commercial health supplements of different mushroom species.

PubMed

López-García, Marta; García, María Sonia Dopico; Vilariño, José Manuel López; Rodríguez, María Victoria González

2016-05-15

In this work MALDI-TOF mass spectroscopy was investigated to characterise the β-glucan profiles of several commercial health supplements, without any derivatisation or purification pre-treatment. The effect of two solvents (water and dimethyl sulfoxide) and two MALDI matrices (2,5-dihydroxybenzoic acid and 2',4',6'-trihydroxyacetophenone) was first evaluated on dextran standards. MALDI-TOF was found as a useful and quick technique to obtain structural information of diverse food supplements based on mushroom extracts. The MALDI polysaccharide profiles of 5 supplements from different mushroom species were qualitatively similar showing [Glucan+Na](+) cations with a peak-to-peak mass difference of 16 Da consistent with the repeating unit of the β-(1→3)-glucan. The profiles strongly depended on the sample solvent used, with m/z values around 5000-8000 for water and 2000 for dimethyl sulfoxide; differences between samples were revealed in the molecular weight of the aqueous preparation, with the highest values for Maitake and Cordyceps species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Long-Chain Fatty Acid as N-(4-Aminomethylphenyl) Pyridinium Derivative by MALDI LIFT-TOF/TOF Mass Spectrometry

NASA Astrophysics Data System (ADS)

Frankfater, Cheryl; Jiang, Xuntian; Hsu, Fong-Fu

2018-05-01

Charge remote fragmentation (CRF) elimination of CnH2n+2 residues along the aliphatic tail of long chain fatty acid is hall mark of keV high-energy CID fragmentation process. It is an important fragmentation pathway leading to structural characterization of biomolecules by CID tandem mass spectrometry. In this report, we describe MALDI LIFT TOF-TOF mass spectrometric approach to study a wide variety of fatty acids (FAs), which were derivatized to N-(4-aminomethylphenyl) pyridinium (AMPP) derivative, and desorbed as M+ ions by laser with or without matrix. The high-energy MALDI LIFT TOF-TOF mass spectra of FA-AMPP contain fragment ions mainly deriving from CRF cleavages of CnH2n+2 residues, as expected. These ions together with ions from specific cleavages of the bond(s) involving the functional group within the molecule provide more complete structural identification than those produced by low-energy CID/HCD using a linear ion-trap instrument. However, this LIFT TOF-TOF mass spectrometric approach inherits low sensitivity, a typical feature of high-energy CID tandem mass spectrometry. Because of the lack of unit mass precursor ion selection with sufficient sensitivity of the current LIFT TOF-TOF technology, product ion spectra from same chain length fatty acids with difference in one or two double bonds in a mixture are not distinguishable.

Application of MALDI-TOF MS Systems in the Rapid Identification of Campylobacter spp. of Public Health Importance.

PubMed

Hsieh, Ying-Hsin; Wang, Yun F; Moura, Hercules; Miranda, Nancy; Simpson, Steven; Gowrishankar, Ramnath; Barr, John; Kerdahi, Khalil; Sulaiman, Irshad M

2018-05-01

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

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

PubMed Central

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. PMID:26300860

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

PubMed

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.

Detection of Rickettsia spp in Ticks by MALDI-TOF MS

PubMed Central

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

2015-01-01

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

Biomarkers of Aspergillus spores

NASA Astrophysics Data System (ADS)

Sulc, Miroslav; Peslova, Katerina; Zabka, Martin; Hajduch, Marian; Havlicek, Vladimir

2009-02-01

We applied both matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric and 1D sodium dodecylsulfate polyacrylamide gel electrophoretic (1D-PAGE) approaches for direct analysis of intact fungal spores of twenty four Aspergillus species. In parallel, we optimized various protocols for protein extraction from Aspergillus spores using acidic conditions, step organic gradient and variable sonication treatment. The MALDI-TOF mass spectra obtained from optimally prepared samples provided a reproducible fingerprint demonstrating the capability of the MALDI-TOF approach to type and characterize different fungal strains within the Aspergillus genus. Mass spectra of intact fungal spores provided signals mostly below 20 kDa. The minimum material amount represented 0.3 [mu]g (10,000 spores). Proteins with higher molecular weight were detected by 1D-PAGEE Eleven proteins were identified from three selected strains in the range 5-25 kDa by the proteomic approach. Hemolysin and hydrophobin have the highest relevance in host-pathogen interactions.

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.

PubMed

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.

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

PubMed

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.

Usefulness and accuracy of MALDI-TOF mass spectrometry as a supplementary tool to identify mosquito vector species and to invest in development of international database.

PubMed

Raharimalala, F N; Andrianinarivomanana, T M; Rakotondrasoa, A; Collard, J M; Boyer, S

2017-09-01

Arthropod-borne diseases are important causes of morbidity and mortality. The identification of vector species relies mainly on morphological features and/or molecular biology tools. The first method requires specific technical skills and may result in misidentifications, and the second method is time-consuming and expensive. The aim of the present study is to assess the usefulness and accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a supplementary tool with which to identify mosquito vector species and to invest in the creation of an international database. A total of 89 specimens belonging to 10 mosquito species were selected for the extraction of proteins from legs and for the establishment of a reference database. A blind test with 123 mosquitoes was performed to validate the MS method. Results showed that: (a) the spectra obtained in the study with a given species differed from the spectra of the same species collected in another country, which highlights the need for an international database; (b) MALDI-TOF MS is an accurate method for the rapid identification of mosquito species that are referenced in a database; (c) MALDI-TOF MS allows the separation of groups or complex species, and (d) laboratory specimens undergo a loss of proteins compared with those isolated in the field. In conclusion, MALDI-TOF MS is a useful supplementary tool for mosquito identification and can help inform vector control. © 2017 The Royal Entomological Society.

Rapid detection of carbapenemase-producing Klebsiella pneumoniae strains derived from blood cultures by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

PubMed

Sakarikou, Christina; Ciotti, Marco; Dolfa, Camilla; Angeletti, Silvia; Favalli, Cartesio

2017-03-08

Carbapenemase-producing Enterobacteriaceae (CPE), particularly carbapenemase-producing Klebsiella pneumoniae isolates, are important causative agents of nosocomial infections associated with significant mortality rates mostly in critical wards. The rapid detection and typing of these strains is critical either for surveillance purposes and to prevent outbreaks and optimize antibiotic therapy. In this study, the MALDI-TOF MS method was used to detect rapidly these isolates from blood cultures (BCs) and to obtain proteomic profiles enable to discriminate between carbapenemase-producing and non-carbapenemase-producing strains. Fifty-five K. pneumoniae strains were tested. Identification and carbapenemase-production detection assay using Ertapenem were performed both from bacterial pellets extracted directly from BCs flasks and from subcultures of these strains. For all isolates, a complete antimicrobial susceptibility testing and a genotypic characterization were performed. We found 100% agreement between the carbapenemase-producing profile generated by MALDI TOF MS and that obtained using conventional methods. The assay detected and discriminated different carbapenemase-producing K. pneumoniae isolates within 30 min to 3 h after incubation with Ertapenem. MALDI-TOF MS is a promising, rapid and economical method for the detection of carbapenemase-producing K. pneumoniae strains that could be successfully introduced into the routine diagnostic workflow of clinical microbiology laboratories.

Intensity fading MALDI-TOF mass spectrometry and functional proteomics assignments to identify protease inhibitors in marine invertebrates.

PubMed

Covaleda, Giovanni; Trejo, Sebastian A; Salas-Sarduy, Emir; Del Rivero, Maday Alonso; Chavez, Maria Angeles; Aviles, Francesc X

2017-08-08

Proteases and their inhibitors have become molecules of increasing fundamental and applicative value. Here we report an integrated strategy to identify and analyze such inhibitors from Caribbean marine invertebrates extracts by a fast and sensitive functional proteomics-like approach. The strategy works in three steps: i) multiplexed enzymatic inhibition kinetic assays, ii) Intensity Fading MALDI-TOF MS to establish a link between inhibitory molecules and the related MALDI signal(s) detected in the extract(s), and iii) ISD-CID-T 3 MS fragmentation on the parent MALDI signals selected in the previous step, enabling the partial or total top-down sequencing of the molecules. The present study has allowed validation of the whole approach, identification of a substantial number of novel protein protease inhibitors, as well as full or partial sequencing of reference molecular species and of many unknown ones, respectively. Such inhibitors correspond to six protease subfamilies (metallocarboxypeptidases-A and -B, pepsin, papain, trypsin and subtilisin), are small (1-10KDa) disulfide-rich proteins, and have been found at diverse frequencies among the invertebrates (13 to 41%). The overall procedure could be tailored to other enzyme-inhibitor and protein interacting systems, analyzing samples at medium-throughput level and leading to the functional and structural characterization of proteinaceous ligands from complex biological extracts. Invertebrate animals, and marine ones among, display a remarkable diversity of species and contained biomolecules. Many of their proteins-peptides have high biological, biotechnological and biomedical potential interest but, because of the lack of sequenced genomes behind, their structural and functional characterization constitutes a great challenge. Here, looking at the small, disulfide-rich, proteinaceous inhibitors of proteases found in them, it is shown that such problem can be significatively facilitated by integrative multiplexed enzymatic assays, affinity-based Intensity-Fading (IF-) MALDI-TOF mass spectrometry (MS), and on-line MS fragmentation, in a fast and easy approach. Copyright © 2017. Published by Elsevier B.V.

Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method.

PubMed

Ferreira, L; Sánchez-Juanes, F; Muñoz-Bellido, J L; González-Buitrago, J M

2011-07-01

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

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

PubMed

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.

Analysis and Quantitation of Glycated Hemoglobin by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry

NASA Astrophysics Data System (ADS)

Hattan, Stephen J.; Parker, Kenneth C.; Vestal, Marvin L.; Yang, Jane Y.; Herold, David A.; Duncan, Mark W.

2016-03-01

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the β-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R2 > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate β-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry (MALDI-TOF MS) Based Microbial Identifications: Challenges and Scopes for Microbial Ecologists

PubMed Central

Rahi, Praveen; Prakash, Om; Shouche, Yogesh S.

2016-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based biotyping is an emerging technique for high-throughput and rapid microbial identification. Due to its relatively higher accuracy, comprehensive database of clinically important microorganisms and low-cost compared to other microbial identification methods, MALDI-TOF MS has started replacing existing practices prevalent in clinical diagnosis. However, applicability of MALDI-TOF MS in the area of microbial ecology research is still limited mainly due to the lack of data on non-clinical microorganisms. Intense research activities on cultivation of microbial diversity by conventional as well as by innovative and high-throughput methods has substantially increased the number of microbial species known today. This important area of research is in urgent need of rapid and reliable method(s) for characterization and de-replication of microorganisms from various ecosystems. MALDI-TOF MS based characterization, in our opinion, appears to be the most suitable technique for such studies. Reliability of MALDI-TOF MS based identification method depends mainly on accuracy and width of reference databases, which need continuous expansion and improvement. In this review, we propose a common strategy to generate MALDI-TOF MS spectral database and advocated its sharing, and also discuss the role of MALDI-TOF MS based high-throughput microbial identification in microbial ecology studies. PMID:27625644

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

PubMed

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.

Diagnostic performance of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry in blood bacterial infections: a systematic review and meta-analysis.

PubMed

Scott, Jamie S; Sterling, Sarah A; To, Harrison; Seals, Samantha R; Jones, Alan E

2016-07-01

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) has shown promise in decreasing time to identification of causative organisms compared to traditional methods; however, the utility of MALDI-TOF MS in a heterogeneous clinical setting is uncertain. To perform a systematic review on the operational performance of the Bruker MALDI-TOF MS system and evaluate published cut-off values compared to traditional blood cultures. A comprehensive literature search was performed. Studies were included if they performed direct MALDI-TOF MS analysis of blood culture specimens in human patients with suspected bacterial infections using the Bruker Biotyper software. Sensitivities and specificities of the combined studies were estimated using a hierarchical random effects linear model (REML) incorporating cut-off scores of ≥1.7 and ≥2.0. Fifty publications were identified, with 11 studies included after final review. The estimated sensitivity utilising a cut-off of ≥2.0 from the combined studies was 74.6% (95% CI = 67.9-89.3%), with an estimated specificity of 88.0% (95% CI = 74.8-94.7%). When assessing a cut-off of ≥1.7, the combined sensitivity increases to 92.8% (95% CI = 87.4-96.0%), but the estimated specificity decreased to 81.2% (95% CI = 61.9-96.6%). In this analysis, MALDI-TOF MS showed acceptable sensitivity and specificity in bacterial speciation with the current recommended cut-off point compared to blood cultures; however, lowering the cut-off point from ≥2.0 to ≥1.7 would increase the sensitivity of the test without significant detrimental effect on the specificity, which could improve clinician confidence in their results.

Effect of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Alone versus MALDI-TOF MS Combined with Real-Time Antimicrobial Stewardship Interventions on Time to Optimal Antimicrobial Therapy in Patients with Positive Blood Cultures.

PubMed

Beganovic, Maya; Costello, Michael; Wieczorkiewicz, Sarah M

2017-05-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) decreases the time to organism identification and improves clinical and financial outcomes. The purpose of this study was to evaluate the impact of MALDI-TOF MS alone versus MALDI-TOF MS combined with real-time, pharmacist-driven, antimicrobial stewardship (AMS) intervention on patient outcomes. This single-center, pre-post, quasiexperimental study evaluated hospitalized patients with positive blood cultures identified via MALDI-TOF MS combined with prospective AMS intervention compared to a control cohort with MALDI-TOF MS identification without AMS intervention. AMS intervention included: real-time MALDI-TOF MS pharmacist notification and prospective AMS provider feedback. The primary outcome was the time to optimal therapy (TTOT). A total of 252 blood cultures, 126 in each group, were included in the final analysis. MALDI-TOF MS plus AMS intervention significantly reduced the overall TTOT (75.17 versus 43.06 h; P < 0.001), the Gram-positive contaminant TTOT (48.21 versus 11.75 h; P < 0.001), the Gram-negative infection (GNI) TTOT (71.83 versus 35.98 h; P < 0.001), and the overall hospital length of stay (LOS; 15.03 versus 9.02 days; P = 0.021). The TTOT for Gram-positive infection (GPI) was improved (64.04 versus 41.61 h; P = 0.082). For GPI, the hospital LOS (14.64 versus 10.31 days; P = 0.002) and length of antimicrobial therapy 24.30 versus 18.97 days; P = 0.018) were reduced. For GNI, the time to microbiologic clearance (51.13 versus 34.51 h; P < 0.001), the hospital LOS (15.40 versus 7.90 days; P = 0.027), and the intensive care unit LOS (5.55 versus 1.19 days; P = 0.035) were reduced. To achieve optimal outcomes, rapid identification with MALDI-TOF MS combined with real-time AMS intervention is more impactful than MALDI-TOF MS alone. Copyright © 2017 American Society for Microbiology.

Novel approaches for the taxonomic and metabolic characterization of lactobacilli: Integration of 16S rRNA gene sequencing with MALDI-TOF MS and 1H-NMR

PubMed Central

Parolin, Carola; Giordani, Barbara; Compri, Monica; Cevenini, Roberto; Vitali, Beatrice

2017-01-01

Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the ecological equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the ‘health-promoting’ significance of Lactobacillus genus, its genotypic and phenotypic characterization still poses several difficulties. The aim of this study was to assess the integration of different approaches, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic and metabolic characterization of Lactobacillus species. For this purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, probiotics), belonging to different species. The high discriminatory power of MALDI-TOF for species identification was underlined by the excellent agreement with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 out of 40 Lactobacillus strains at the species level, with an overall concordance of 97.5%. In the perspective to simplify the MALDI TOF sample preparation, especially for routine practice, we demonstrated the perfect agreement of the colony-picking from agar plates with the protein extraction protocol. 1H-NMR analysis, applied to both culture supernatants and bacterial lysates, identified a panel of metabolites whose variations in concentration were associated with the taxonomy, but also revealed a high intra-species variability that did not allow a species-level identification. Therefore, despite not suitable for mere taxonomic purposes, metabolomics can be useful to correlate particular biological activities with taxonomy and to understand the mechanisms related to the antimicrobial effect shown by some Lactobacillus species. PMID:28207855

MALDI-TOF Mass Spectrometry for the Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar

PubMed Central

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

2015-01-01

Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson’s medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples. PMID:25874612

High-throughput matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative approach to monitoring drug resistance of hepatitis B virus.

PubMed

Rybicka, Magda; Stalke, Piotr; Dreczewski, Marcin; Smiatacz, Tomasz; Bielawski, Krzysztof Piotr

2014-01-01

Long-term antiviral therapy of chronic hepatitis B virus (HBV) infection can lead to the selection of drug-resistant HBV variants and treatment failure. Moreover, these HBV strains are possibly present in treatment-naive patients. Currently available assays for the detection of HBV drug resistance can identify mutants that constitute ≥5% of the viral population. Furthermore, drug-resistant HBV variants can be detected when a viral load is >10(4) copies/ml (1,718 IU/ml). The aim of this study was to compare matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multitemperature single-strand conformation polymorphism (MSSCP) with commercially available assays for the detection of drug-resistant HBV strains. HBV DNA was extracted from 87 serum samples acquired from 45 chronic hepatitis B (CHB) patients. The 37 selected HBV variants were analyzed in 4 separate primer extension reactions on the MALDI-TOF MS. Moreover, MSSCP for identifying drug-resistant HBV YMDD variants was developed and turned out to be more sensitive than INNOLiPA HBV DR and direct sequencing. MALDI-TOF MS had the capability to detect mutant strains within a mixed viral population occurring with an allelic frequency of approximately 1% (with a specific value of ≥10(2) copies/ml, also expressed as ≥17.18 IU/ml). In our study, MSSCP detected 98% of the HBV YMDD variants among strains detected by the MALDI-TOF MS assay. The routine tests revealed results of 40% and 11%, respectively, for INNOLiPA and direct sequencing. The commonly available HBV tests are less sensitive than MALDI-TOF MS in the detection of HBV-resistant variants, including quasispecies.

Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS.

PubMed

Shubhakar, Archana; Kozak, Radoslaw P; Reiding, Karli R; Royle, Louise; Spencer, Daniel I R; Fernandes, Daryl L; Wuhrer, Manfred

2016-09-06

Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.

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

PubMed

Levander, Fredrik; James, Peter

2005-01-01

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

[Molecular characterization of pathogenic bacteria of the respiratory tract in peruvian patients with cystic fibrosis].

PubMed

Aquino, Ruth; Gonzáles, Emely; Samaniego, Sol; Rivera, Juan; Cedeño, Virna; Urbina, Yrene; Diringer, Benoit

2017-01-01

To molecularly characterize the pathogenic bacteria of the respiratory tract isolated from patients with cystic fibrosis (CF) in Peru. Bacterial communities cultured from sputum samples of pediatric and adult patients with CF admitted to the Edgardo Rebagliati Martins National Hospital and the National Institute of Child Health were characterized. Standard microbiological techniques were used for bacterial culture, and gene sequencing of 16S rRNA and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and tandem MALDI-TOF mass spectrometry (MALDI TOF/TOF) were used for molecular characterization. Seventeen bacterial strains were characterized by 16S rRNA sequencing, and the identified pathogenic bacteria were Pseudomonas aeruginosa (31.5%), Staphylococcus aureus (12.6%), Pseudomonas spp. (11.8%), and Klebsiella oxytoca (3.1%). MALDI-TOF analysis generated a series of spectra representative of each isolated bacterial species, whereas MALDI TOF/TOF analysis identified the peptides and proteins of the most common strains and provided data on pathogenicity and sensitivity to antibiotics. The primary pathogenic microorganisms found in the respiratory tract of patients with CF in Peru were the same as those found in other countries. This study is the first to perform 16S rRNA sequencing as well as MALDI-TOF and MALDI-TOF/TOF analysis of the bacterial pathogens circulating in Peru. The inclusion of proteomic analysis further allowed for the identification of native microorganisms involved in CF.

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

PubMed

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.

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

PubMed

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

2015-06-01

Utilizing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra for Candida glabrata typing would be a cost-effective and easy-to-use alternative to classical DNA-based typing methods. This study aimed to use MALDI-TOF for the typing of C. glabrata clinical isolates from various geographical origins and test its capacity to differentiate between fluconazole-sensitive and -resistant strains.Both microsatellite length polymorphism (MLP) and MALDI-TOF mass spectra of 58 C. glabrata isolates originating from Marseilles (France) and Tunis (Tunisia) as well as collection strains from diverse geographic origins were analyzed. The same analysis was conducted on a subset of C. glabrata isolates that were either susceptible (MIC ≤ 8 mg/l) or resistant (MIC ≥ 64 mg/l) to fluconazole.According to the seminal results, both MALDI-TOF and MLP classifications could highlight C. glabrata population structures associated with either geographical dispersal barriers (p < 10(-5)) or the selection of antifungal drug resistance traits (<10(-5)).In conclusion, MALDI-TOF geographical clustering was congruent with MPL genotyping and highlighted a significant population genetic structure according to fluconazole susceptibility in C. glabrata. Furthermore, although MALDI-TOF and MLP resulted in distinct classifications, MALDI-TOF also classified the isolates with respect to their fluconazole susceptibility profile. Further prospective studies are required to evaluate the capacity of MALDI-TOF typing to investigate C. glabrata infection outbreaks and predict the antifungal susceptibility profile of clinical laboratory isolates. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories ▿

PubMed Central

van Veen, S. Q.; Claas, E. C. J.; Kuijper, Ed J.

2010-01-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory. PMID:20053859

High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories.

PubMed

van Veen, S Q; Claas, E C J; Kuijper, Ed J

2010-03-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.

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

NASA Astrophysics Data System (ADS)

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.

Solvent-based and solvent-free characterization of low solubility and low molecular weight polyamides by mass spectrometry: a complementary approach.

PubMed

Barrère, Caroline; Hubert-Roux, Marie; Lange, Catherine M; Rejaibi, Majed; Kebir, Nasreddine; Désilles, Nicolas; Lecamp, Laurence; Burel, Fabrice; Loutelier-Bourhis, Corinne

2012-06-15

Polyamides (PA) belong to the most used classes of polymers because of their attractive chemical and mechanical properties. In order to monitor original PA design, it is essential to develop analytical methods for the characterization of these compounds that are mostly insoluble in usual solvents. A low molecular weight polyamide (PA11), synthesized with a chain limiter, has been used as a model compound and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In the solvent-based approach, specific solvents for PA, i.e. trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP), were tested. Solvent-based sample preparation methods, dried-droplet and thin layer, were optimized through the choice of matrix and salt. Solvent-based (thin layer) and solvent-free methods were then compared for this low solubility polymer. Ultra-high-performance liquid chromatography/electrospray ionization (UHPLC/ESI)-TOF-MS analyses were then used to confirm elemental compositions through accurate mass measurement. Sodium iodide (NaI) and 2,5-dihydroxybenzoic acid (2,5-DHB) are, respectively, the best cationizing agent and matrix. The dried-droplet sample preparation method led to inhomogeneous deposits, but the thin-layer method could overcome this problem. Moreover, the solvent-free approach was the easiest and safest sample preparation method giving equivalent results to solvent-based methods. Linear as well as cyclic oligomers were observed. Although the PA molecular weights obtained by MALDI-TOF-MS were lower than those obtained by (1)H NMR and acido-basic titration, this technique allowed us to determine the presence of cyclic and linear species, not differentiated by the other techniques. TFA was shown to induce modification of linear oligomers that permitted cyclic and linear oligomers to be clearly highlighted in spectra. Optimal sample preparation conditions were determined for the MALDI-TOF-MS analysis of PA11, a model of polyamide analogues. The advantages of the solvent-free and solvent-based approaches were shown. Molecular weight determination using MALDI was discussed. Copyright © 2012 John Wiley & Sons, Ltd.

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

PubMed

Wang, Aili; Liu, Li; Peng, Yanchun; Islam, Shahidul; Applebee, Marie; Appels, Rudi; Yan, Yueming; Ma, Wujun

2015-01-01

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

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

PubMed Central

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

2015-01-01

Low molecular weight glutenin subunits (LMW-GS) play an important role in determining dough properties and breadmaking quality. However, resolution of the currently used methodologies for analyzing LMW-GS is rather low which prevents an efficient use of genetic variations associated with these alleles in wheat breeding. The aim of the current study is to evaluate and develop a rapid, simple, and accurate method to differentiate LMW-GS alleles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A set of standard single LMW-GS allele lines as well as a suite of well documented wheat cultivars were collected from France, CIMMYT, and Canada. Method development and optimization were focused on protein extraction procedures and MALDI-TOF instrument settings to generate reproducible diagnostic spectrum peak profiles for each of the known wheat LMW-GS allele. Results revealed a total of 48 unique allele combinations among the studied genotypes. Characteristic MALDI-TOF peak patterns were obtained for 17 common LMW-GS alleles, including 5 (b, a or c, d, e, f), 7 (a, b, c, d or i, f, g, h) and 5 (a, b, c, d, f) patterns or alleles for the Glu-A3, Glu-B3, and Glu-D3 loci, respectively. In addition, some reproducible MALDI-TOF peak patterns were also obtained that did not match with any known alleles. The results demonstrated a high resolution and throughput nature of MALDI-TOF technology in analyzing LMW-GS alleles, which is suitable for application in wheat breeding programs in processing a large number of wheat lines with high accuracy in limited time. It also suggested that the variation of LMW-GS alleles is more abundant than what has been defined by the current nomenclature system that is mainly based on SDS-PAGE system. The MALDI-TOF technology is useful to differentiate these variations. An international joint effort may be needed to assign allele symbols to these newly identified alleles and determine their effects on end-product quality attributes. PMID:26407296

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

PubMed

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.

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.

PubMed

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.

Comparative evaluation of the identification of rapidly growing non-tuberculous mycobacteria by mass spectrometry (MALDI-TOF MS), GenoType Mycobacterium CM/AS assay and partial sequencing of the rpoβ gene with phylogenetic analysis as a reference method.

PubMed

Costa-Alcalde, José Javier; Barbeito-Castiñeiras, Gema; González-Alba, José María; Aguilera, Antonio; Galán, Juan Carlos; Pérez-Del-Molino, María Luisa

2018-06-02

The American Thoracic Society and the Infectious Diseases Society of America recommend that clinically significant non-tuberculous mycobacteria (NTM) should be identified to the species level in order to determine their clinical significance. The aim of this study was to evaluate identification of rapidly growing NTM (RGM) isolated from clinical samples by using MALDI-TOF MS and a commercial molecular system. The results were compared with identification using a reference method. We included 46 clinical isolates of RGM and identified them using the commercial molecular system GenoType ® CM/AS (Hain, Lifescience, Germany), MALDI-TOF MS (Bruker) and, as reference method, partial rpoβ gene sequencing followed by BLAST and phylogenetic analysis with the 1093 sequences available in the GeneBank. The degree of agreement between GenoType ® and MALDI-TOF MS and the reference method, partial rpoβ sequencing, was 27/43 (62.8%) and 38/43 cases (88.3%) respectively. For all the samples correctly classified by GenoType ® , we obtained the same result with MALDI-TOF MS (27/27). However, MALDI-TOF MS also correctly identified 68.75% (11/16) of the samples that GenoType ® had misclassified (p=0.005). MALDI-TOF MS classified significantly better than GenoType ® . When a MALDI-TOF MS score >1.85 was achieved, MALDI-TOF MS and partial rpoβ gene sequencing were equivalent. GenoType ® was not able to distinguish between species belonging to the M. fortuitum complex. MALDI-TOF MS methodology is simple, rapid and associated with lower consumable costs than GenoType ® . The partial rpoβ sequencing methods with BLAST and phylogenetic analysis were not able to identify some RGM unequivocally. Therefore, sequencing of additional regions would be indicated in these cases. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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...

MALDI-TOF MS of Trichoderma: A model system for the identification of microfungi

USDA-ARS?s Scientific Manuscript database

This investigation aimed to assess whether MALDI-TOF MS analysis of proteomics could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of proteomics would reveal ap...

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

PubMed

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 < 0.0001). None of the isolates was discordantly identified at the genus level using MALDI-TOF MS and only 9 of them could not be identified using the method. Thus, our results show that MALDI-TOF MS is a robust and reliable tool for the identification of 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.

Top-down proteomic identification of bacterial protein biomarkers and toxins using MALDI-TOF-TOF-MS/MS and post-source decay

USDA-ARS?s Scientific Manuscript database

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight mass spectrometry(MALDI-TOF-TOF-MS)has provided new capabilities for the rapid identification of digested and non-digested proteins. The tandem (MS/MS) capability of TOF-TOF instruments allows precursor ion selection/isolation...

Analysis of Bacterial Lipooligosaccharides by MALDI-TOF MS with Traveling Wave Ion Mobility

NASA Astrophysics Data System (ADS)

Phillips, Nancy J.; John, Constance M.; Jarvis, Gary A.

2016-07-01

Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source "prompt" fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis.

Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry

PubMed Central

Wang, Da-Zhi; Dong, Hong-Po; Li, Cheng; Xie, Zhang-Xian; Lin, Lin; Hong, Hua-Sheng

2011-01-01

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions. PMID:21904561

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

PubMed

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.

Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Schmidt, V; Jarosch, A; März, P; Sander, C; Vacata, V; Kalka-Moll, W

2012-03-01

Blood culture is probably the most significant specimen used for the diagnosis of bacterial infections, especially for bloodstream infections. In the present study, we compared the resin-containing BD BACTEC™ Plus-Aerobic (Becton Dickinson), non-charcoal-containing BacT/Alert(®) SA (bioMérieux), and charcoal-containing BacT/Alert(®) FA (bioMérieux) blood culture bottles with direct identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 103 bacterial isolates, from clinical blood cultures, representing the most frequent 13 genera and 24 species were examined. Bacteria were extracted from positive blood culture broth by density centrifugation and then subjected to identification by MALDI-TOF MS using two different volumes and chemical treatments. Overall, correct identification by MALDI-TOF MS was obtained for the BD BACTEC™ Plus-Aerobic, BacT/Alert(®) SA, and BacT/Alert(®) FA blood culture bottles in 72%, 45.6%, and 23%, respectively, for gram-negative bacteria in 86.6%, 69.2%, and 47.1%, respectively, and for gram-positive bacteria in 60.0%, 28.8%, and 5.4%, respectively. The lack of identification was observed mainly with viridans streptococci. Depending on the blood culture bottles used in routine diagnostic procedures and the protocol for bacterial preparation, the applied MALDI-TOF MS represents an efficient and rapid method for direct bacterial identification.

MALDI-TOF Mass Spectrometry: A Powerful Tool for Clinical Microbiology at Hôpital Principal de Dakar, Senegal (West Africa)

PubMed Central

Lo, Cheikh I.; Fall, Bécaye; Sambe-Ba, Bissoume; Diawara, Silman; Gueye, Mamadou W.; Mediannikov, Oleg; Sokhna, Cheikh; Faye, Ngor; Diemé, Yaya; Wade, Boubacar; Raoult, Didier; Fenollar, Florence

2015-01-01

Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, “Necropsobacter massiliensis.” The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries. PMID:26716681

Prospective Evaluation of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System in a Hospital Clinical Microbiology Laboratory for Identification of Bacteria and Yeasts: a Bench-by-Bench Study for Assessing the Impact on Time to Identification and Cost-Effectiveness

PubMed Central

Tan, K. E.; Ellis, B. C.; Lee, R.; Stamper, P. D.; Zhang, S. X.

2012-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories. PMID:22855510

Prospective evaluation of a matrix-assisted laser desorption ionization-time of flight mass spectrometry system in a hospital clinical microbiology laboratory for identification of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identification and cost-effectiveness.

PubMed

Tan, K E; Ellis, B C; Lee, R; Stamper, P D; Zhang, S X; Carroll, K C

2012-10-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories.

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

PubMed

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

PubMed

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.

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

PubMed

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.

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▿

PubMed Central

Uhlik, Ondrej; Strejcek, Michal; Junkova, Petra; Sanda, Miloslav; Hroudova, Miluse; Vlcek, Cestmir; Mackova, Martina; Macek, Tomas

2011-01-01

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases. PMID:21821747

Evaluation of the Andromas Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Aerobically Growing Gram-Positive Bacilli

PubMed Central

Farfour, E.; Leto, J.; Barritault, M.; Barberis, C.; Meyer, J.; Dauphin, B.; Le Guern, A.-S.; Leflèche, A.; Badell, E.; Guiso, N.; Leclercq, A.; Le Monnier, A.; Lecuit, M.; Rodriguez-Nava, V.; Bergeron, E.; Raymond, J.; Vimont, S.; Bille, E.; Carbonnelle, E.; Guet-Revillet, H.; Lécuyer, H.; Beretti, J.-L.; Vay, C.; Berche, P.; Ferroni, A.; Nassif, X.

2012-01-01

Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry. PMID:22692743

[MALDI-TOF mass spectrometry: Evaluation of the preanalytical phase for identification of molds].

PubMed

Maldonado, Ivana; García Ramírez, Dolores; Striebeck, Pablo; Lafage, Marcelo; Fernández Canigia, Liliana

In order to optimize the identification of molds with MALDI-TOF MS, three protein extraction-methodologies were evaluated against 44 isolates: water extraction (WE), zirconium extraction (ZE) and the provider's recommended method (PRM). Two data bases were compared, Bruker (BK) and Bruker+National Institutes of Health. Considering both databases, results were respectively as follows: correct identification (CI) at gender level, 10 and 16 by WE; 27 and 32 by ZE and 18 and 23 by PRM; CI at species level, 5 and 7 by WE; 17 and 20 by ZE and 11 and 14 by PRM; non-reliable identification, 18 and 12 by WE; 9 and 4 by ZE and by PRM. No peaks were observed in 16 by WE, 8 by ZE and 17 by PRM. ZE showed the best perfomance (p<0.05). Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

A designed experiments approach to optimizing MALDI-TOF MS spectrum processing parameters enhances detection of antibiotic resistance in Campylobacter jejuni

USDA-ARS?s Scientific Manuscript database

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this...

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

PubMed

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

2014-01-01

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

Identification of urinary tract pathogens after 3-hours urine culture by MALDI-TOF mass spectrometry.

PubMed

Haiko, Johanna; Savolainen, Laura E; Hilla, Risto; Pätäri-Sampo, Anu

2016-10-01

Complicated urinary tract infections, such as pyelonephritis, may lead to sepsis. Rapid diagnosis is needed to identify the causative urinary pathogen and to verify the appropriate empirical antimicrobial therapy. We describe here a rapid identification method for urinary pathogens: urine is incubated on chocolate agar for 3h at 35°C with 5% CO2 and subjected to MALDI-TOF MS analysis by VITEK MS. Overall 207 screened clinical urine samples were tested in parallel with conventional urine culture. The method, called U-si-MALDI-TOF (urine short incubation MALDI-TOF), showed correct identification for 86% of Gram-negative urinary tract pathogens (Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae), when present at >10(5)cfu/ml in culture (n=107), compared with conventional culture method. However, Gram-positive bacteria (n=28) were not successfully identified by U-si-MALDI-TOF. This method is especially suitable for rapid identification of E. coli, the most common cause of urinary tract infections and urosepsis. Turnaround time for identification using U-si-MALDI-TOF compared with conventional urine culture was improved from 24h to 4-6h. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

NASA Astrophysics Data System (ADS)

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.

Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing.

PubMed

Lawton, Samantha J; Weis, Allison M; Byrne, Barbara A; Fritz, Heather; Taff, Conor C; Townsend, Andrea K; Weimer, Bart C; Mete, Aslı; Wheeler, Sarah; Boyce, Walter M

2018-05-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates.

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.

PubMed

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 (<20 minutes) bacterial identification 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.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry for the identification of Neisseria gonorrhoeae.

PubMed

Buchanan, R; Ball, D; Dolphin, H; Dave, J

2016-09-01

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared with the API NH biochemical method for the identification of Neisseria gonorrhoeae in routine clinical samples. A retrospective review of laboratory records for 1090 isolates for which both biochemical and MALDI-TOF MS identifications were available was performed. Cases of discrepant results were examined in detail for evidence supportive of a particular organism identification. Of 1090 isolates, 1082 were identified as N. gonorrhoeae by API NH. MALDI-TOF MS successfully identified 984 (91%) of these after one analysis, rising to 1081 (99.9%) after two analyses, with a positive predictive value of 99.3%. For those isolates requiring a repeat analysis, failure to generate an identifiable proteomic signature was the reason in 76% of cases, with alternative initial identifications accounting for the remaining 24%. MALDI-TOF MS identified eight isolates as N. gonorrhoeae that were not identified as such by API NH-examination of these discrepant results suggested that the MALDI-TOF MS identification may be the more reliable. MALDI-TOF MS is at least as accurate and reliable a method of identifying N. gonorrhoeae as API NH. We propose that MALDI-TOF MS could potentially be used as a single method for N. gonorrhoeae identification in routine cases by laboratories with access to this technology. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Safety and Accuracy of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Highly Pathogenic Organisms.

PubMed

Rudrik, James T; Soehnlen, Marty K; Perry, Michael J; Sullivan, Maureen M; Reiter-Kintz, Wanda; Lee, Philip A; Pettit, Denise; Tran, Anthony; Swaney, Erin

2017-12-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) sample preparation methods, including the direct, on-plate formic acid, and ethanol/formic acid tube extraction methods, were evaluated for their ability to render highly pathogenic organisms nonviable and safe for handling in a biosafety level 2 laboratory. Of these, the tube extraction procedure was the most successful, with none of the tested strains surviving this sample preparation method. Tube extracts from several agents of bioterrorism and their near neighbors were analyzed in an eight-laboratory study to examine the utility of the Bruker Biotyper and Vitek MS MALDI-TOF MS systems and their in vitro diagnostic (IVD), research-use-only, and Security-Relevant databases, as applicable, to accurately identify these agents. Forty-six distinct strains of Bacillus anthracis , Yersinia pestis , Francisella tularensis , Burkholderia mallei , Burkholderia pseudomallei , Clostridium botulinum , Brucella melitensis , Brucella abortus , Brucella suis , and Brucella canis were extracted and distributed to participating laboratories for analysis. A total of 35 near-neighbor isolates were also analyzed. Copyright © 2017 Rudrik et al.

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

PubMed

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.

Unlocking the proteomic information encoded in MALDI-TOF-MS data used for microbial identification and characterization.

PubMed

Fagerquist, Clifton K

2017-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is increasingly utilized as a rapid technique to identify microorganisms including pathogenic bacteria. However, little attention has been paid to the significant proteomic information encoded in the MS peaks that collectively constitute the MS 'fingerprint'. This review/perspective is intended to explore this topic in greater detail in the hopes that it may spur interest and further research in this area. Areas covered: This paper examines the recent literature on utilizing MALDI-TOF for bacterial identification. Critical works highlighting protein biomarker identification of bacteria, arguments for and against protein biomarker identification, proteomic approaches to biomarker identification, emergence of MALDI-TOF-TOF platforms and their use for top-down proteomic identification of bacterial proteins, protein denaturation and its effect on protein ion fragmentation, collision cross-sections and energy deposition during desorption/ionization are also explored. Expert commentary: MALDI-TOF and TOF-TOF mass spectrometry platforms will continue to provide chemical analyses that are rapid, cost-effective and high throughput. These instruments have proven their utility in the taxonomic identification of pathogenic bacteria at the genus and species level and are poised to more fully characterize these microorganisms to the benefit of clinical microbiology, food safety and other fields.

Time-resolved imaging of the MALDI linear-TOF ion cloud: direct visualization and exploitation of ion optical phenomena using a position- and time-sensitive detector.

PubMed

Ellis, Shane R; Soltwisch, Jens; Heeren, Ron M A

2014-05-01

In this study, we describe the implementation of a position- and time-sensitive detection system (Timepix detector) to directly visualize the spatial distributions of the matrix-assisted laser desorption ionization ion cloud in a linear-time-of-flight (MALDI linear-ToF) as it is projected onto the detector surface. These time-resolved images allow direct visualization of m/z-dependent ion focusing effects that occur within the ion source of the instrument. The influence of key parameters, namely extraction voltage (E(V)), pulsed-ion extraction (PIE) delay, and even the matrix-dependent initial ion velocity was investigated and were found to alter the focusing properties of the ion-optical system. Under certain conditions where the spatial focal plane coincides with the detector plane, so-called x-y space focusing could be observed (i.e., the focusing of the ion cloud to a small, well-defined spot on the detector). Such conditions allow for the stigmatic ion imaging of intact proteins for the first time on a commercial linear ToF-MS system. In combination with the ion-optical magnification of the system (~100×), a spatial resolving power of 11–16 μm with a pixel size of 550 nm was recorded within a laser spot diameter of ~125 μm. This study demonstrates both the diagnostic and analytical advantages offered by the Timepix detector in ToF-MS.

What Is New in Clinical Microbiology—Microbial Identification by MALDI-TOF Mass Spectrometry

PubMed Central

Murray, Patrick R.

2012-01-01

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) offers the possibility of accurate, rapid, inexpensive identification of bacteria, fungi, and mycobacteria isolated in clinical microbiology laboratories. The procedures for preanalytic processing of organisms and analysis by MALDI-TOF MS are technically simple and reproducible, and commercial databases and interpretive algorithms are available for the identification of a wide spectrum of clinically significant organisms. Although only limited work has been reported on the use of this technique to identify molds, perform strain typing, or determine antibiotic susceptibility results, these are fruitful areas of promising research. As experience is gained with MALDI-TOF MS, it is expected that the databases will be expanded to resolve many of the current inadequate identifications (eg, no identification, genus-level identification) and algorithms for potential misidentification will be developed. The current lack of Food and Drug Administration approval of any MALDI-TOF MS system for organism identification limits widespread use in the United States. PMID:22795961

Evaluation of capacity to detect ability to form biofilm in Candida parapsilosis sensu stricto strains by MALDI-TOF MS.

PubMed

Mlynáriková, Katarína; Šedo, Ondrej; Růžička, Filip; Zdráhal, Zbyněk; Holá, Veronika; Mahelová, Martina

2016-11-01

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is, currently, used as a rapid and reliable tool in microbial diagnostics. The discriminatory power of the method extends its applicability also beyond species level. This study examined the possibility to use MALDI-TOF MS to differentiate between Candida parapsilosis sensu stricto biofilm-positive (n = 12) and biofilm-negative (n = 9) strains. The results indicated a grouping trend within MALDI-TOF mass spectra belonging to each of the tested groups. However, these trends were eclipsed by mass spectral variations resulting from limited repeatability of the method, making its application for the selected purpose impossible. Improvement in the discriminatory power of the method was not obtained neither by using different matrices (α-cyano-4-hydroxycinnamic acid, ferulic acid, 5-chloro-2-mercaptobenzothionazole) for MALDI-TOF MS analysis nor by testing different culture conditions (cultivation length, culture media).

Peptidylation for the determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Tang, Feng; Cen, Si-Ying; He, Huan; Liu, Yi; Yuan, Bi-Feng; Feng, Yu-Qi

2016-05-23

Determination of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been a great challenge in the analytical research field. Here we developed a universal peptide-based derivatization (peptidylation) strategy for the sensitive analysis of low-molecular-weight compounds by MALDI-TOF-MS. Upon peptidylation, the molecular weights of target analytes increase, thus avoiding serious matrix ion interference in the low-molecular-weight region in MALDI-TOF-MS. Since peptides typically exhibit good signal response during MALDI-TOF-MS analysis, peptidylation endows high detection sensitivities of low-molecular-weight analytes. As a proof-of-concept, we analyzed low-molecular-weight compounds of aldehydes and thiols by the developed peptidylation strategy. Our results showed that aldehydes and thiols can be readily determined upon peptidylation, thus realizing the sensitive and efficient determination of low-molecular-weight compounds by MALDI-TOF-MS. Moreover, target analytes also can be unambiguously detected in biological samples using the peptidylation strategy. The established peptidylation strategy is a universal strategy and can be extended to the sensitive analysis of various low-molecular-weight compounds by MALDI-TOF-MS, which may be potentially used in areas such as metabolomics.

Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

PubMed

Barnini, Simona; Ghelardi, Emilia; Brucculeri, Veronica; Morici, Paola; Lupetti, Antonella

2015-06-18

Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

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.

PubMed

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.

Identification of bacteria isolated from veterinary clinical specimens using MALDI-TOF MS.

PubMed

Pavlovic, Melanie; Wudy, Corinna; Zeller-Peronnet, Veronique; Maggipinto, Marzena; Zimmermann, Pia; Straubinger, Alix; Iwobi, Azuka; Märtlbauer, Erwin; Busch, Ulrich; Huber, Ingrid

2015-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged as a rapid and accurate identification method for bacterial species. Although it has been successfully applied for the identification of human pathogens, it has so far not been well evaluated for routine identification of veterinary bacterial isolates. This study was performed to compare and evaluate the performance of MALDI-TOF MS based identification of veterinary bacterial isolates with commercially available conventional test systems. Discrepancies of both methods were resolved by sequencing 16S rDNA and, if necessary, the infB gene for Actinobacillus isolates. A total of 375 consecutively isolated veterinary samples were collected. Among the 357 isolates (95.2%) correctly identified at the genus level by MALDI-TOF MS, 338 of them (90.1% of the total isolates) were also correctly identified at the species level. Conventional methods offered correct species identification for 319 isolates (85.1%). MALDI-TOF identification therefore offered more accurate identification of veterinary bacterial isolates. An update of the in-house mass spectra database with additional reference spectra clearly improved the identification results. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for classification and identification of veterinary bacterial isolates.

How to: identify non-tuberculous Mycobacterium species using MALDI-TOF mass spectrometry.

PubMed

Alcaide, F; Amlerová, J; Bou, G; Ceyssens, P J; Coll, P; Corcoran, D; Fangous, M-S; González-Álvarez, I; Gorton, R; Greub, G; Hery-Arnaud, G; Hrábak, J; Ingebretsen, A; Lucey, B; Marekoviċ, I; Mediavilla-Gradolph, C; Monté, M R; O'Connor, J; O'Mahony, J; Opota, O; O'Reilly, B; Orth-Höller, D; Oviaño, M; Palacios, J J; Palop, B; Pranada, A B; Quiroga, L; Rodríguez-Temporal, D; Ruiz-Serrano, M J; Tudó, G; Van den Bossche, A; van Ingen, J; Rodriguez-Sanchez, B

2018-06-01

The implementation of MALDI-TOF MS for microorganism identification has changed the routine of the microbiology laboratories as we knew it. Most microorganisms can now be reliably identified within minutes using this inexpensive, user-friendly methodology. However, its application in the identification of mycobacteria isolates has been hampered by the structure of their cell wall. Improvements in the sample processing method and in the available database have proved key factors for the rapid and reliable identification of non-tuberculous mycobacteria isolates using MALDI-TOF MS. The main objective is to provide information about the proceedings for the identification of non-tuberculous isolates using MALDI-TOF MS and to review different sample processing methods, available databases, and the interpretation of the results. Results from relevant studies on the use of the available MALDI-TOF MS instruments, the implementation of innovative sample processing methods, or the implementation of improved databases are discussed. Insight about the methodology required for reliable identification of non-tuberculous mycobacteria and its implementation in the microbiology laboratory routine is provided. Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Prediction of fetal lung maturity using the lecithin/sphingomyelin (L/S) ratio analysis with a simplified sample preparation, using a commercial microtip-column combined with mass spectrometric analysis.

PubMed

Kwak, Ho-Seok; Chung, Hee-Jung; Choi, Young Sik; Min, Won-Ki; Jung, So Young

2015-07-01

Fetal lung maturity is estimated using the lecithin/sphingomyelin ratio (L/S ratio) in amniotic fluid and it is commonly measured with thin-layer chromatography (TLC). The TLC method is time consuming and technically difficult; however, it is widely used because there is no alternative. We evaluated a novel method for measuring the L/S ratio, which involves a tip-column with a cation-exchange resin and mass spectrometry. Phospholipids in the amniotic fluid were extracted using methanol and chloroform. Choline-containing phospholipids such as lecithin and sphingomyelin were purified by passing them through the tip-column. LC-MS/MS and MALDI-TOF were used to directly analyze the purified samples. The L/S ratio by mass spectrometry was calculated from the sum peak intensity of the six lecithin, and that of sphingomyelin 34:1. In 20 samples, the L/S ratio determined with TLC was significantly correlated with that obtained by LC-MS/MS and MALDI-TOF. There was a 100% concordance between the L/S ratio by TLC and that by LC-MS/MS (kappa value=1.0). The concordance between the L/S ratio by TLC and that by MALDI-TOF was also 100% (kappa value=1.0). Our method provides a faster, simpler, and more reliable assessment of fetal lung maturity. The L/S ratio measured by LC-MS/MS and MALDI-TOF offers a compelling alternative method to traditional TLC. Copyright © 2015 Elsevier B.V. All rights reserved.

MALDI-TOF for the rapid detection of carbapenemase-producing Enterobacteriaceae: comparison of the commercialized MBT STAR®-Carba IVD Kit with two in-house MALDI-TOF techniques and the RAPIDEC® CARBA NP.

PubMed

Dortet, Laurent; Tandé, Didier; de Briel, Dominique; Bernabeu, Sandrine; Lasserre, Camille; Gregorowicz, Guillaume; Jousset, Agnès B; Naas, Thierry

2018-06-11

There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here, we have evaluated three MALDI-TOF-based techniques to detect carbapenemase-producing Enterobacteriaceae (CPE) from cultured colonies. The performance of three MALDI-TOF-based techniques, including the commercialized MBT STAR®-Carba IVD Kit (Bruker Daltonics) and two in-house protocols performed on the Microflex LT Biotyper (Bruker Daltonics) and the VITEK® MS Plus (bioMérieux), were compared with those of the RAPIDEC® CARBA NP (bioMérieux). A collection of 175 isolates including 120 carbapenemase producers and 55 non-carbapenemase producers was tested. Samples were tested blind in the three participating centres. The repeatability of the MBT STAR®-Carba IVD Kit was also evaluated. The three MALDI-TOF techniques possess sensitivities ranging from 95% to 100% and specificities from 98.2% to 100% compared with 99.2% and 100%, respectively, for the RAPIDEC® CARBA NP. The MBT STAR®-Carba IVD Kit gave highly reproducible results and is the only technique able to provide a concomitant identification of the bacterial isolate. The three MALDI-TOF techniques possess a fast turnaround time (less than 1.5 h). Overall, MALDI-TOF is a reliable technique for the rapid detection of CPE from cultured colonies. MBT STAR®-Carba IVD Kit, the only commercially available assay, could easily be implemented in a clinical microbiology laboratory if it is already equipped with a Microflex LT Biotyper mass spectrometer.

MALDI-TOF mass spectrometry provides high accuracy in identification of Salmonella at species level but is limited to type or subtype Salmonella serovars.

PubMed

Kang, Lin; Li, Nan; Li, Ping; Zhou, Yang; Gao, Shan; Gao, Hongwei; Xin, Wenwen; Wang, Jinglin

2017-04-01

Salmonella can cause global foodborne illnesses in humans and many animals. The current diagnostic gold standard used for detecting Salmonella infection is microbiological culture followed by serological confirmation tests. However, these methods are complicated and time-consuming. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis offers some advantages in rapid identification, for example, simple and fast sample preparation, fast and automated measurement, and robust and reliable identification up to genus and species levels, possibly even to the strain level. In this study, we established a reference database for species identification using whole-cell MALDI-TOF MS; the database consisted of 12 obtained main spectra of the Salmonella culture collection strains belonged to seven serotypes. Eighty-two clinical isolates of Salmonella were identified using established database, and partial 16S rDNA gene sequencing and serological method were used as comparison. We found that MALDI-TOF mass spectrometry provided high accuracy in identification of Salmonella at species level but was limited to type or subtype Salmonella serovars. We also tried to find serovar-specific biomarkers and failed. Our study demonstrated that (a) MALDI-TOF MS was suitable for identification of Salmonella at species level with high accuracy and (b) that MALDI-TOF MS method presented in this study was not useful for serovar assignment of Salmonella currently, because of its low matching with serological method and (c) MALDI-TOF MS method presented in this study was not suitable to subtype S. typhimurium because of its low discriminatory ability.

Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard; Raoult, Didier

2013-07-01

During the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard

2013-01-01

During the past 5 years, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories. PMID:23637301

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

PubMed

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.

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis.

PubMed

Kim, Young Eun; Yi, So Yeon; Lee, Chang-Soo; Jung, Yongwon; Chung, Bong Hyun

2012-01-21

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.

A new calibrant for MALDI-TOF-TOF-PSD-MS/MS of non-digested proteins for top-down proteomic analysis

USDA-ARS?s Scientific Manuscript database

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight (TOF-TOF) tandem mass spectrometry (MS/MS) has seen increasing use for post-source decay (PSD)-MS/MS analysis of non-digested protein ions for top-down proteomic identification. However, there is no commonl...

High sensitive and throughput screening of Aflatoxin using MALDI-TOF-TOF-PSD-MS/MS

USDA-ARS?s Scientific Manuscript database

We have achieved sensitive and efficient detection of aflatoxin B1(AFB1) through matrix-assisted laser desorption/ionization time-of-flight-time-of-flight mass spectrometry (MALDI-TOF-TOF) and post-source decay (PSD) tandem mass spectrometry (MS/MS) using an acetic acid – a-cyano-4-hydroxycinnamic a...

Short-term incubation of positive blood cultures in brain-heart infusion broth accelerates identification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry.

PubMed

Torres, Ignacio; Gimenez, Estela; Pascual, Tania; Bueno, Felipe; Huntley, Dixie; Martínez, Mireia; Navarro, David

2017-12-01

Fast identification of bacteria directly from positive blood cultures (BCs) by matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) can be achieved either using the MALDI Sepsityper kit (protein extraction method) or after a short-term pre-cultivation step on solid medium. We developed a new method that involves short-term enrichment of positive BCs in brain-heart infusion broth (BHI) prior to MALDI-TOF MS analysis. Eighty-four BCs flagged as positive were included in this study; these were processed in parallel either directly using the MALDI Sepsityper kit or following a short-term culture either in BHI or on Columbia blood agar with 5 % sheep blood (CBA). Bacterial species were successfully identified in 91.6, 89.2 and 65.4 % of cases after pre-cultivation for 4 h in BHI, on CBA, or by using the MALDI Sepsityper kit, respectively. Overall, the mean incubation time to correct identification was shorter when pre-cultures were performed in BHI; the mean time for Gram-negative rods was 78.2 min in BHI and 108.2 min on CBA (P=0.045), and the mean time for Gram-positive cocci was 128.5 min in BHI and 169.6 min on CBA (P=0.013). Short-term enrichment of BCs in BHI accelerates identification of a number of bacterial species by MALDI-TOF MS. Further prospective studies are needed to validate our method and gauge its potential clinical impact on the management of bloodstream bacterial infections.

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

PubMed

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.

MALDI-TOF-mass spectrometry applications in clinical microbiology.

PubMed

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.

Application of MALDI-TOF MS for the Identification of Food Borne Bacteria

PubMed Central

Pavlovic, Melanie; Huber, Ingrid; Konrad, Regina; Busch, Ulrich

2013-01-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS based identification of bacteria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality. This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed. PMID:24358065

Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOF.

PubMed Central

Tolson, D A; Nicholson, N H

1998-01-01

The determination of DNA sequences by partial exonuclease digestion followed by Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF) is a well established method. When the same procedure is applied to RNA, difficulties arise due to the small (1 Da) mass difference between the nucleotides U and C, which makes unambiguous assignment difficult using a MALDI-TOF instrument. Here we report our experiences with sequence specific endonucleases and chemical methods followed by MALDI-TOF to resolve these sequence ambiguities. We have found chemical methods superior to endonucleases both in terms of correct specificity and extent of sequence coverage. This methodology can be used in combination with exonuclease digestion to rapidly assign RNA sequences. PMID:9421498

Identification of Lactobacillus from the Saliva of Adult Patients with Caries Using Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry

PubMed Central

Ma, Qingwei; Song, Yeqing; Zhang, Qian; Wang, Xiaoyan; Chen, Feng

2014-01-01

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been presented as a superior method for the detection of microorganisms in body fluid samples (e.g., blood, saliva, pus, etc.) However, the performance of MALDI-TOF MS in routine identification of caries-related Lactobacillus isolates from saliva of adult patients with caries has not been determined. In the present study, we introduced a new MALDI-TOF MS system for identification of lactobacilli. Saliva samples were collected from 120 subjects with caries. Bacteria were isolated and cultured, and each isolate was identified by both 16S rRNA sequencing and MALDI-TOF MS. The identification results obtained by MALDI-TOF MS were concordant at the genus level with those of conventional 16S rRNA-based sequencing for 88.6% of lactobacilli (62/70) and 95.5% of non-lactobacilli (21/22). Up to 96 results could be obtained in parallel on a single MALDI target, suggesting that this is a reliable high-throughput approach for routine identification of lactobacilli. However, additional reference strains are necessary to increase the sensitivity and specificity of species-level identification. PMID:25166027

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

PubMed

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 approach allowed an optimized treatment recommendation. MALDI-TOF MS following 4h pre-culture is a valuable tool for rapid pathogen identification from positive blood cultures, allowing easy integration in diagnostic routine and the opportunity of considerably earlier treatment adaptation. Copyright © 2015 Elsevier GmbH. All rights reserved.

YahO protein as a calibrant for top-down proteomic identification of Shiga toxin using MALDI-TOF-TOF-MS/MS and post-source decay

USDA-ARS?s Scientific Manuscript database

Matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF-TOF) mass spectrometry is increasingly utilized for rapid top-down proteomic identification of proteins. This identification may involve analysis of either a pure protein or a protein mixture. For analysis of a pure protein...

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

PubMed

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.

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.

PubMed

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.

Species Identification and Delineation of Pathogenic Mucorales by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

PubMed

Shao, Jin; Wan, Zhe; Li, Ruoyu; Yu, Jin

2018-04-01

This study aimed to validate the effectiveness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification of filamentous fungi of the order Mucorales. A total of 111 isolates covering six genera preserved at the Research Center for Medical Mycology of Peking University were selected for MALDI-TOF MS analysis. We emphasized the study of 23 strains of Mucor irregularis predominantly isolated from patients in China. We first used the Bruker Filamentous Fungi library (v1.0) to identify all 111 isolates. To increase the identification rate, we created a compensatory in-house database, the Beijing Medical University (BMU) database, using 13 reference strains covering 6 species, including M. irregularis , Mucor hiemalis , Mucor racemosus , Cunninghamella bertholletiae , Cunninghamella phaeospora , and Cunninghamella echinulata All 111 isolates were then identified by MALDI-TOF MS using a combination of the Bruker library and BMU database. MALDI-TOF MS identified 55 (49.5%) and 74 (66.7%) isolates at the species and genus levels, respectively, using the Bruker Filamentous Fungi library v1.0 alone. A combination of the Bruker library and BMU database allowed MALDI-TOF MS to identify 90 (81.1%) and 111 (100%) isolates at the species and genus levels, respectively, with a significantly increased accuracy rate. MALDI-TOF MS poorly identified Mucorales when the Bruker library was used alone due to its lack of some fungal species. In contrast, this technique perfectly identified M. irregularis after main spectrum profiles (MSPs) of relevant reference strains were added to the Bruker library. With an expanded Bruker library, MALDI-TOF MS is an effective tool for the identification of pathogenic Mucorales. Copyright © 2018 American Society for Microbiology.

Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

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

2009-08-15

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification. MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in <1 h using a database comprising > or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.

Identification and differentiation of food-related bacteria: A comparison of FTIR spectroscopy and MALDI-TOF mass spectrometry.

PubMed

Wenning, Mareike; Breitenwieser, Franziska; Konrad, Regina; Huber, Ingrid; Busch, Ulrich; Scherer, Siegfried

2014-08-01

The food industry requires easy, accurate, and cost-effective techniques for microbial identification to ensure safe products and identify microbial contaminations. In this work, FTIR spectroscopy and MALDI-TOF mass spectrometry were assessed for their suitability and applicability for routine microbial diagnostics of food-related microorganisms by analyzing their robustness according to changes in incubation time and medium, identification accuracy and their ability to differentiate isolates down to the strain level. Changes in the protocol lead to a significantly impaired performance of FTIR spectroscopy, whereas they had only little effects on MALDI-TOF MS. Identification accuracy was tested using 174 food-related bacteria (93 species) from an in-house strain collection and 40 fresh isolates from routine food analyses. For MALDI-TOF MS, weaknesses in the identification of bacilli and pseudomonads were observed; FTIR spectroscopy had most difficulties in identifying pseudomonads and enterobacteria. In general, MALDI-TOF MS obtained better results (52-85% correct at species level), since the analysis of mainly ribosomal proteins is more robust and seems to be more reliable. FTIR spectroscopy suffers from the fact that it generates a whole-cell fingerprint and intraspecies diversity may lead to overlapping species borders which complicates identification. In the present study values between 56% and 67% correct species identification were obtained. On the opposite, this high sensitivity offers the opportunity of typing below the species level which was not possible using MALDI-TOF MS. Using fresh isolates from routine diagnostics, both techniques performed well with 88% (MALDI-TOF) and 75% (FTIR) correct identifications at species level, respectively. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of Candida species.

PubMed

Lacroix, C; Gicquel, A; Sendid, B; Meyer, J; Accoceberry, I; François, N; Morio, F; Desoubeaux, G; Chandenier, J; Kauffmann-Lacroix, C; Hennequin, C; Guitard, J; Nassif, X; Bougnoux, M-E

2014-02-01

Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas™ and Bruker Biotyper™, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas™; 1360/1383 for Bruker Biotyper™) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Early identification of microorganisms in blood culture prior to the detection of a positive signal in the BACTEC FX system using matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

PubMed

Wang, Ming-Cheng; Lin, Wei-Hung; Yan, Jing-Jou; Fang, Hsin-Yi; Kuo, Te-Hui; Tseng, Chin-Chung; Wu, Jiunn-Jong

2015-08-01

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a valuable method for rapid identification of blood stream infection (BSI) pathogens. Integration of MALDI-TOF MS and blood culture system can speed the identification of causative BSI microorganisms. We investigated the minimal microorganism concentrations of common BSI pathogens required for positive blood culture using BACTEC FX and for positive identification using MALDI-TOF MS. The time to detection with positive BACTEC FX and minimal incubation time with positive MALDI-TOF MS identification were determined for earlier identification of common BSI pathogens. The minimal microorganism concentrations required for positive blood culture using BACTEC FX were >10(7)-10(8) colony forming units/mL for most of the BSI pathogens. The minimal microorganism concentrations required for identification using MALDI-TOF MS were > 10(7) colony forming units/mL. Using simulated BSI models, one can obtain enough bacterial concentration from blood culture bottles for successful identification of five common Gram-positive and Gram-negative bacteria using MALDI-TOF MS 1.7-2.3 hours earlier than the usual time to detection in blood culture systems. This study provides an approach to earlier identification of BSI pathogens prior to the detection of a positive signal in the blood culture system using MALDI-TOF MS, compared to current methods. It can speed the time for identification of BSI pathogens and may have benefits of earlier therapy choice and on patient outcome. Copyright © 2013. Published by Elsevier B.V.

Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

EPA Science Inventory

Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

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

PubMed

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.

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

PubMed

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.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology.

PubMed

Clark, Andrew E; Kaleta, Erin J; Arora, Amit; Wolk, Donna M

2013-07-01

Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the "nuts and bolts" of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

PubMed Central

Clark, Andrew E.; Kaleta, Erin J.; Arora, Amit

2013-01-01

SUMMARY Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the “nuts and bolts” of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care. PMID:23824373

Accurate differentiation of Mycobacterium chimaera from Mycobacterium intracellulare by MALDI-TOF MS analysis.

PubMed

Pranada, Arthur B; Witt, Ellen; Bienia, Michael; Kostrzewa, Markus; Timke, Markus

2017-05-01

The increasing number of infections caused by nontuberculous mycobacteria (NTM) has prompted the need for rapid and precise identification methods of these pathogens. Several studies report the applicability of MALDI-TOF mass spectrometry (MS) for identification of NTM. However, some closely related species have very similar spectral mass fingerprints, and until recently, Mycobacterium chimaera and M. intracellulare could not be separated from each other by MALDI-TOF MS. The conventional identification methods used in routine diagnostics have similar limitations. Recently, the differentiation of these two species within the Mycobacterium avium complex has become increasingly important due to reports of M. chimaera infections related to open heart surgery in Europe and in the USA. In this report, a method for the distinct differentiation of M. chimaera and M. intracellulare using a more detailed analysis of MALDI-TOF mass spectra is presented. Species-specific peaks could be identified and it was possible to assign all isolates (100 %) from reference strain collections as well as clinical isolates to the correct species. We have developed a model for the accurate identification of M. chimaera and M. intracellulare by MALDI-TOF MS. This approach has the potential for routine use in microbiology laboratories, as the model itself can be easily implemented into the software of the currently available systems by MALDI-TOF MS manufacturers.

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

PubMed

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 (p<10-4) and strains for a given species (p<10-4) in a multivariate analysis. Addressing the heterogeneity of MALDI-TOF spectra derived from filamentous fungi by increasing the number of RMS obtained from distinct subcultures of strains included in the reference spectra library markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi.

Comprehensive Characterization of Extractable and Nonextractable Phenolic Compounds by High-Performance Liquid Chromatography-Electrospray Ionization-Quadrupole Time-of-Flight of a Grape/Pomegranate Pomace Dietary Supplement.

PubMed

Pérez-Ramírez, Iza F; Reynoso-Camacho, Rosalía; Saura-Calixto, Fulgencio; Pérez-Jiménez, Jara

2018-01-24

Grape and pomegranate are rich sources of phenolic compounds, and their derived products could be used as ingredients for the development of functional foods and dietary supplements. However, the profile of nonextractable or macromolecular phenolic compounds in these samples has not been evaluated. Here, we show a comprehensive characterization of extractable and nonextractable phenolic compounds of a grape/pomegranate pomace dietary supplement using high-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight (HPLC-ESI-QTOF) and matrix-assisted laser desorption/ionization (MALDI)-TOF techniques. The main extractable phenolic compounds were several anthocyanins (principally malvidin 3-O-glucoside) as well as gallotannins and gallagyl derivatives; some phenolic compounds were reported in grape or pomegranate for the first time. Additionally, there was a high proportion of nonextractable phenolic compounds, including vanillic acid, and dihydroxybenzoic acid. Unidentified polymeric structures were detected by MALDI-TOF MS analysis. This study shows that mixed grape and pomegranate pomaces are a source of different classes of phenolic compounds including a high proportion of nonextractable phenolic compounds.

The Ongoing Revolution of MALDI-TOF Mass Spectrometry for Microbiology Reaches Tropical Africa

PubMed Central

Fall, Bécaye; Lo, Cheikh Ibrahima; Samb-Ba, Bissoume; Perrot, Nadine; Diawara, Silman; Gueye, Mamadou Wague; Sow, Kowry; Aubadie-Ladrix, Maxence; Mediannikov, Oleg; Sokhna, Cheikh; Diemé, Yaya; Chatellier, Sonia; Wade, Boubacar; Raoult, Didier; Fenollar, Florence

2015-01-01

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa. PMID:25601995

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

PubMed

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.

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences*

PubMed Central

Hessling, Bernd; Büttner, Knut; Hecker, Michael; Becher, Dörte

2013-01-01

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC–electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments. PMID:23788530

Global relative quantification with liquid chromatography-matrix-assisted laser desorption ionization time-of-flight (LC-MALDI-TOF)--cross-validation with LTQ-Orbitrap proves reliability and reveals complementary ionization preferences.

PubMed

Hessling, Bernd; Büttner, Knut; Hecker, Michael; Becher, Dörte

2013-10-01

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.

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

PubMed Central

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

2016-01-01

Background 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. Material/Methods 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). Results 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. Conclusions 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. PMID:27798637

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

PubMed

Schubert, Sören; Kostrzewa, Markus

2017-01-01

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

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

PubMed

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 direct identification of pathogens from positive BC vials, with a lower cost ($1.5 vs. $ 7) albeit a slightly more laborious extracting process (an extra 15 min) compared with Sepsityper™ kit.

Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics.

PubMed

Sebela, Marek; Rehulka, Pavel; Kábrt, Jaromír; Rehulková, Helena; Ozdian, Tomás; Raus, Martin; Franc, Vojtech; Chmelík, Josef

2009-11-01

An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis. Copyright 2009 John Wiley & Sons, Ltd.

Fifteen novel immunoreactive proteins of Chinese virulent Haemophilus parasuis serotype 5 verified by an immunoproteomic assay.

PubMed

Yu, Yanfei; Wu, Guangyan; Zhai, Zhipeng; Yao, Huochun; Lu, Chengping; Zhang, Wei

2015-01-01

Haemophilus parasuis (H. parasuis) is associated with meningitis, polyserositis, polyarthritis and bacterial pneumonia. At present, its prevention and control is difficult because of the lack of suitable subunit vaccines. Nowadays, high-throughput methods, immunoproteomics, are available to screen for more vaccine candidates. A protein extraction method for H. parasuis and two-dimensional electrophoresis (2-DE) were optimized to provide high-resolution profiles covering pH 3 to 10. Twenty immunoreactive spots were excised from gels after strict comparison between 2-DE Western blot membranes and the relevant gels. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-TOF-MS successfully identified 16 different proteins. Fifteen of them were reported as immunoreactive proteins in H. parasuis for the first time. In addition, recombinant HP5-7 (ABC transporter, periplasmic-binding protein) showed immunoreactivity both with hyperimmune rabbit serum and convalescent swine serum. Four recombinants of the 14 successfully expressed genes showed immunoreactivity with hyperimmune rabbit serum.

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

PubMed

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.

High-throughput identification of the microbial biodiversity of cocoa bean fermentation by MALDI-TOF MS.

PubMed

Miescher Schwenninger, S; Freimüller Leischtfeld, S; Gantenbein-Demarchi, C

2016-11-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful biotyping tool increasingly used for high-throughput identification of clinical microbial isolates, however, in food fermentation research this approach is still not well established. This study examines the microbial biodiversity of cocoa bean fermentation based on the isolation of micro-organisms in cocoa-producing regions, followed by MALDI-TOF MS in Switzerland. A preceding 6-week storage test to mimic lengthy transport of microbial samples from cocoa-producing regions to Switzerland was performed with strains of Lactobacillus plantarum, Acetobacter pasteurianus and Saccharomyces cerevisiae. Weekly MALDI-TOF MS analysis was able to successfully identify microbiota to the species level after storing live cultures on slant agar at mild temperatures (7°C) and/or in 75% aqueous ethanol at differing temperatures (-20, 7 and 30°C). The efficacy of this method was confirmed by on-site recording of the microbial biodiversity in cocoa bean fermentation in Bolivia and Brazil, with a total of 1126 randomly selected isolates. MALDI-TOF MS analyses revealed known dominant cocoa bean fermentation species with Lact. plantarum and Lactobacillus fermentum in the lactic acid bacteria taxon, Hanseniaspora opuntiae and S. cerevisiae in the yeast taxon, and Acet. pasteurianus, Acetobacter fabarum, Acetobacter ghanensis and Acetobacter senegalensis in the acetic acid bacteria taxon. Microbial identification with MALDI-TOF MS has increased the number of samples that can be analysed in a given time, a prerequisite for high-throughput methods. This method is already widely used for the identification of clinical microbial isolates, whereas in food fermentation research, including cocoa bean fermentation, microbiota is mostly identified by time-consuming, biochemical-based phenotyping and molecular approaches. This study presents the use of MALDI-TOF MS for characterizing the microbial biodiversity of cocoa bean fermentation. The feasibility of MALDI-TOF MS identification of cocoa-specific microbiota has been shown with samples collected during on-site studies in two countries of origin, Bolivia and Brazil. © 2016 The Society for Applied Microbiology.

MALDI-TOF MS identification of Anopheles gambiae Giles blood meal crushed on Whatman filter papers.

PubMed

Niare, Sirama; Almeras, Lionel; Tandina, Fatalmoudou; Yssouf, Amina; Bacar, Affane; Toilibou, Ali; Doumbo, Ogobara; Raoult, Didier; Parola, Philippe

2017-01-01

Identification of the source of mosquito blood meals is an important component for disease control and surveillance. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as an effective tool for mosquito blood meal identification, using the abdomens of freshly engorged mosquitoes. In the field, mosquito abdomens are crushed on Whatman filter papers to determine the host feeding patterns by identifying the origin of their blood meals. The aim of this study was to test whether crushing engorged mosquito abdomens on Whatman filter papers was compatible with MALDI-TOF MS for mosquito blood meal identification. Both laboratory reared and field collected mosquitoes were tested. Sixty Anopheles gambiae Giles were experimentally engorged on the blood of six distinct vertebrate hosts (human, sheep, rabbit, dog, chicken and rat). The engorged mosquito abdomens were crushed on Whatman filter papers for MALDI-TOF MS analysis. 150 Whatman filter papers, with mosquitoes engorged on cow and goat blood, were preserved. A total of 77 engorged mosquito abdomens collected in the Comoros Islands and crushed on Whatman filter papers were tested with MALDI-TOF MS. The MS profiles generated from mosquito engorged abdomens crushed on Whatman filter papers exhibited high reproducibility according to the original host blood. The blood meal host was correctly identified from mosquito abdomens crushed on Whatman filter papers by MALDI-TOF MS. The MS spectra obtained after storage were stable regardless of the room temperature and whether or not they were frozen. The MS profiles were reproducible for up to three months. For the Comoros samples, 70/77 quality MS spectra were obtained and matched with human blood spectra. This was confirmed by molecular tools. The results demonstrated that MALDI-TOF MS could identify mosquito blood meals from Whatman filter papers collected in the field during entomological surveys. The application of MALDI-TOF MS has proved to be rapid and successful, making it a new and efficient tool for mosquito-borne disease surveillance.

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

PubMed

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 (<5 ppm) and resolving power (∼75 000 at m/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.

Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC-ESI-MS/MS and LC-MALDI-TOF/TOF.

PubMed

Ujang, Jorim Anak; Kwan, Soon Hong; Ismail, Mohd Nazri; Lim, Boon Huat; Noordin, Rahmah; Othman, Nurulhasanah

2016-01-01

Excretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa. E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC-ESI-MS/MS and LC-MALDI-TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB). A complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC-ESI-MS/MS and 107 proteins by LC-MALDI-TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC-ESI-MS/MS and LC-MALDI-TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts. This study showed that complementary use of LC-ESI-MS/MS and LC-MALDI-TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.

Electrospun polystyrene/oxidized carbon nanotubes film as both sorbent for thin film microextraction and matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

He, Xiao-Mei; Zhu, Gang-Tian; Yin, Jia; Zhao, Qin; Yuan, Bi-Feng; Feng, Yu-Qi

2014-07-18

In the current study, polystyrene/oxidized carbon nanotubes (PS/OCNTs) film was prepared and applied as both an adsorbent of thin film microextraction (TFME) and matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the first time. The uniform size of PS/OCNTs film with OCNTs evenly and firmly immobilized in PS was obtained by electrospinning. And a novel TFME device was developed using the prepared PS/OCNTs film to enrich benzo[a]pyrene (BaP) from water, and also BaP and 1-hydroxypyrene (1-OHP) from urine sample. Then the extracted analytes on the PS/OCNTs film were directly applied to MALDI-MS analysis with PS/OCNTs film as the MALDI matrix. Our results show that PS/OCNTs film is a good TFME adsorbent toward the analytes and an excellent matrix for the sensitive determination of BaP and 1-OHP using MALDI-TOF-MS. The employment of PS/OCNTs as the matrix for MALDI can effectively avoid the large variation of signal intensity normally resulting from heterogeneous distribution of the adsorbed analyte on matrix layer, which therefore significantly improve spot-to-spot reproducibility. The introduction of PS in the film can prevent OCNTs from flying out of MALDI plate to damage the equipment. In addition, PS/OCNTs film also largely extended the duration of ion signal of target analyte compared to OCNTs matrix. The developed method was further successfully used to quantitatively determine BaP in environmental water and 1-OHP in urine samples. The results show that BaP and 1-OHP could be easily detected at concentrations of 50pgmL(-1) and 500pgmL(-1), respectively, indicating the high detection sensitivity of this method. For BaP analysis, the linear range was 0.1-20ngmL(-1) with a correlation coefficient of 0.9970 and the recoveries were in the range of 81.3 to 123.4% with the RSD≤8.5% (n=3); for urinary 1-OHP analysis, the linear range was 0.5-20ngmL(-1) with a correlation coefficient of 0.9937 and the recoveries were in the range of 79.2 to 103.4% with the RSD≤7.6% (n=3). Taken together, the developed method provides a simple, rapid, cost-effective and high-throughput approach for the analysis of BaP in environmental water and endogenous 1-OHP in urine samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry.

PubMed

Wang, Michael Z; Howard, Brandon; Campa, Michael J; Patz, Edward F; Fitzgerald, Michael C

2003-09-01

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.

High-energy and low-energy collision-induced dissociation of protonated flavonoids generated by MALDI and by electrospray ionization

NASA Astrophysics Data System (ADS)

March, Raymond E.; Li, Hongxia; Belgacem, Omar; Papanastasiou, Dimitris

2007-04-01

Product ion mass spectra of a series of nine protonated flavonoids have been observed by electrospray ionization combined with quadrupole/time-of-flight (ESI QTOF), and matrix-assisted laser desorption ionization combined either with quadrupole ion trap (MALDI QIT) tandem mass spectrometry or time-of-flight tandem mass spectrometry (MALDI TOF ReTOF). The compounds examined are 3,6-, 3,2'-, and 3,3'-dihydoxyflavone, apigenin (5,7,4'-trihydroxyflavone), luteolin (5,7,3',4'-tetrahydroxyflavone), apigenin-7-O-glucoside, hesperidin (5,7,3'-trihydroxy-4'-methoxyflavanone), daidzen (7,4'-dihydroxyisoflavone), and rutin (quercitin-3-O-rutinoside) where quercitin is 3,5,7,3',4'-pentahydroxyflavone; sodiated rutin was examined also. The center-of-mass energies in ESI QTOF and MALDI QIT are similar (1-4 eV) and their product ion mass spectra are virtually identical. In the MALDI TOF ReTOF instrument, center-of-mass energies range from 126-309 eV for sodiated rutin to protonated dihydroxyflavones, respectively. Due to the high center-of-mass energies available with the MALDI TOF ReTOF instrument, some useful structural information may be obtained; however, with increasing precursor mass/charge ratio, product ion mass spectra become simplified so as to be of limited structural value. Electronic excitation of the protonated (and sodiated) species examined here offers an explanation for the very simple product ion mass spectra observed particularly for glycosylated flavonoids.

Identification of Cronobacter species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with an optimized analysis method.

PubMed

Wang, Qi; Zhao, Xiao-Juan; Wang, Zi-Wei; Liu, Li; Wei, Yong-Xin; Han, Xiao; Zeng, Jing; Liao, Wan-Jin

2017-08-01

Rapid and precise identification of Cronobacter species is important for foodborne pathogen detection, however, commercial biochemical methods can only identify Cronobacter strains to genus level in most cases. To evaluate the power of mass spectrometry based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) for Cronobacter species identification, 51 Cronobacter strains (eight reference and 43 wild strains) were identified by both MALDI-TOF MS and 16S rRNA gene sequencing. Biotyper RTC provided by Bruker identified all eight reference and 43 wild strains as Cronobacter species, which demonstrated the power of MALDI-TOF MS to identify Cronobacter strains to genus level. However, using the Bruker's database (6903 main spectra products) and Biotyper software, the MALDI-TOF MS analysis could not identify the investigated strains to species level. When MALDI-TOF MS analysis was performed using the combined in-house Cronobacter database and Bruker's database, bin setting, and unweighted pair group method with arithmetic mean (UPGMA) clustering, all the 51 strains were clearly identified into six Cronobacter species and the identification accuracy increased from 60% to 100%. We demonstrated that MALDI-TOF MS was reliable and easy-to-use for Cronobacter species identification and highlighted the importance of establishing a reliable database and improving the current data analysis methods by integrating the bin setting and UPGMA clustering. Copyright © 2017. Published by Elsevier B.V.

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

PubMed Central

2013-01-01

Background 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. Results 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 (p<10-4) and strains for a given species (p<10-4) in a multivariate analysis. Conclusion Addressing the heterogeneity of MALDI-TOF spectra derived from filamentous fungi by increasing the number of RMS obtained from distinct subcultures of strains included in the reference spectra library markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi. PMID:23565856

The potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of biogroups of Cronobacter sakazakii.

PubMed

Karamonová, Ludmila; Junková, Petra; Mihalová, Denisa; Javůrková, Barbora; Fukal, Ladislav; Rauch, Pavel; Blažková, Martina

2013-02-15

The bacterial genus Cronobacter was established quite recently, in 2008. Therefore, its systematic classification is still in progress as well as the risk assessment of Cronobacter strains. The possibility of rapid identification within the biogroup level has an essential epidemiological significance. We examined the potential of mass spectrometry to accomplish this task on species Cronobacter sakazakii comprising eight different biogroups. Members of all Cronobacter sakazakii biogroups were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using intact cells. Analyses were performed on a Biflex IV MALDI-TOF mass spectrometer in the range of 2000 to 20 000 Da in linear mode with an accelerated voltage of 19 kV. Optimal conditions for a proper identification of biogroups, such as suitable cultivation media or growth time of bacteria, were investigated. The biomarker patterns characterizing each of the Cronobacter sakazakii biogroups were obtained. The established identification protocol was applied to ten previously non-identified strains and their biogroups were successfully determined. The presented work is the first report of successful and rapid bacterial biogroup taxonomy classification using MALDI-TOF-MS that could substitute demanding biochemical testing. Copyright © 2012 John Wiley & Sons, Ltd.

Application of porous metal enrichment probe sampling to single cell analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

PubMed

Fu, Qiang; Tang, Jun; Cui, Meng; Xing, Junpeng; Liu, Zhiqiang; Liu, Shuying

2016-01-01

There is an increasing need for analyzing metabolism in a single cell, which is important to understand the nature of cellular heterogeneity, disease, growth and specialization, etc. However, single cell analysis is often challenging for the traces of samples. In the present study, porous metal enrichment probe sampling combined with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has been applied for in situ analysis of live onion epidemic cell. Porous probe, treated by corroding copper wire with HCl, was directly inserted into a single cell to get cell solution. A self-made linear actuator was enough to control the penetration of probe into the target cell accurately. Then samples on the tip of probe were eluted and detected by a commercial MALDI-TOF-MS directly. The formation of porous microstructure on the probe surface increased the adsorptive capacity of cell solution. The sensitivity of porous probe sampling was 6 times higher than uncorroded probes generally. This method provides a sensitive and convenient way for the sampling and detection of single cell solution. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Shiga toxin 2 subtypes of enterohemorrhagic E. coli O157:H- E32511 analyzed by RT-qPCR and top-down proteomics using MALDI-TOF-TOF-MS

USDA-ARS?s Scientific Manuscript database

We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-M...

Development of soft extraction method for structural characterization of boreal forest soil proteins with MALDI-TOF/MS

NASA Astrophysics Data System (ADS)

Kanerva, Sanna; Ketola, Raimo A.; Kitunen, Veikko; Smolander, Aino; Kotiaho, Tapio

2010-05-01

Nitrogen (N) is usually the nutrient restricting productivity in boreal forests. Forest soils contain a great amount of nitrogen, but only a small part of it is in mineral form. Most part of soil N is bound in the structures of different organic compounds such as proteins, peptides, amino acids and more stabilized, refractory compounds. Due to the fact that soil organic N has a very important role in soil nutrient cycling and in plant nutrition, there is a need for more detailed knowledge of its chemistry in soil. Conventional methods to extract and analyze soil organic N are usually very destructive for structures of higher molecular weight organic compounds, such as proteins. The aim of this study was to characterize proteins extracted from boreal forest soil by "soft" extraction methods in order to maintain their molecular structure. The organic layer (F) from birch forest floor containing 78% of organic matter was sieved, freeze dried, pulverized, and extracted with a citrate or phosphate buffer (pH 6 or 8). Sequential extraction with the citrate or phosphate buffer and an SDS buffer (pH 6.8), slightly modified from the method of Chen et al. (2009, Proteomics 9: 4970-4973), was also done. Proteins were purified from the soil extract by extraction with buffered phenol and precipitated with methanol + 0.1M ammonium acetate at -20°C. Characterization of proteins was performed with matrix assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF/MS) and the concentration of total proteins was measured using Bradford's method. Bovine serum albumin (BSA) was used as a positive control in the extractions and as a standard protein in Bradford's method. Our results showed that sequential extraction increased the amount of extracted proteins compared to the extractions without the SDS-buffer; however, it must be noted that the use of SDS-buffer very probably increased denaturization of proteins. Purification of proteins from crude soil extracts by phenol extraction was essential prior to measurement of total proteins; there seemed to be a lot of compounds in crude soil extracts that interfere with the analysis of total proteins, causing overestimation in protein concentration. pH of the buffer solution did not seem to be very crucial for the extractability of soil natural proteins, but at the higher pH, the amount of interfering compounds increased. However, the recovery of BSA added was clearly higher at the higher pH. When the protein precipitates were analyzed with MALDI-TOF/MS, a large curve, most likely formed from wide peaks of several compounds, indicate that most of the compounds in the precipitate were <15 kDa or ~20-50 kDa in molecular weight. It seems that in order to identify individual proteins from mass spectra, a separation of compounds with varying molecular weight is needed before the MALDI-TOF/MS analysis. Due to the fact that a relatively high amount of BSA added was not recovered by the extractions and that the intensity of the signals observed in mass spectra was low, it is questionable whether it is possible to extract soil natural proteins effectively from soils containing a high amount of organic matter without destructing the structures of proteins.

Localization of ginsenosides in Panax ginseng with different age by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry imaging.

PubMed

Bai, Hangrui; Wang, Shujuan; Liu, Jianjun; Gao, Dan; Jiang, Yuyang; Liu, Hongxia; Cai, Zongwei

2016-07-15

The root of Panax ginseng C.A. Mey. (P. ginseng) is one of the most popular traditional Chinese medicines, with ginsenosides as its main bioactive components. Because different ginsenosides have varied pharmacological effects, extraction and separation of ginsenosides are usually required for the investigation of pharmacological effects of different ginsenosides. However, the contents of ginsenosides vary with the ages and tissues of P. ginseng root. In this research, an efficient method to explore the distribution of ginsenosides and differentiate P. ginseng roots with different ages was developed based on matrix assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI). After a simple sample preparation, there were 18 peaks corresponding to 31 ginsenosides with distinct localization in the mass range of m/z 700-1400 identified by MALDI-TOF-MSI and MALDI-TOF-MS/MS. All the three types of ginsenosides were successfully detected and visualized in images, which could be correlated with anatomical features. The P. ginseng at the ages of 2, 4 and 6 could be differentiated finely through the principal component analysis of data collected from the cork based on the ion images but not data from the whole tissue. The experimental result implies that the established method for the direct analysis of metabolites in plant tissues has high potential for the rapid identification of metabolites and analysis of their localizations in medicinal herbs. Furthermore, this technique also provides valuable information for the component-specific extraction and pharmacological research of herbs. Copyright © 2015 Elsevier B.V. All rights reserved.

The use of MALDI-TOF ICMS as an alternative tool for Trichophyton rubrum identification and typing.

PubMed

Pereira, Leonel; Dias, Nicolina; Santos, Cledir; Lima, Nelson

2014-01-01

In this study, the potential of matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) was investigated for the identification of clinical isolates. The isolates were analyzed at the species and strain level. Spectral identification by MALDI-TOF ICMS was performed for all strains, and compared with the results of sequencing of the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA region. PCR fingerprinting analysis using primers M13, (GACA)4, and (AC)10 was performed in order to assess the intra-specific variability of Trichophyton rubrum strains. The identification of strains at species level by MALDI-TOF ICMS was in agreement with the previously performed morphological and biochemical analysis. Sequence data confirmed spectral mass identification at species level. Intra-specific variability was assessed. Within the T. rubrum cluster, strains were distributed into smaller highly related sub-groups with a similarity values above 85%. MALDI-TOF ICMS was shown to be a rapid, low-cost and accurate alternative tool for the identification and strain typing of T. rubrum. Copyright © 2012 Elsevier España, S.L. All rights reserved.

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry for Detection of Antibiotic Resistance Mechanisms: from Research to Routine Diagnosis

PubMed Central

Chudáčková, Eva; Walková, Radka

2013-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied as an identification procedure in clinical microbiology and has been widely used in routine laboratory practice because of its economical and diagnostic benefits. The range of applications of MALDI-TOF MS has been growing constantly, from rapid species identification to labor-intensive proteomic studies of bacterial physiology. The purpose of this review is to summarize the contribution of the studies already performed with MALDI-TOF MS concerning antibiotic resistance and to analyze future perspectives in this field. We believe that current research should continue in four main directions, including the detection of antibiotic modifications by degrading enzymes, the detection of resistance mechanism determinants through proteomic studies of multiresistant bacteria, and the analysis of modifications of target sites, such as ribosomal methylation. The quantification of antibiotics is suggested as a new approach to study influx and efflux in bacterial cells. The results of the presented studies demonstrate that MALDI-TOF MS is a relevant tool for the detection of antibiotic resistance and opens new avenues for both clinical and experimental microbiology. PMID:23297261

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

PubMed

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.

Short communication: Evaluation of MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci.

PubMed

Cameron, M; Perry, J; Middleton, J R; Chaffer, M; Lewis, J; Keefe, G P

2018-01-01

This study evaluated MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci (CNS). A total of 861 CNS isolates were used in the study, covering 21 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 804) and the remainder were identified by sequencing of hsp60 (n = 56) and tuf (n = 1). The genotypic identification was considered the gold standard identification. Using a direct transfer protocol and the existing commercial database, MALDI-TOF mass spectrometry showed a typeability of 96.5% (831/861) and an accuracy of 99.2% (824/831). Using a custom reference spectra expanded database, which included an additional 13 in-house created reference spectra, isolates were identified by MALDI-TOF mass spectrometry with 99.2% (854/861) typeability and 99.4% (849/854) accuracy. Overall, MALDI-TOF mass spectrometry using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

PubMed

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.

Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

PubMed

Hettick, Justin M; Green, Brett J; Buskirk, Amanda D; Kashon, Michael L; Slaven, James E; Janotka, Erika; Blachere, Francoise M; Schmechel, Detlef; Beezhold, Donald H

2008-09-15

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI-TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI-TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI-TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI-TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.

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

PubMed

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 (<2 %) and human errors (<1 %) suggested that the system was sufficiently robust to be implemented in a network. With a median time-to-identification of 5 h and 11 min (78 min, min-max: 154-547), MALDI-TOF MS networking always provided a faster identification result than conventional techniques, except when chromogenic culture media and oxidase tests were used (p < 0.0001). However, the limited clinical benefits of the chromogenic culture media do not support their extra cost. Our financial analysis also suggested that MALDI-TOF 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.

Extracting Both Peptide Sequence and Glycan Structural Information by 157 nm Photodissociation of N-Linked Glycopeptides

PubMed Central

Zhang, Liangyi; Reilly, James P.

2009-01-01

157 nm photodissociation of N-linked glycopeptides was investigated in MALDI tandem time-of-flight (TOF) and linear ion trap mass spectrometers. Singly-charged glycopeptides yielded abundant peptide and glycan fragments. The peptide fragments included a series of x-, y-, v- and w- ions with the glycan remaining intact. These provide information about the peptide sequence and the glycosylation site. In addition to glycosidic fragments, abundant cross-ring glycan fragments that are not observed in low-energy CID were detected. These fragments provide insight into the glycan sequence and linkages. Doubly-charged glycopeptides generated by nanospray in the linear ion trap mass spectrometer also yielded peptide and glycan fragments. However, the former were dominated by low-energy fragments such as b- and y- type ions while glycan was primarily cleaved at glycosidic bonds. PMID:19113943

Performance and cost analysis of matrix-assisted laser desorption ionization-time of flight mass spectrometry for routine identification of yeast.

PubMed

Dhiman, Neelam; Hall, Leslie; Wohlfiel, Sherri L; Buckwalter, Seanne P; Wengenack, Nancy L

2011-04-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was compared to phenotypic testing for yeast identification. MALDI-TOF mass spectrometry yielded 96.3% and 84.5% accurate species level identifications (spectral scores, ≥ 1.8) for 138 common and 103 archived strains of yeast. MALDI-TOF mass spectrometry is accurate, rapid (5.1 min of hands-on time/identification), and cost-effective ($0.50/sample) for yeast identification in the clinical laboratory.

Pushing the Limits of MALDI-TOF Mass Spectrometry: Beyond Fungal Species Identification

PubMed Central

Rizzato, Cosmeri; Lombardi, Lisa; Zoppo, Marina; Lupetti, Antonella; Tavanti, Arianna

2015-01-01

Matrix assisted laser desorption ionization time of flight (MALDI-TOF) is a powerful analytical tool that has revolutionized microbial identification. Routinely used for bacterial identification, MALDI-TOF has recently been applied to both yeast and filamentous fungi, confirming its pivotal role in the rapid and reliable diagnosis of infections. Subspecies-level identification holds an important role in epidemiological investigations aimed at tracing virulent or drug resistant clones. This review focuses on present and future applications of this versatile tool in the clinical mycology laboratory. PMID:29376916

Current status of matrix-assisted laser desorption ionisation-time of flight mass spectrometry in the clinical microbiology laboratory.

PubMed

Kok, Jen; Chen, Sharon C A; Dwyer, Dominic E; Iredell, Jonathan R

2013-01-01

The integration of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) into many clinical microbiology laboratories has revolutionised routine pathogen identification. MALDI-TOF MS complements and has good potential to replace existing phenotypic identification methods. Results are available in a more clinically relevant timeframe, particularly in bacteraemic septic shock. Novel applications include strain typing and the detection of antimicrobial resistance, but these are not widely used. This review discusses the technical aspects, current applications, and limitations of MALDI-TOF MS.

A systematic review of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry compared to routine microbiological methods for the time taken to identify microbial organisms from positive blood cultures.

PubMed

Dixon, P; Davies, P; Hollingworth, W; Stoddart, M; MacGowan, A

2015-05-01

Bloodstream infections are a significant source of mortality and morbidity. Patient outcomes are improved by rapid identification of the causative pathogen and administration of appropriate antimicrobial therapy. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry has recently emerged as an alternative to microbiological identification. It is important to establish whether the costs of MALDI-TOF are justified by more timely identification and appropriate therapy, reduced length of stay and reduced hospital costs. We undertook a systematic review of the literature comparing MALDI-TOF and routine methods for the identification of the aetiological agent in patients with known or suspected bloodstream infection. The primary outcome of the review was the 'time to identify' organisms. Information on related measures such as 'time to appropriate antimicrobial treatment' and downstream hospital cost was also collected where reported. Ten of 775 articles identified met the inclusion criteria. All included studies were observational. MALDI-TOF identification was at least 24 h faster than routine methods in most circumstances. MADLI-TOF was associated with a reduction in downstream hospital costs and length of stay in studies reporting these outcomes. The observational studies reviewed provide evidence of potentially substantial time savings of MALDI-TOF in pathogen identification and instigation of appropriate therapy, which may also reduce hospital stay. Due to the small number of studies, all at relatively high risk of bias, this cannot be considered as definitive evidence of the impact of MALDI-TOF. More and better evidence, including impact on patient health and cost-effectiveness, is required.

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

USDA-ARS?s Scientific Manuscript database

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...

Identification and structural characterization of avian beta-defensin 2 peptides from pheasant and quail

USDA-ARS?s Scientific Manuscript database

Pheasant and quail orthologues of avian ß-defensin 2 (AVBD2) were identified in methanol extracts of heterophil and bone marrow using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). We used comparative pattern profiling before and after reduction/alkyla...

2-DE analysis indicates that Acinetobacter baumannii displays a robust and versatile metabolism

PubMed Central

Soares, Nelson C; Cabral, Maria P; Parreira, José R; Gayoso, Carmen; Barba, Maria J; Bou, Germán

2009-01-01

Background Acinetobacter baumannii is a nosocomial pathogen that has been associated with outbreak infections in hospitals. Despite increasing awareness about this bacterium, its proteome remains poorly characterised, however recently the complete genome of A. baumannii reference strain ATCC 17978 has been sequenced. Here, we have used 2-DE and MALDI-TOF/TOF approach to characterise the proteome of this strain. Results The membrane and cytoplasmatic protein extracts were analysed separately, these analyses revealed the reproducible presence of 239 and 511 membrane and cytoplamatic protein spots, respectively. MALDI-TOF/TOF characterisation identified a total of 192 protein spots (37 membrane and 155 cytoplasmatic) and revealed that the identified membrane proteins were mainly transport-related proteins, whereas the cytoplasmatic proteins were of diverse nature, although mainly related to metabolic processes. Conclusion This work indicates that A. baumannii has a versatile and robust metabolism and also reveal a number of proteins that may play a key role in the mechanism of drug resistance and virulence. The data obtained complements earlier reports of A. baumannii proteome and provides new tools to increase our knowledge on the protein expression profile of this pathogen. PMID:19785748

Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

PubMed Central

2014-01-01

Background Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms. Methods Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry. Results A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Conclusions Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations. PMID:24904238

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

PubMed

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 comparable discriminatory indices of 0.89 and 0.86, respectively. MALDI-TOF MS thus represents a rapid and cost-effective source-tracking technique for L. monocytogenes. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2016-01-01

Background 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. Methodology/Principal Findings 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. Conclusion The present study highlights the application of MALDI-TOF MS for monitoring sand fly fauna captured in the field. The low cost, reliability and rapidity of MALDI-TOF MS analyses opens up new ways in the management of phlebotomine sand fly-borne diseases. PMID:26771833

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

PubMed Central

Khennouchi, Nour Chems el Houda; Loucif, Lotfi; Boutefnouchet, Nafissa; Allag, Hamoudi

2015-01-01

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. PMID:26239991

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

PubMed

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

2016-01-01

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

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

PubMed

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.

Mass spectrometric imaging of metabolites in kidney tissues from rats treated with furosemide.

PubMed

Jung, Jin Woo; Lee, Mi Suk; Choi, Hyo-Jung; Jung, Sunhee; Lee, Yu-Jung; Hwang, Geum-Sook; Kwon, Tae-Hwan

2016-06-01

In the kidney, metabolic processes are different among the cortex (COR), outer medulla (OM), and inner medulla (IM). Using matrix-assisted laser desorption/ionization (MALDI) and imaging mass spectrometry (IMS), we examined the change of metabolites in the COR, OM, and IM of the rat kidney after furosemide treatment compared with vehicle-treated controls. Osmotic minipumps were implanted in male Sprague-Dawley rats to deliver 12 mg·day(-1)·rat(-1) of furosemide. Vehicle-treated (n = 14) and furosemide-treated (furosemide rats, n = 15) rats in metabolic cages received a fixed amount of rat chow (15 g·220 g body wt(-1)·day(-1) for each rat) with free access to water intake for 6 days. At day 6, higher urine output (32 ± 4 vs. 9 ± 1 ml/day) and lower urine osmolality (546 ± 44 vs. 1,677 ± 104 mosmol/kgH2O) were observed in furosemide rats. Extracts of COR, OM, and IM were analyzed by ultraperformance liquid chromatography coupled with quadrupole time-of-flight (TOF) mass spectrometry, where multivariate analysis revealed significant differences between the two groups. Several metabolites, including acetylcarnitine, betaine, carnitine, choline, and glycerophosphorylcholine (GPC), were significantly changed. The changes of metabolites were further identified by MALDI-TOF/TOF and IMS. Their spatial distribution and relative quantitation in the kidneys were analyzed by IMS. Carnitine compounds were increased in COR and IM, whereas carnitine and acetylcarnitine were decreased in OM. Choline compounds were increased in COR and OM but decreased in IM from furosemide rats. Betaine and GPC were decreased in OM and IM. Taken together, MALDI-TOF/TOF and IMS successfully provide the spatial distribution and relative quantitation of metabolites in the kidney. Copyright © 2016 the American Physiological Society.

Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization-time of flight mass spectrometry and cydB real-time quantitative PCR.

PubMed

Pizauro, Lucas J L; de Almeida, Camila C; Soltes, Glenn A; Slavic, Durda; Rossi-Junior, Oswaldo D; de Ávila, Fernando A; Zafalon, Luiz F; MacInnes, Janet I

2017-05-01

Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species. Copyright © 2017 Elsevier B.V. All rights reserved.

The use of Matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis.

PubMed

Karatuna, Onur; Celebi, Bekir; Can, Simge; Akyar, Isin; Kilic, Selcuk

2016-01-15

Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institute of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica due to RD1 subspecies-specific PCR result. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of fungal rhinosinusitis pathogens.

PubMed

Huang, Yanfei; Wang, Jinglin; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Lu, Xinxin

2017-03-01

Filamentous fungi are among the most important pathogens, causing fungal rhinosinusitis (FRS). Current laboratory diagnosis of FRS pathogens mainly relies on phenotypic identification by culture and microscopic examination, which is time consuming and expertise dependent. Although matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has been employed to identify various fungi, its efficacy in the identification of FRS fungi is less clear. A total of 153 FRS isolates obtained from patients were analysed at the Clinical Laboratory at the Beijing Tongren Hospital affiliated to the Capital Medical University, between January 2014 and December 2015. They were identified by traditional phenotypic methods and Bruker MALDI-TOF MS (Bruker, Biotyper version 3.1), respectively. Discrepancies between the two methods were further validated by sequencing. Among the 153 isolates, 151 had correct species identification using MALDI-TOF MS (Bruker, Biot 3.1, score ≥2.0 or 2.3). MALDI-TOF MS enabled identification of some very closely related species that were indistinguishable by conventional phenotypic methods, including 1/10 Aspergillus versicolor, 3/20 Aspergillus flavus, 2/30 Aspergillus fumigatus and 1/20 Aspergillus terreus, which were misidentified by conventional phenotypic methods as Aspergillus nidulans, Aspergillus oryzae, Aspergillus japonicus and Aspergillus nidulans, respectively. In addition, 2/2 Rhizopus oryzae and 1/1 Rhizopus stolonifer that were identified only to the genus level by the phenotypic method were correctly identified by MALDI-TOF MS. MALDI-TOF MS is a rapid and accurate technique, and could replace the conventional phenotypic method for routine identification of FRS fungi in clinical microbiology laboratories.

The use of matrix-assisted laser desorption ionization-time of flight mass spectrometry in the identification of Francisella tularensis

PubMed Central

Karatuna, Onur; Çelebi, Bekir; Can, Simge; Akyar, Işın; Kiliç, Selçuk

2016-01-01

Francisella tularensis is the cause of the zoonotic disease tularemia and is classified among highly pathogenic bacteria (HPB) due to its low infection dose and potential for airborne transmission. In the case of HBP, there is a pressing need for rapid, accurate and reliable identification. Phenotypic identification of Francisella species is inappropriate for clinical microbiology laboratories because it is time-consuming, hazardous and subject to variable interpretation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently evaluated as a useful tool for the rapid identification of a variety of microorganisms. In this study, we evaluated the use of MALDI-TOF MS for the rapid identification of Francisella tularensis and differentiation of its subspecies. Using national collection of Francisella isolates from the National Tularemia Reference Laboratory (Public Health Institution of Turkey, Ankara), a total of 75 clinical isolates were investigated by species and subspecies-specific polymerase chain reaction (PCR) test and MALDI-TOF MS. All isolates were originally identified as F. tularensis subsp. holarctica according to region of difference 1 (RD1) subspecies-specific PCR results. For all isolates MALDI-TOF MS provided results in concordance with subspecies-specific PCR analysis. Although PCR-based methods are effective in identifying Francisella species, they are labor-intensive and take longer periods of time to obtain the results when compared with MALDI-TOF MS. MALDI-TOF MS appeared to be a rapid, reliable and cost-effective identification technique for Francisella spp. Shorter analysis time and low cost make this an appealing new option in microbiology laboratories. PMID:26773181

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

PubMed

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.

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

PubMed Central

Martin, Misty S.; Carlton, Doug D.; Castro, Paula M. L.; Hildenbrand, Zacariah L.; Schug, Kevin A.

2017-01-01

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. PMID:28796186

Critical factors determining the quantification capability of matrix-assisted laser desorption/ionization– time-of-flight mass spectrometry

PubMed Central

Wang, Chia-Chen; Lai, Yin-Hung; Ou, Yu-Meng; Chang, Huan-Tsung; Wang, Yi-Sheng

2016-01-01

Quantitative analysis with mass spectrometry (MS) is important but challenging. Matrix-assisted laser desorption/ionization (MALDI) coupled with time-of-flight (TOF) MS offers superior sensitivity, resolution and speed, but such techniques have numerous disadvantages that hinder quantitative analyses. This review summarizes essential obstacles to analyte quantification with MALDI-TOF MS, including the complex ionization mechanism of MALDI, sensitive characteristics of the applied electric fields and the mass-dependent detection efficiency of ion detectors. General quantitative ionization and desorption interpretations of ion production are described. Important instrument parameters and available methods of MALDI-TOF MS used for quantitative analysis are also reviewed. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644968

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

PubMed Central

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

Whole-cell MALDI-TOF MS: a new tool to assess the multifaceted activation of macrophages.

PubMed

Ouedraogo, Richard; Daumas, Aurélie; Ghigo, Eric; Capo, Christian; Mege, Jean-Louis; Textoris, Julien

2012-10-22

Whole-cell MALDI-TOF MS is routinely used to identify bacterial species in clinical samples. This technique has also proven to allow identification of intact mammalian cells, including macrophages. Here, we wondered whether this approach enabled the assessment human macrophages plasticity. The whole-cell MALDI-TOF spectra of macrophages stimulated with IFN-γ and IL-4, two inducers of M1 and M2 macrophage polarisation, consisted of peaks ranging from 2 to 12 kDa. The spectra of unstimulated and stimulated macrophages were clearly different. The fingerprints induced by the M1 agonists, IFN-γ, TNF, LPS and LPS+IFN-γ, and the M2 agonists, IL-4, TGF-β1 and IL-10, were specific and readily identifiable. Thus, whole-cell MALDI-TOF MS was able to characterise M1 and M2 macrophage subtypes. In addition, the fingerprints induced by extracellular (group B Streptococcus, Staphylococcus aureus) or intracellular (BCG, Orientia tsutsugamushi, Coxiella burnetii) bacteria were bacterium-specific. The whole-cell MALDI-TOF MS fingerprints therefore revealed the multifaceted activation of human macrophages. This approach opened a new avenue of studies to assess the immune response in the clinical setting, by monitoring the various activation patterns of immune cells in pathological conditions. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of phenotypic methods and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for the identification of aero-tolerant Actinomyces spp. isolated from soft-tissue infections.

PubMed

Ng, L S Y; Sim, J H C; Eng, L C; Menon, S; Tan, T Y

2012-08-01

Aero-tolerant Actinomyces spp. are an under-recognised cause of cutaneous infections, in part because identification using conventional phenotypic methods is difficult and may be inaccurate. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a promising new technique for bacterial identification, but with limited data on the identification of aero-tolerant Actinomyces spp. This study evaluated the accuracy of a phenotypic biochemical kit, MALDI-TOF MS and genotypic identification methods for the identification of this problematic group of organisms. Thirty aero-tolerant Actinomyces spp. were isolated from soft-tissue infections over a 2-year period. Species identification was performed by 16 s rRNA sequencing and genotypic results were compared with results obtained by API Coryne and MALDI-TOF MS. There was poor agreement between API Coryne and genotypic identification, with only 33% of isolates correctly identified to the species level. MALDI-TOF MS correctly identified 97% of isolates to the species level, with 33% of identifications achieved with high confidence scores. MALDI-TOF MS is a promising new tool for the identification of aero-tolerant Actinomyces spp., but improvement of the database is required in order to increase the confidence level of identification.

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry improves appropriateness of antibiotic treatment of bacteremia.

PubMed

Vlek, Anne L M; Bonten, Marc J M; Boel, C H Edwin

2012-01-01

Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification of microorganisms directly from positive blood culture broths. Use of the MALDI-TOF MS for rapid identification of microorganisms from blood culture broths can reduce the turnaround time to identification and may lead to earlier appropriate treatment of bacteremia. During February and April 2010, direct MALDI-TOF MS was routinely performed on all positive blood cultures. During December 2009 and March 2010 no direct MALDI-TOF MS was used. Information on antibiotic therapy was collected from the hospital and intensive care units' information systems from all positive blood cultures during the study period. In total, 253 episodes of bacteremia were included of which 89 during the intervention period and 164 during the control period. Direct performance of MALDI-TOF MS on positive blood culture broths reduced the time till species identification by 28.8-h and was associated with an 11.3% increase in the proportion of patients receiving appropriate antibiotic treatment 24 hours after blood culture positivity (64.0% in the control period versus 75.3% in the intervention period (p0.01)). Routine implementation of this technique increased the proportion of patients on adequate antimicrobial treatment within 24 hours.

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

PubMed

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.

In situ analysis of plant tissue underivatized carbohydrates and on-probe enzymatic degraded starch by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry by using carbon nanotubes as matrix.

PubMed

Gholipour, Yousef; Nonami, Hiroshi; Erra-Balsells, Rosa

2008-12-15

Underivatized carbohydrates of tulip bulb and leaf tissues were characterized in situ by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by using carbon nanotubes (CNTs) as matrix. Two sample preparation methods--(i) depositing CNTs on the fresh tissue slices placed on the probe and (ii) locating semitransparent tissues on a dried layer of CNTs on the probe--were examined. Furthermore, practicability of in situ starch analysis by MALDI-TOF MS was examined by detection of glucose originated from on-probe amyloglucosidase-catalyzed degradation of starch on the tissue surface. Besides, CNTs could efficiently desorb/ionize natural mono-, di-, and oligosaccharides extracted from tulip bulb tissues as well as glucose resulting from starch enzymatic degradation in vitro. These results were compared with those obtained by in situ MALDI-TOF MS analysis of similar tissues. Positive ion mode showed superior signal reproducibility. CNTs deposited under semitransparent tissue could also desorb/ionize neutral carbohydrates, leading to nearly complete elimination of matrix cluster signals but with an increase in tissue-originated signals. Furthermore, several experiments were carried out to compare the efficiency of 2,5-dihydroxybenzoic acid, nor-harmane, alpha-cyano-4-hydroxycinnamic acid, and CNTs as matrices for MALDI of neutral carbohydrates from the intact plant tissue surface and for enzymatic tissue starch degradation; these results are discussed in brief. Among matrices studied, the lowest laser power was needed to acquire carbohydrate signals with high signal-to-noise ratio and resolution when CNTs were used.

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

PubMed Central

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

[FEATURES OF MASS-SPECTROMETRIC PROTEIN PROFILES OF STRAINS OF BRUCELLOSIS CAUSATIVE AGENT DURING PREPARATION OF CULTURE ON VARIOUS NUTRIENT MEDIA].

PubMed

Ulshina, D V; Kovalev, D A; Zhirov, A M; Zharinova, N V; Khudoleev, A A; Kogotkova, O I; Efremenko, V I; Evchenko, N I; Kulichenko, A N

2016-01-01

Carry out comparative analysis using time-of-flight mass-spectrometry with matrix laser desorption/ionization (MALDI-TOF MS) of protein profiles of brucellosis causative agents (Brucella melitensis Rev-1 and Brucella abortus 19BA), cultivated in various nutrient media: Albimi agar, brucellagar and erythrit-agar. Vaccine,strains: Brucella melitensis Rev-1 and Brucella abortus 19BA. Protein profiling in linear mode on Microflex "Bruker Daltonics" MALDI-TOF mass-spectrometer. A number of characteristic features of brucella mass-spectra was detected: in particular, preservation of the total qualitative composition of protein profiles of cultures and significant differences in the intensity of separate peaks depending on the nutrient medium used. Based on the analysis of the data obtained, use of Albimi agar as the nutrient medium for preparation of brucella culture samples for mass-spectrometric analysis was shown to be optimal.

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

PubMed Central

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

PubMed

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 value<1.7. Two strains could not be identified at first as they represented a mix of two different species. These mixes were Rhodotorula babjevae with Meyerozyma caribbica and Clavispora lusitaniae with Debaryomyces hansenii. After separation, all four species could be correctly identified with scores>1.7. 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.

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

PubMed

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

2014-02-01

Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique. To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS. An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand. MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA. Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks. © 2013 British Association of Dermatologists.

Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali

PubMed Central

Diarra, Adama Zan; Almeras, Lionel; Berenger, Jean-Michel; Koné, Abdoulaye K.; Bocoum, Zakaria; Dabo, Abdoulaye; Doumbo, Ogobara; Raoult, Didier; Parola, Philippe

2017-01-01

Ticks are considered the second vector of human and animal diseases after mosquitoes. Therefore, identification of ticks and associated pathogens is an important step in the management of these vectors. In recent years, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of arthropods including ticks. The objective of this study was to improve the conditions for the preparation of tick samples for their identification by MALDI-TOF MS from field-collected ethanol-stored Malian samples and to evaluate the capacity of this technology to distinguish infected and uninfected ticks. A total of 1,333 ticks were collected from mammals in three distinct sites from Mali. Morphological identification allowed classification of ticks into 6 species including Amblyomma variegatum, Hyalomma truncatum, Hyalomma marginatum rufipes, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi and Rhipicephalus sanguineus sl. Among those, 471 ticks were randomly selected for molecular and proteomic analyses. Tick legs submitted to MALDI-TOF MS revealed a concordant morpho/molecular identification of 99.6%. The inclusion in our MALDI-TOF MS arthropod database of MS reference spectra from ethanol-preserved tick leg specimens was required to obtain reliable identification. When tested by molecular tools, 76.6%, 37.6%, 20.8% and 1.1% of the specimens tested were positive for Rickettsia spp., Coxiella burnetii, Anaplasmataceae and Borrelia spp., respectively. These results support the fact that MALDI-TOF is a reliable tool for the identification of ticks conserved in alcohol and enhances knowledge about the diversity of tick species and pathogens transmitted by ticks circulating in Mali. PMID:28742123

Molecular and MALDI-TOF identification of ticks and tick-associated bacteria in Mali.

PubMed

Diarra, Adama Zan; Almeras, Lionel; Laroche, Maureen; Berenger, Jean-Michel; Koné, Abdoulaye K; Bocoum, Zakaria; Dabo, Abdoulaye; Doumbo, Ogobara; Raoult, Didier; Parola, Philippe

2017-07-01

Ticks are considered the second vector of human and animal diseases after mosquitoes. Therefore, identification of ticks and associated pathogens is an important step in the management of these vectors. In recent years, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of arthropods including ticks. The objective of this study was to improve the conditions for the preparation of tick samples for their identification by MALDI-TOF MS from field-collected ethanol-stored Malian samples and to evaluate the capacity of this technology to distinguish infected and uninfected ticks. A total of 1,333 ticks were collected from mammals in three distinct sites from Mali. Morphological identification allowed classification of ticks into 6 species including Amblyomma variegatum, Hyalomma truncatum, Hyalomma marginatum rufipes, Rhipicephalus (Boophilus) microplus, Rhipicephalus evertsi evertsi and Rhipicephalus sanguineus sl. Among those, 471 ticks were randomly selected for molecular and proteomic analyses. Tick legs submitted to MALDI-TOF MS revealed a concordant morpho/molecular identification of 99.6%. The inclusion in our MALDI-TOF MS arthropod database of MS reference spectra from ethanol-preserved tick leg specimens was required to obtain reliable identification. When tested by molecular tools, 76.6%, 37.6%, 20.8% and 1.1% of the specimens tested were positive for Rickettsia spp., Coxiella burnetii, Anaplasmataceae and Borrelia spp., respectively. These results support the fact that MALDI-TOF is a reliable tool for the identification of ticks conserved in alcohol and enhances knowledge about the diversity of tick species and pathogens transmitted by ticks circulating in Mali.

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

PubMed

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.

Use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analyser in a diagnostic microbiology laboratory in a developing country.

PubMed

Bulane, Atang; Hoosen, Anwar

2017-01-01

Rapid and accurate identification of pathogens is of utmost importance for management of patients. Current identification relies on conventional phenotypic methods which are time consuming. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) is based on proteomic profiling and allows for rapid identification of pathogens. We compared MALDI-TOF MS against two commercial systems, MicroScan Walkaway and VITEK 2 MS. Over a three-month period from July 2013 to September 2013, a total of 227 bacteria and yeasts were collected from an academic microbiology laboratory ( N = 121; 87 Gram-negatives, seven Gram-positives, 27 yeasts) and other laboratories ( N = 106; 35 Gram-negatives, 34 Gram-positives, 37 yeasts). Sixty-five positive blood cultures were initially processed with Bruker Sepsityper kit for direct identification. From the 65 blood culture bottles, four grew more than one bacterial pathogen and MALDI-TOF MS identified only one isolate. The blood cultures yielded 21 Gram-negatives, 43 Gram-positives and one Candida . There were 21 Escherirchia coli isolates which were reported by the MALDI-TOF MS as E. coli / Shigella . Of the total 292 isolates, discrepant results were found for one bacterial and three yeast isolates. Discrepant results were resolved by testing with the API system with MALDI-TOF MS showing 100% correlation. The MALDI-TOF MS proved to be very useful for rapid and reliable identification of bacteria and yeasts directly from blood cultures and after culture of other specimens. The difference in time to identification was significant for all isolates. However, for positive blood cultures with minimal sample preparation time there was a massive difference in turn-around time with great appreciation by clinicians.

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

PubMed

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.

Performance of two MALDI-TOF MS systems for the identification of yeasts isolated from bloodstream infections and cerebrospinal fluids using a time-saving direct transfer protocol.

PubMed

Hamprecht, Axel; Christ, Sara; Oestreicher, Tanja; Plum, Georg; Kempf, Volkhard A J; Göttig, Stephan

2014-04-01

The rapid and correct identification of pathogens is of paramount importance for the treatment of patients with invasive infections. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can speed up the identification of bacteria and fungi and has quickly been embraced by medical microbiology laboratories worldwide. Different MALDI-TOF systems have been compared in studies focussing on identification rates of different pathogens. Another aspect that has not been systematically assessed is the performance in daily routine and handling, which is important especially for microbiology routine laboratories. We compared two widespread commercial systems, Microflex LT Biotyper (Bruker) and VitekMS (bioMérieux), for the identification of 210 relevant clinical yeasts under routine conditions, using a time-saving direct transfer protocol. We assessed the need for an additional extraction step, the threshold for species identification and the duration of measurements with the two systems. The tested yeasts included 34 Candida albicans isolates, 144 non-albicans Candida spp. and 32 yeasts of different genera. The results of the two MS systems were compared with that of biochemical identification and, in case of discrepancies, DNA sequencing of the internal transcribed spacer or the large subunit of ribosomal DNA. Both systems correctly identified 96.2 % of isolates [202/210, non-significant (n.s.)]. Misidentifications were observed for VitekMS only (n = 5, no major errors, n.s.). VitekMS was the slower system (19.8 vs. 8.0 min for 10 samples, p = 0.002) but had the advantage of a more effective direct transfer protocol with less need for an additional extraction step.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

Interlaboratory Comparison of Sample Preparation Methods, Database Expansions, and Cutoff Values for Identification of Yeasts by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Using a Yeast Test Panel

PubMed Central

Vlek, Anneloes; Kolecka, Anna; Khayhan, Kantarawee; Theelen, Bart; Groenewald, Marizeth; Boel, Edwin

2014-01-01

An interlaboratory study using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts (n = 35) was performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases. Logistic regression analysis was performed to analyze the association between the number of spectra in the database and the percentage of strains that were correctly identified. A total of 5,460 MALDI-TOF MS results were obtained. Using all results, the area under the ROC curve was 0.95 (95% confidence interval [CI], 0.94 to 0.96). With a sensitivity of 0.84 and a specificity of 0.97, a cutoff value of 1.7 was considered optimal. The overall percentage of correct identifications (formic acid-ethanol extraction method, score ≥ 1.7) was 61.5% when the commercial Bruker Daltonics database (BDAL) was used, and it increased to 86.8% by using an extended BDAL supplemented with a Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre in-house database (BDAL+CBS in-house). A greater number of main spectra (MSP) in the database was associated with a higher percentage of correct identifications (odds ratio [OR], 1.10; 95% CI, 1.05 to 1.15; P < 0.01). The results from the direct transfer method ranged from 0% to 82.9% correct identifications, with the results of the top four centers ranging from 71.4% to 82.9% correct identifications. This study supports the use of a cutoff value of 1.7 for the identification of yeasts using MALDI-TOF MS. The inclusion of enough isolates of the same species in the database can enhance the proportion of correctly identified strains. Further optimization of the preparation methods, especially of the direct transfer method, may contribute to improved diagnosis of yeast-related infections. PMID:24920782

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

PubMed

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 permissions, please e-mail: journals.permissions@oup.com.

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

PubMed

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.

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

PubMed

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.

Semi Quantitative MALDI TOF for Antimicrobial Susceptibility Testing in Staphylococcus aureus

DTIC Science & Technology

2017-08-31

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

Fast identification of dermatophytes by MALDI-TOF/MS using direct transfer of fungal cells on ground steel target plates.

PubMed

da Cunha, Keith C; Riat, Arnaud; Normand, Anne-Cecile; Bosshard, Philipp P; de Almeida, Margarete T G; Piarroux, Renaud; Schrenzel, Jacques; Fontao, Lionel

2018-05-15

Dermatophytes cause human infections limited to keratinized tissues. We showed that the direct transfer method allows reliable identification of non-dermatophytes mould and yeast by MALDI-TOF/MS. We aimed at assessing whether the direct transfer method can be used for dermatophytes and whether an own mass spectra library would be superior to the Bruker library. We used the Bruker Biotyper to build a dermatophyte mass spectra library and assessed its performance by 1/ testing a panel of mass spectrum produced with strains genotypically identified and, 2/ comparing MALDI-TOF/MS identification to morphology-based methods. Identification of dermatophytes using the Bruker library is poor. Our library provided 97% concordance between ITS sequencing and MALDI-TOF/MS analysis with a panel of 1104 spectra corresponding to 276 strains. Direct transfer method using unpolished target plates allowed proper identification of 85% of dermatophytes clinical isolates most of which were common dermatophytes. A homemade dermatophyte MSP library is a prerequisite for accurate identification of species absent in the Bruker library but it also improves identification of species already listed in the database. The direct deposit method can be used to identify the most commonly found dermatophytes such as T. rubrum and T. interdigitale/mentagrophytes by MALDI-TOF/MS. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

PubMed

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.

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

PubMed Central

Chang, Susane; Porto Carneiro-Leão, Mariele; Ferreira de Oliveira, Benny; Souza-Motta, Cristina; Lima, Nelson; Santos, Cledir; Tinti de Oliveira, Neiva

2016-01-01

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 (GTG)5 and (GACA)4. 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). PMID:26927172

Application of proteotyping Strain Solution™ ver. 2 software and theoretically calculated mass database in MALDI-TOF MS typing of Salmonella serotype.

PubMed

Ojima-Kato, Teruyo; Yamamoto, Naomi; Nagai, Satomi; Shima, Keisuke; Akiyama, Yumi; Ota, Junji; Tamura, Hiroto

2017-12-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification is a popular analytical method. Strain Solution proteotyping software available for MALDI-TOF MS has great potential for the precise and detailed discrimination of microorganisms at serotype- or strain-level, beyond the conventional mass fingerprinting approaches. Here, we constructed a theoretically calculated mass database of Salmonella enterica subspecies enterica consisting of 12 biomarker proteins: ribosomal proteins S8, L15, L17, L21, L25, and S7, Mn-cofactor-containing superoxide dismutase (SodA), peptidyl-prolyl cis-trans isomerase C (PPIase C), and protein Gns, and uncharacterized proteins YibT, YaiA, and YciF, that can allow serotyping of Salmonella. Strain Solution ver. 2 software with the novel database constructed in this study demonstrated that 109 strains (94%), including the major outbreak-associated serotypes, Enteritidis, Typhimurium, and Infantis, could be correctly identified from others by colony-directed MALDI-TOF MS using 116 strains belonging to 23 kinds of typed and untyped serotypes of S. enterica from culture collections, patients, and foods. We conclude that Strain Solution ver. 2 software integrated with the accurate mass database will be useful for the bacterial proteotyping by MALDI-TOF MS-based microbial classification in the clinical and food safety fields.

MALDI-TOF Mass Spectrometry as a Useful Tool for Identification of Enterococcus spp. from Wild Birds and Differentiation of Closely Related Species.

PubMed

Stępień-Pyśniak, Dagmara; Hauschild, Tomasz; Różański, Paweł; Marek, Agnieszka

2017-06-28

The aim of this study was to explore the accuracy and feasibility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying bacteria from environmental sources, as compared with rpoA gene sequencing, and to evaluate the occurrence of bacteria of the genus Enterococcus in wild birds. In addition, a phyloproteomic analysis of certain Enterococcus species with spectral relationships was performed. The enterococci were isolated from 25 species of wild birds in central Europe (Poland). Proteomic (MALDI-TOF MS) and genomic ( rpoA gene sequencing) methods were used to identify all the isolates. Using MALDI-TOF MS, all 54 (100%) isolates were identified as Enterococcus spp. Among these, 51 (94.4%) isolates were identified to the species level (log(score) > or =2.0), and three isolates (5.6%) were identified at a level of probable genus identification (log(score) 1.88-1.927). Phylogenetic analysis based on rpoA sequences confirmed that all enterococci had been correctly identified. Enterococcus faecalis was the most prevalent enterococcal species (50%) and Enterococcus faecium (33.3%) the second most frequent species, followed by Enterococcus hirae (9.3%), Enterococcus durans (3.7%), and Enterococcus casseliflavus (3.7%). The phyloproteomic analysis of the spectral profiles of the isolates showed that MALDI-TOF MS is able to differentiate among similar species of the genus Enterococcus .

Analysis of protein glycation products by MALDI-TOF/MS.

PubMed

Kislinger, Thomas; Humeny, Andreas; Peich, Carlo C; Becker, Cord-Michael; Pischetsrieder, Monika

2005-06-01

Matrix-assisted laser desorption ionization-mass spectrometry with time-of-flight detection (MALDI-TOF/MS) is a promising tool to analyze advanced glycation end product (AGE)-modified proteins. The combination of soft ionization (MALDI) with time-of-flight mass detection allows analysis of peptides and proteins of a molecular mass up to 300 kDa with minimal sample workup. Because the direct structural analysis of intact AGE proteins is not possible due to the formation of broad and poorly resolved peaks, peptide mapping was introduced into the analysis of AGE proteins by MALDI-TOF/MS, allowing site-specific analysis of defined AGEs. When methylglyoxal-modified lysozyme was subjected to MALDI-TOF/MS peptide mapping, methylimidazolone and argpyrimidine attached to the arginine residue and carboxyethyl (CEL) bound to the lysine were detected on peptide(aa1-7) (KVFGRCE). In contrast, only one methylimidazolone was found on peptide(aa8-35) (LAAAMKRHGLDNYRGYSLGNWVCAAKFE) and peptide(aa120-129) (VQAWIRGCRL), respectively. The analysis of AGE protein, which had been incubated with glucose, revealed the presence of an Amadori product and a carboxymethyl residue (CML) on peptide(aa1-7) and peptide(aa8-35), as well as an imidazolone A on peptide(aa120-129). Furthermore, the early Maillard reaction of lysozyme, which had been glycated by seven different sugars, was monitored by MALDI-TOF/MS peptide mapping. Finally, this approach was successfully applied for site- and product-specific relative quantification of AGEs. For example, kinetics of CML and Amadori product formation on peptide(aa1-7), as well as imidazolone A formation on peptide(aa120-129), were determined.

Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cells.

PubMed

Ha, Moon Kyung; Chung, Kee Yang; Lee, Ju Hee; Bang, Dongsik; Park, Yoon Kee; Lee, Kwang Hoon

2004-09-01

Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3-10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14-15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging.

Effects of solid-medium type on routine identification of bacterial isolates by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Anderson, Neil W; Buchan, Blake W; Riebe, Katherine M; Parsons, Lauren N; Gnacinski, Stacy; Ledeboer, Nathan A

2012-03-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.

Forensic determination of ricin and the alkaloid marker ricinine from castor bean extracts.

PubMed

Darby, S M; Miller, M L; Allen, R O

2001-09-01

Liquid chromatography/mass spectrometry (LC/ MS) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS methods were developed for the presumptive identification of ricin toxin and the alkaloid marker ricinine from crude plant materials. Ricin is an extremely potent poison, which is of forensic interest due to its appearance in terrorism literature and its potential for use as a homicide agent. Difficulties arise in attempting to analyze ricin because it is a large heterogeneous protein with glycosylation. The general protein identification scheme developed uses LC/MS or MALDI-TOF for size classification followed by the use of the same instrumentation for the analysis of the tryptic digest. Fragments of the digest can be searched in an online database for tentative identification of the unknown protein and then followed by comparison to authentic reference materials. LC fractionation or molecular weight cutoff filtration was used for preparation of the intact toxin before analysis. Extracts from two types of castor beans were prepared using a terrorist handbook procedure and determined to contain 1% ricin. Additionally, a forensic sample suspected to contain ricin was analyzed using the presented identification scheme (data not shown). The identification of the alkaloid ricinine by GC/MS and LC/MS was shown to be a complementary technique for the determination of castor bean extracts.

Identification of Molds by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Posteraro, Brunella

2016-01-01

ABSTRACT Although to a lesser extent than diagnostic bacteriology, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has recently revolutionized the diagnostic mycology workflow. With regard to filamentous fungi (or molds), the precise recognition of pathogenic species is important for rapid diagnosis and appropriate treatment, especially for invasive diseases. This review summarizes the current experience with MALDI-TOF MS-based identification of common and uncommon mold species of Aspergillus, Fusarium, Mucorales, dimorphic fungi, and dermatophytes. This experience clearly shows that MALDI-TOF MS holds promise as a fast and accurate identification tool, particularly with common species or typical strains of filamentous fungi. PMID:27807151

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.

PubMed

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.

DIGE Analysis Software and Protein Identification Approaches.

PubMed

Hmmier, Abduladim; Dowling, Paul

2018-01-01

DIGE is a high-resolution two-dimensional gel electrophoresis method, with excellent dynamic range obtained by fluorescent tag labeling of protein samples. Scanned images of DIGE gels show thousands of protein spots, each spot representing a single or a group of protein isoforms. By using commercially available software, each protein spot is defined by an outline, which is digitized and correlated with the quantity of proteins present in each spot. Software packages include DeCyder, SameSpots, and Dymension 3. In addition, proteins of interest can be excised from post-stained gels and identified with conventional mass spectrometry techniques. High-throughput mass spectrometry is performed using sophisticated instrumentation including matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), MALDI-TOF/TOF, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Tandem MS (MALDI-TOF/TOF or LC-MS/MS), analyzes fragmented peptides, resulting in amino acid sequence information, especially useful when protein spots are low abundant or where a mixture of proteins is present.

MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex

USDA-ARS?s Scientific Manuscript database

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a powerful tool for taxonomic resolution of microorganisms. In this proof-of-concept study, we assessed the effectiveness of this technique to track the current gene sequence-based phylogenet...

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

PubMed

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.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?

PubMed Central

Welker, Martin; Pincus, David; Charrier, Jean-Philippe; Girard, Victoria

2017-01-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microbial species in clinical microbiology laboratories. MALDI-TOF-MS has swiftly become the new gold-standard method owing to its key advantages of simplicity and robustness. However, as with all new methods, adoption of the MALDI-TOF MS approach is still not widespread. Optimal sample preparation has not yet been achieved for several applications, and there are continuing discussions on the need for improved database quality and the inclusion of additional microbial species. New applications such as in the field of antimicrobial susceptibility testing have been proposed but not yet translated to the level of ease and reproducibility that one should expect in routine diagnostic systems. Finally, during routine identification testing, unexpected results are regularly obtained, and the best methods for transmitting these results into clinical care are still evolving. We here discuss the success of MALDI-TOF MS in clinical microbiology and highlight fields of application that are still amenable to improvement. PMID:28840984

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?

PubMed

van Belkum, Alex; Welker, Martin; Pincus, David; Charrier, Jean Philippe; Girard, Victoria

2017-11-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microbial species in clinical microbiology laboratories. MALDI-TOF-MS has swiftly become the new gold-standard method owing to its key advantages of simplicity and robustness. However, as with all new methods, adoption of the MALDI-TOF MS approach is still not widespread. Optimal sample preparation has not yet been achieved for several applications, and there are continuing discussions on the need for improved database quality and the inclusion of additional microbial species. New applications such as in the field of antimicrobial susceptibility testing have been proposed but not yet translated to the level of ease and reproducibility that one should expect in routine diagnostic systems. Finally, during routine identification testing, unexpected results are regularly obtained, and the best methods for transmitting these results into clinical care are still evolving. We here discuss the success of MALDI-TOF MS in clinical microbiology and highlight fields of application that are still amenable to improvement. © The Korean Society for Laboratory Medicine.

Gain Switching for a Detection System to Accommodate a Newly Developed MALDI-Based Quantification Method

NASA Astrophysics Data System (ADS)

Ahn, Sung Hee; Hyeon, Taeghwan; Kim, Myung Soo; Moon, Jeong Hee

2017-09-01

In matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), matrix-derived ions are routinely deflected away to avoid problems with ion detection. This, however, limits the use of a quantification method that utilizes the analyte-to-matrix ion abundance ratio. In this work, we will show that it is possible to measure this ratio by a minor instrumental modification of a simple form of MALDI-TOF. This involves detector gain switching. [Figure not available: see fulltext.

Bacillus Classification Based on Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry-Effects of Culture Conditions.

PubMed

Shu, Lin-Jie; Yang, Yu-Liang

2017-11-14

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a reliable and rapid technique applied widely in the identification and classification of microbes. MALDI-TOF MS has been used to identify many endospore-forming Bacillus species; however, endospores affect the identification accuracy when using MALDI-TOF MS because they change the protein composition of samples. Since culture conditions directly influence endospore formation and Bacillus growth, in this study we clarified how culture conditions influence the classification of Bacillus species by using MALDI-TOF MS. We analyzed members of the Bacillus subtilis group and Bacillus cereus group using different incubation periods, temperatures and media. Incubation period was found to affect mass spectra due to endospores which were observed mixing with vegetative cells after 24 hours. Culture temperature also resulted in different mass spectra profiles depending on the temperature best suited growth and sporulation. Conversely, the four common media for Bacillus incubation, Luria-Bertani agar, nutrient agar, plate count agar and brain-heart infusion agar did not result in any significant differences in mass spectra profiles. Profiles in the range m/z 1000-3000 were found to provide additional data to the standard ribosomal peptide/protein region m/z 3000-15000 profiles to enable easier differentiation of some highly similar species and the identification of new strains under fresh culture conditions. In summary, control of culture conditions is vital for Bacillus identification and classification by MALDI-TOF MS.

Utilization of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry for identification of infantile seborrheic dermatitis-causing Malassezia and incidence of culture-based cutaneous Malassezia microbiota of 1-month-old infants.

PubMed

Yamamoto, Mikachi; Umeda, Yoshiko; Yo, Ayaka; Yamaura, Mariko; Makimura, Koichi

2014-02-01

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been utilized for identification of various microorganisms. Malassezia species, including Malassezia restricta, which is associated with seborrheic dermatitis, has been difficult to identify by traditional means. This study was performed to develop a system for identification of Malassezia species with MALDI-TOF-MS and to investigate the incidence and variety of cutaneous Malassezia microbiota of 1-month-old infants using this technique. A Malassezia species-specific MALDI-TOF-MS database was developed from eight standard strains, and the availability of this system was assessed using 54 clinical strains isolated from the skin of 1-month-old infants. Clinical isolates were cultured initially on CHROMagar Malassezia growth medium, and the 28S ribosomal DNA (D1/D2) sequence was analyzed for confirmatory identification. Using this database, we detected and analyzed Malassezia species in 68% and 44% of infants with and without infantile seborrheic dermatitis, respectively. The results of MALDI-TOF-MS analysis were consistent with those of rDNA sequencing identification (100% accuracy rate). To our knowledge, this is the first report of a MALDI-TOF-MS database for major skin pathogenic Malassezia species. This system is an easy, rapid and reliable method for identification of Malassezia. © 2014 Japanese Dermatological Association.

The Exploration of Peptide Biomarkers in Malignant Pleural Effusion of Lung Cancer Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

PubMed Central

Xu, Jing; Xu, Bin; Tang, Chuanhao; Li, Xiaoyan; Qin, Haifeng; Wang, Weixia; Wang, Hong; Wang, Zhongyuan; Li, Liangliang; Li, Zhihua; Gao, Hongjun

2017-01-01

Background. Diagnoses of malignant pleural effusion (MPE) are a crucial problem in clinics. In our study, we compared the peptide profiles of MPE and tuberculosis pleural effusion (TPE) to investigate the value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in diagnosis of MPE. Material and Methods. The 46 MPE and 32 TPE were randomly assigned to training set and validation set. Peptides were isolated by weak cation exchange magnetic beads and peaks in the m/z range of 800–10000 Da were analyzed. Comparing the peptide profile between 30 MPE and 22 TPE samples in training set by ClinProTools software, we screened the specific biomarkers and established a MALDI-TOF-MS classification of MPE. Finally, the other 16 MPE and 10 TPE were included to verify the model. We additionally determined carcinoembryonic antigen (CEA) in MPE and TPE samples using electrochemiluminescent immunoassay method. Results. Five peptide peaks (917.37 Da, 4469.39 Da, 1466.5 Da, 4585.21 Da, and 3216.87 Da) were selected to separate MPE and TPE by MALDI-TOF-MS. The sensitivity, specificity, and accuracy of the classification were 93.75%, 100%, and 96.15%, respectively, after blinded test. The sensitivity of CEA was significantly lower than MALDI-TOF-MS classification (P = 0.035). Conclusions. The results indicate MALDI-TOF-MS is a potential method for diagnosing MPE. PMID:28386154

The Exploration of Peptide Biomarkers in Malignant Pleural Effusion of Lung Cancer Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

PubMed

Xu, Jing; Xu, Bin; Tang, Chuanhao; Li, Xiaoyan; Qin, Haifeng; Wang, Weixia; Wang, Hong; Wang, Zhongyuan; Li, Liangliang; Li, Zhihua; Gao, Hongjun; He, Kun; Liu, Xiaoqing

2017-01-01

Background . Diagnoses of malignant pleural effusion (MPE) are a crucial problem in clinics. In our study, we compared the peptide profiles of MPE and tuberculosis pleural effusion (TPE) to investigate the value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in diagnosis of MPE. Material and Methods . The 46 MPE and 32 TPE were randomly assigned to training set and validation set. Peptides were isolated by weak cation exchange magnetic beads and peaks in the m / z range of 800-10000 Da were analyzed. Comparing the peptide profile between 30 MPE and 22 TPE samples in training set by ClinProTools software, we screened the specific biomarkers and established a MALDI-TOF-MS classification of MPE. Finally, the other 16 MPE and 10 TPE were included to verify the model. We additionally determined carcinoembryonic antigen (CEA) in MPE and TPE samples using electrochemiluminescent immunoassay method. Results . Five peptide peaks (917.37 Da, 4469.39 Da, 1466.5 Da, 4585.21 Da, and 3216.87 Da) were selected to separate MPE and TPE by MALDI-TOF-MS. The sensitivity, specificity, and accuracy of the classification were 93.75%, 100%, and 96.15%, respectively, after blinded test. The sensitivity of CEA was significantly lower than MALDI-TOF-MS classification ( P = 0.035). Conclusions . The results indicate MALDI-TOF-MS is a potential method for diagnosing MPE.

Direct identification of bacteria causing urinary tract infections by combining matrix-assisted laser desorption ionization-time of flight mass spectrometry with UF-1000i urine flow cytometry.

PubMed

Wang, X-H; Zhang, G; Fan, Y-Y; Yang, X; Sui, W-J; Lu, X-X

2013-03-01

Rapid identification of bacterial pathogens from clinical specimens is essential to establish an adequate empirical antibiotic therapy to treat urinary tract infections (UTIs). We used matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) combined with UF-1000i urine flow cytometry of urine specimens to quickly and accurately identify bacteria causing UTIs. We divided each urine sample into three aliquots for conventional identification, UF-1000i, and MALDI-TOF MS, respectively. We compared the results of the conventional method with those of MALDI-TOF MS combined with UF-1000i, and discrepancies were resolved by 16S rRNA gene sequencing. We analyzed 1456 urine samples from patients with UTI symptoms, and 932 (64.0%) were negative using each of the three testing methods. The combined method used UF-1000i to eliminate negative specimens and then MALDI-TOF MS to identify the remaining positive samples. The combined method was consistent with the conventional method in 1373 of 1456 cases (94.3%), and gave the correct result in 1381 of 1456 cases (94.8%). Therefore, the combined method described here can directly provide a rapid, accurate, definitive bacterial identification for the vast majority of urine samples, though the MALDI-TOF MS software analysis capabilities should be improved, with regard to mixed bacterial infection. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

PubMed

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.

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

PubMed Central

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. PMID:29020109

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.

PubMed

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.

MALDI-TOF MS Distinctly Differentiates Nontypable Haemophilus influenzae from Haemophilus haemolyticus

PubMed Central

Zhang, Huifang; Zhang, Yongchan; Gao, Yuan; Xu, Li; Lv, Jing; Wang, Yingtong; Zhang, Jianzhong; Shao, Zhujun

2013-01-01

Nontypable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus exhibit different pathogenicities, but to date, there remains no definitive and reliable strategy for differentiating these strains. In this study, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a potential method for differentiating NTHi and H. haemolyticus. The phylogenetic analysis of concatenated 16S rRNA and recombinase A (recA) gene sequences, outer membrane protein P6 gene sequencing and single-gene PCR were used as reference methods. The original reference database (ORD, provided with the Biotyper software) and new reference database (NRD, extended with Chinese strains) were compared for the evaluation of MALDI-TOF MS. Through a search of the ORD, 76.9% of the NTHi (40/52) and none of the H. haemolyticus (0/20) strains were identified at the species level. However, all NTHi and H. haemolyticus strains used for identification were accurately recognized at the species level when searching the NRD. From the dendrogram clustering of the main spectra projections, the Chinese and foreign H. influenzae reference strains were categorized into two distinct groups, and H. influenzae and H. haemolyticus were also separated into two categories. Compared to the existing methods, MALDI-TOF MS has the advantage of integrating high throughput, accuracy and speed. In conclusion, MALDI-TOF MS is an excellent method for differentiating NTHi and H. haemolyticus. This method can be recommended for use in appropriately equipped laboratories. PMID:23457514

Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

PubMed Central

2010-01-01

Background Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. Results When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. Conclusion These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates. PMID:21073689

Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Aspergillus Species Directly from Growth on Solid Agar Media

PubMed Central

Li, Ying; Wang, He; Zhao, Yu-Pei; Xu, Ying-Chun; Hsueh, Po-Ren

2017-01-01

We evaluated the accuracy of the Bruker Biotyper matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) system at identifying clinical isolates of Aspergillus species that were grown on agar media. A total of 381 non-duplicate Aspergillus isolates representing 21 different Aspergillus species identified by molecular analysis were included in this study. The Bruker Biotyper MALDI-TOF MS system was able to identify 30.2% (115/381) of the isolates to the species level (score values of ≥2.000) and 49.3% to the genus level (score values of 1.700–1.999). When the identification cutoff value was lowered from ≥2.000 to ≥1.700, the species-level identification rate increased to 79.5% with a slight rise of false identification from 2.6 to 5.0%. From another aspect, a correct species-level identification rate of 89% could be reached by the Bruker Biotyper MALDI-TOF MS system regardless of the score values obtained. The Bruker Biotyper MALDI-TOF MS system had a moderate performance in identification of Aspergillus directly inoculated on solid agar media. Continued expansion of the Bruker Biotyper MALDI-TOF MS database and adoption of alternative cutoff values for interpretation are required to improve the performance of the system for identifying highly diverse species of clinically encountered Aspergillus isolates. PMID:28706514

Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Aspergillus Species Directly from Growth on Solid Agar Media.

PubMed

Li, Ying; Wang, He; Zhao, Yu-Pei; Xu, Ying-Chun; Hsueh, Po-Ren

2017-01-01

We evaluated the accuracy of the Bruker Biotyper matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) system at identifying clinical isolates of Aspergillus species that were grown on agar media. A total of 381 non-duplicate Aspergillus isolates representing 21 different Aspergillus species identified by molecular analysis were included in this study. The Bruker Biotyper MALDI-TOF MS system was able to identify 30.2% (115/381) of the isolates to the species level (score values of ≥2.000) and 49.3% to the genus level (score values of 1.700-1.999). When the identification cutoff value was lowered from ≥2.000 to ≥1.700, the species-level identification rate increased to 79.5% with a slight rise of false identification from 2.6 to 5.0%. From another aspect, a correct species-level identification rate of 89% could be reached by the Bruker Biotyper MALDI-TOF MS system regardless of the score values obtained. The Bruker Biotyper MALDI-TOF MS system had a moderate performance in identification of Aspergillus directly inoculated on solid agar media. Continued expansion of the Bruker Biotyper MALDI-TOF MS database and adoption of alternative cutoff values for interpretation are required to improve the performance of the system for identifying highly diverse species of clinically encountered Aspergillus isolates.

Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

PubMed

Ayyadurai, Saravanan; Flaudrops, Christophe; Raoult, Didier; Drancourt, Michel

2010-11-12

Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates.

Evaluation of mass spectrometric data using principal component analysis for determination of the effects of organic lakes on protein binder identification.

PubMed

Hrdlickova Kuckova, Stepanka; Rambouskova, Gabriela; Hynek, Radovan; Cejnar, Pavel; Oltrogge, Doris; Fuchs, Robert

2015-11-01

Matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry is commonly used for the identification of proteinaceous binders and their mixtures in artworks. The determination of protein binders is based on a comparison between the m/z values of tryptic peptides in the unknown sample and a reference one (egg, casein, animal glues etc.), but this method has greater potential to study changes due to ageing and the influence of organic/inorganic components on protein identification. However, it is necessary to then carry out statistical evaluation on the obtained data. Before now, it has been complicated to routinely convert the mass spectrometric data into a statistical programme, to extract and match the appropriate peaks. Only several 'homemade' computer programmes without user-friendly interfaces are available for these purposes. In this paper, we would like to present our completely new, publically available, non-commercial software, ms-alone and multiMS-toolbox, for principal component analyses of MALDI-TOF MS data for R software, and their application to the study of the influence of heterogeneous matrices (organic lakes) for protein identification. Using this new software, we determined the main factors that influence the protein analyses of artificially aged model mixtures of organic lakes and fish glue, prepared according to historical recipes that were used for book illumination, using MALDI-TOF peptide mass mapping. Copyright © 2015 John Wiley & Sons, Ltd.

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

PubMed

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 .

Optimizing identification of clinically relevant Gram-positive organisms by use of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system.

PubMed

McElvania Tekippe, Erin; Shuey, Sunni; Winkler, David W; Butler, Meghan A; Burnham, Carey-Ann D

2013-05-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identification of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical specimens. We evaluated 4 direct-smear methods, including "heavy" (H) and "light" (L) smears, with and without a 1-μl direct formic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or "score." We found that a heavy smear with a formic acid overlay (H+FA) produced optimal MALDI-TOF MS identification scores and the highest percentage of correctly identified organisms. Using a score of ≥2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of ≥1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identification of Gram-positive bacteria by use of MALDI-TOF MS.

Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF mass spectrometry.

PubMed

Angelakis, Emmanouil; Million, Matthieu; Henry, Mireille; Raoult, Didier

2011-10-01

Probiotic food is manufactured by adding probiotic strains simultaneously with starter cultures in fermentation tanks. Here, we investigate the accuracy and feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification at the species level in probiotic food and yoghurts. Probiotic food and yoghurts were cultured in Columbia and Lactobacillus specific agar and tested by quantitative real-time PCR (qPCR) for the detection and quantification of Lactobacillus sp. Bacterial identification was performed by MALDI-TOF analysis and by amplification and sequencing of tuf and 16S rDNA genes. We tested 13 probiotic food and yoghurts and we identified by qPCR that they presented 10(6) to 10(7) copies of Lactobacillus spp. DNA/g. All products contained very large numbers of living bacteria varying from 10(6) to 10(9) colony forming units/g. These bacteria were identified as Lactobacillus casei, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus delbrueckii, and Streptococcus thermophilus. MALDI-TOF MS presented 92% specificity compared to the molecular assays. In one product we found L. lactis, instead of Bifidus spp. which was mentioned on the label and for another L. delbrueckii and S. thermophilus instead of Bifidus spp. MALDI-TOF MS allows a rapid and accurate bacterial identification at the species level in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. Practical Application:  MALDI-TOF MS is rapid and specific for the identification of bacteria in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. © 2011 Institute of Food Technologists®

Rapid identification of oral Actinomyces species cultivated from subgingival biofilm by MALDI-TOF-MS

PubMed Central

Stingu, Catalina S.; Borgmann, Toralf; Rodloff, Arne C.; Vielkind, Paul; Jentsch, Holger; Schellenberger, Wolfgang; Eschrich, Klaus

2015-01-01

Background Actinomyces are a common part of the residential flora of the human intestinal tract, genitourinary system and skin. Isolation and identification of Actinomyces by conventional methods is often difficult and time consuming. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a rapid and simple method to identify bacteria. Objective The present study evaluated a new in-house algorithm using MALDI-TOF-MS for rapid identification of different species of oral Actinomyces cultivated from subgingival biofilm. Design Eleven reference strains and 674 clinical strains were used in this study. All the strains were preliminarily identified using biochemical methods and then subjected to MALDI-TOF-MS analysis using both similarity-based analysis and classification methods (support vector machine [SVM]). The genotype of the reference strains and of 232 clinical strains was identified by sequence analysis of the 16S ribosomal RNA (rRNA). Results The sequence analysis of the 16S rRNA gene of all references strains confirmed their previous identification. The MALDI-TOF-MS spectra obtained from the reference strains and the other clinical strains undoubtedly identified as Actinomyces by 16S rRNA sequencing were used to create the mass spectra reference database. Already a visual inspection of the mass spectra of different species reveals both similarities and differences. However, the differences between them are not large enough to allow a reliable differentiation by similarity analysis. Therefore, classification methods were applied as an alternative approach for differentiation and identification of Actinomyces at the species level. A cross-validation of the reference database representing 14 Actinomyces species yielded correct results for all species which were represented by more than two strains in the database. Conclusions Our results suggest that a combination of MALDI-TOF-MS with powerful classification algorithms, such as SVMs, provide a useful tool for the differentiation and identification of oral Actinomyces. PMID:25597306

Cost Savings Realized by Implementation of Routine Microbiological Identification by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Alby, Kevin; Kerr, Alan; Jones, Melissa; Gilligan, Peter H.

2015-01-01

Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is an emerging technology for rapid identification of bacterial and fungal isolates. In comparison to conventional methods, this technology is much less labor intensive and can provide accurate and reliable results in minutes from a single isolated colony. We compared the cost of performing the bioMérieux Vitek MALDI-TOF MS with conventional microbiological methods to determine the amount saved by the laboratory by converting to the new technology. Identification costs for 21,930 isolates collected between April 1, 2013, and March 31, 2014, were directly compared for MALDI-TOF MS and conventional methodologies. These isolates were composed of commonly isolated organisms, including commonly encountered aerobic and facultative bacteria and yeast but excluding anaerobes and filamentous fungi. Mycobacterium tuberculosis complex and rapidly growing mycobacteria were also evaluated for a 5-month period during the study. Reagent costs and a total cost analysis that included technologist time in addition to reagent expenses and maintenance service agreement costs were analyzed as part of this study. The use of MALDI-TOF MS equated to a net savings of $69,108.61, or 87.8%, in reagent costs annually compared to traditional methods. When total costs are calculated to include technologist time and maintenance costs, traditional identification would have cost $142,532.69, versus $68,886.51 with the MALDI-TOF MS method, resulting in a laboratory savings of $73,646.18, or 51.7%, annually by adopting the new technology. The initial cost of the instrument at our usage level would be offset in about 3 years. MALDI-TOF MS not only represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates, it also provides a significant cost savings for the laboratory. PMID:25994167

Optimizing Identification of Clinically Relevant Gram-Positive Organisms by Use of the Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System

PubMed Central

McElvania TeKippe, Erin; Shuey, Sunni; Winkler, David W.; Butler, Meghan A.

2013-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be used as a method for the rapid identification of microorganisms. This study evaluated the Bruker Biotyper (MALDI-TOF MS) system for the identification of clinically relevant Gram-positive organisms. We tested 239 aerobic Gram-positive organisms isolated from clinical specimens. We evaluated 4 direct-smear methods, including “heavy” (H) and “light” (L) smears, with and without a 1-μl direct formic acid (FA) overlay. The quality measure assigned to a MALDI-TOF MS identification is a numerical value or “score.” We found that a heavy smear with a formic acid overlay (H+FA) produced optimal MALDI-TOF MS identification scores and the highest percentage of correctly identified organisms. Using a score of ≥2.0, we identified 183 of the 239 isolates (76.6%) to the genus level, and of the 181 isolates resolved to the species level, 141 isolates (77.9%) were correctly identified. To maximize the number of correct identifications while minimizing misidentifications, the data were analyzed using a score of ≥1.7 for genus- and species-level identification. Using this score, 220 of the 239 isolates (92.1%) were identified to the genus level, and of the 181 isolates resolved to the species level, 167 isolates (92.2%) could be assigned an accurate species identification. We also evaluated a subset of isolates for preanalytic factors that might influence MALDI-TOF MS identification. Frequent subcultures increased the number of unidentified isolates. Incubation temperatures and subcultures of the media did not alter the rate of identification. These data define the ideal bacterial preparation, identification score, and medium conditions for optimal identification of Gram-positive bacteria by use of MALDI-TOF MS. PMID:23426925

Use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry for bacterial monitoring in routine analysis at a drinking water treatment plant.

PubMed

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

2016-10-01

The study of bacterial communities throughout a drinking water treatment plant could provide a basic understanding of the effects of water processing that could then be used to improve the management of such plants. However, it is necessary to develop new analytical techniques that are sufficiently efficient, robust and fast for their effective and useful application in routine analysis. The aim of this study is therefore to assess the performance of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), as compared to the PhenePlate™ system, for routine analysis in a drinking water treatment plant. To this end we studied a total of 277 colonies isolated in different seasons and from different points throughout the water treatment process, including: raw water, sand filtration, ultrafiltration, reverse osmosis and chlorination. The colonies were analysed using MALDI-TOF MS by direct deposition of the cells on the plate. The colonies were also biochemically fingerprinted using the PhenePlate™ system, clustered according to their similarity and a representative strain was selected for 16S rRNA gene sequencing and API ® gallery-based identification. The use of MALDI-TOF MS was reliable compared to the PhenePlate™ system and has the advantage of being faster and relatively cheap. Bacteria typing by MALDI-TOF MS is therefore a promising method to replace conventional routine phenotypic methods for the identification of bacteria in drinking water laboratories, thanks to its robustness. The major limiting factor for MALDI-TOF MS is the lack of a suitable mass spectra database; although each laboratory can develop its own library. This methodology will provide a tracking tool for companies to use in risk management and the detection of possible failures in both the water treatment processes and the distribution network, as well as offering characterization of the intrinsic microbial populations. Copyright © 2016 Elsevier GmbH. All rights reserved.

Direct Detection and Identification of Bacterial Pathogens from Urine with Optimized Specimen Processing and Enhanced Testing Algorithm

PubMed Central

Huang, Bin; Zhang, Lei; Zhang, Weizheng; Liao, Kang; Zhang, Shihong; Zhang, Zhiquan; Ma, Xingyan; Chen, Jialong; Zhang, Xiuhong; Qu, Pinghua; Wu, Shangwei

2017-01-01

ABSTRACT Rapid and accurate detection and identification of microbial pathogens causing urinary tract infections allow prompt and specific treatment. We optimized specimen processing to maximize the limit of detection (LOD) by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and evaluated the capacity of combination of MALDI-TOF MS and urine analysis (UA) for direct detection and identification of bacterial pathogens from urine samples. The optimal volumes of processed urine, formic acid/acetonitrile, and supernatant spotted onto the target plate were 15 ml, 3 μl, and 3 μl, respectively, yielding a LOD of 1.0 × 105 CFU/ml. Among a total of 1,167 urine specimens collected from three hospital centers, 612 (52.4%) and 351 (30.1%) were, respectively, positive by UA and urine culture. Compared with a reference method comprised of urine culture and 16S rRNA gene sequencing, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MALDI-TOF MS alone and MALDI-TOF MS coupled with UA were 86.6% versus 93.4% (χ2 = 8.93; P < 0.01), 91.5% versus 96.3% (χ2 = 7.06; P < 0.01), 81.5% versus 96.4% (χ2 = 37.32; P < 0.01), and 94.1% versus 93.1% (χ2 = 0.40; P > 0.05), respectively. No significant performance differences were revealed among the three sites, while specificity and NPV of MALDI-TOF MS for males were significantly higher than those for females (specificity, 94.3% versus 77.3%, χ2 = 44.90, P < 0.01; NPV, 95.5% versus 86.1%, χ2 = 18.85, P < 0.01). Our results indicated that the optimization of specimen processing significantly enhanced analytical sensitivity and that the combination of UA and MALDI-TOF MS provided an accurate and rapid detection and identification of bacterial pathogens directly from urine. PMID:28249997

Direct Detection and Identification of Bacterial Pathogens from Urine with Optimized Specimen Processing and Enhanced Testing Algorithm.

PubMed

Huang, Bin; Zhang, Lei; Zhang, Weizheng; Liao, Kang; Zhang, Shihong; Zhang, Zhiquan; Ma, Xingyan; Chen, Jialong; Zhang, Xiuhong; Qu, Pinghua; Wu, Shangwei; Chen, Cha; Tang, Yi-Wei

2017-05-01

Rapid and accurate detection and identification of microbial pathogens causing urinary tract infections allow prompt and specific treatment. We optimized specimen processing to maximize the limit of detection (LOD) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and evaluated the capacity of combination of MALDI-TOF MS and urine analysis (UA) for direct detection and identification of bacterial pathogens from urine samples. The optimal volumes of processed urine, formic acid/acetonitrile, and supernatant spotted onto the target plate were 15 ml, 3 μl, and 3 μl, respectively, yielding a LOD of 1.0 × 10 5 CFU/ml. Among a total of 1,167 urine specimens collected from three hospital centers, 612 (52.4%) and 351 (30.1%) were, respectively, positive by UA and urine culture. Compared with a reference method comprised of urine culture and 16S rRNA gene sequencing, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MALDI-TOF MS alone and MALDI-TOF MS coupled with UA were 86.6% versus 93.4% (χ 2 = 8.93; P < 0.01), 91.5% versus 96.3% (χ 2 = 7.06; P < 0.01), 81.5% versus 96.4% (χ 2 = 37.32; P < 0.01), and 94.1% versus 93.1% (χ 2 = 0.40; P > 0.05), respectively. No significant performance differences were revealed among the three sites, while specificity and NPV of MALDI-TOF MS for males were significantly higher than those for females (specificity, 94.3% versus 77.3%, χ 2 = 44.90, P < 0.01; NPV, 95.5% versus 86.1%, χ 2 = 18.85, P < 0.01). Our results indicated that the optimization of specimen processing significantly enhanced analytical sensitivity and that the combination of UA and MALDI-TOF MS provided an accurate and rapid detection and identification of bacterial pathogens directly from urine. Copyright © 2017 American Society for Microbiology.

Cost Savings Realized by Implementation of Routine Microbiological Identification by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

PubMed

Tran, Anthony; Alby, Kevin; Kerr, Alan; Jones, Melissa; Gilligan, Peter H

2015-08-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is an emerging technology for rapid identification of bacterial and fungal isolates. In comparison to conventional methods, this technology is much less labor intensive and can provide accurate and reliable results in minutes from a single isolated colony. We compared the cost of performing the bioMérieux Vitek MALDI-TOF MS with conventional microbiological methods to determine the amount saved by the laboratory by converting to the new technology. Identification costs for 21,930 isolates collected between April 1, 2013, and March 31, 2014, were directly compared for MALDI-TOF MS and conventional methodologies. These isolates were composed of commonly isolated organisms, including commonly encountered aerobic and facultative bacteria and yeast but excluding anaerobes and filamentous fungi. Mycobacterium tuberculosis complex and rapidly growing mycobacteria were also evaluated for a 5-month period during the study. Reagent costs and a total cost analysis that included technologist time in addition to reagent expenses and maintenance service agreement costs were analyzed as part of this study. The use of MALDI-TOF MS equated to a net savings of $69,108.61, or 87.8%, in reagent costs annually compared to traditional methods. When total costs are calculated to include technologist time and maintenance costs, traditional identification would have cost $142,532.69, versus $68,886.51 with the MALDI-TOF MS method, resulting in a laboratory savings of $73,646.18, or 51.7%, annually by adopting the new technology. The initial cost of the instrument at our usage level would be offset in about 3 years. MALDI-TOF MS not only represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates, it also provides a significant cost savings for the laboratory. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

De Carolis, E; Posteraro, B; Lass-Flörl, C; Vella, A; Florio, A R; Torelli, R; Girmenia, C; Colozza, C; Tortorano, A M; Sanguinetti, M; Fadda, G

2012-05-01

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Improved bacterial identification directly from urine samples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Kitagawa, Koichi; Shigemura, Katsumi; Onuma, Ken-Ichiro; Nishida, Masako; Fujiwara, Mayu; Kobayashi, Saori; Yamasaki, Mika; Nakamura, Tatsuya; Yamamichi, Fukashi; Shirakawa, Toshiro; Tokimatsu, Issei; Fujisawa, Masato

2018-03-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) contributes to rapid identification of pathogens in the clinic but has not yet performed especially well for Gram-positive cocci (GPC) causing complicated urinary tract infection (UTI). The goal of this study was to investigate the possible clinical use of MALDI-TOF MS as a rapid method for bacterial identification directly from urine in complicated UTI. MALDI-TOF MS was applied to urine samples gathered from 142 suspected complicated UTI patients in 2015-2017. We modified the standard procedure (Method 1) for sample preparation by adding an initial 10 minutes of ultrasonication followed by centrifugation at 500 g for 1 minutes to remove debris such as epithelial cells and leukocytes from the urine (Method 2). In 133 urine culture-positive bacteria, the rate of corresponded with urine culture in GPC by MALDI-TOF MS in urine with standard sample preparation (Method 1) was 16.7%, but the modified sample preparation (Method 2) significantly improved that rate to 52.2% (P=.045). Method 2 also improved the identification accuracy for Gram-negative rods (GNR) from 77.1% to 94.2% (P=.022). The modified Method 2 significantly improved the average MALDI score from 1.408±0.153 to 2.166±0.045 (P=.000) for GPC and slightly improved the score from 2.107±0.061 to 2.164±0.037 for GNR. The modified sample preparation for MALDI-TOF MS can improve identification accuracy for complicated UTI causative bacteria. This simple modification offers a rapid and accurate routine diagnosis for UTI, and may possibly be a substitute for urine cultures. © 2017 Wiley Periodicals, Inc.

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

PubMed

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 proteic profile and not on the basis of single markers, by using a "new range setting" different from that developed for bacteria and fungi.

Detection of Biomarkers of Pathogenic Naegleria fowleri Through Mass Spectrometry and Proteomics

PubMed Central

Moura, Hercules; Izquierdo, Fernando; Woolfitt, Adrian R.; Wagner, Glauber; Pinto, Tatiana; del Aguila, Carmen; Barr, John R.

2017-01-01

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry MALDI-TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF-TOF instrument. MALDI-TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI-TOF MS fingerprinting is a rapid, reproducible, high-throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents. PMID:25231600

A simple protocol for Matrix Assisted Laser Desorption Ionization- time of flight-mass spectrometry (MALDI-TOF-MS) analysis of lipids and proteins in single microsamples of paintings.

PubMed

van der Werf, Inez D; Calvano, Cosima D; Palmisano, Francesco; Sabbatini, Luigia

2012-03-09

A simple protocol, based on Bligh-Dyer (BD) extraction followed by MALDI-TOF-MS analysis, for fast identification of paint binders in single microsamples is proposed. For the first time it is demonstrated that the BD method is effective for the simultaneous extraction of lipids and proteins from complex, and atypical matrices, such as pigmented paint layers. The protocol makes use of an alternative denaturing anionic detergent (RapiGest™) in order to improve efficiency of protein digestion and purification step. Detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products was accomplished, whereas proteins could be identified by peptide mass fingerprinting. The effect of pigments on ageing of lipids and proteins was also investigated. Finally, the proposed protocol was successfully applied to the study of a late-15th century Italian panel painting allowing the identification of various proteinaceous and lipid sections in organic binders, such as egg yolk, egg white, animal glue, casein, and drying oil. Copyright © 2011 Elsevier B.V. All rights reserved.

[MALDI-TOF mass spectrometry in the investigation of large high-molecular biological compounds].

PubMed

Porubl'ova, L V; Rebriiev, A V; Hromovyĭ, T Iu; Minia, I I; Obolens'ka, M Iu

2009-01-01

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry has become, in the recent years, a tool of choice for analyses of biological polymers. The wide mass range, high accuracy, informativity and sensitivity make it a superior method for analysis of all kinds of high-molecular biological compounds including proteins, nucleic acids and lipids. MALDI-TOF-MS is particularly suitable for the identification of proteins by mass fingerprint or microsequencing. Therefore it has become an important technique of proteomics. Furthermore, the method allows making a detailed analysis of post-translational protein modifications, protein-protein and protein-nucleic acid interactions. Recently, the method was also successfully applied to nucleic acid sequencing as well as screening for mutations.

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

PubMed

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.

Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.

PubMed

Park, Kyung-Eui; Kim, Jun-Dal; Nagashima, Yusuke; Kako, Koichiro; Daitoku, Hiroaki; Matsui, Motoki; Park, Gwi Gun; Fukamizu, Akiyoshi

2014-01-01

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.

The effects of frozen tissue storage conditions on the integrity of RNA and protein.

PubMed

Auer, H; Mobley, J A; Ayers, L W; Bowen, J; Chuaqui, R F; Johnson, L A; Livolsi, V A; Lubensky, I A; McGarvey, D; Monovich, L C; Moskaluk, C A; Rumpel, C A; Sexton, K C; Washington, M K; Wiles, K R; Grizzle, W E; Ramirez, N C

2014-10-01

Unfixed tissue specimens most frequently are stored for long term research uses at either -80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at -80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA integrity and mRNA integrity. Protein stability was analyzed by surface-enhanced or matrix-assisted laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS, MALDI-TOF-MS) and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RNA yield and total RNA integrity showed significantly better results for -80° C storage compared to VPLN storage; the transcripts that were preferentially degraded during VPLN storage were these involved in antigen presentation and processing. No consistent differences were found in the SELDI-TOF-MS, MALDI-TOF-MS or nLC-ESI-MS/MS analyses of specimens stored for more than 8 years at -80° C compared to those stored in VPLN. Long term storage of human research tissues at -80° C provides at least the same quality of RNA and protein as storage in VPLN.

Monitoring Peptidase Activities in Complex Proteomes by MALDI-TOF Mass Spectrometry

PubMed Central

Villanueva, Josep; Nazarian, Arpi; Lawlor, Kevin; Tempst, Paul

2009-01-01

Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semi-quantitative in vitro assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide-substrates using robotic extraction and a MALDI-TOF mass spectrometric read-out. Relative quantitation of the peptide metabolites is done by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be employed for diagnostic or predictive purposes and enables profiling of 96 samples in 30 hours. It could be tailored to many diagnostic and pharmaco-dynamic purposes, as a read-out of catalytic and metabolic activities in body fluids or tissues. PMID:19617888

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing.

PubMed

Karger, Axel; Stock, Rüdiger; Ziller, Mario; Elschner, Mandy C; Bettin, Barbara; Melzer, Falk; Maier, Thomas; Kostrzewa, Markus; Scholz, Holger C; Neubauer, Heinrich; Tomaso, Herbert

2012-10-10

Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

PubMed Central

2012-01-01

Background Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. Results A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. Conclusions Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed. PMID:23046611

MALDI-TOF mass spectrometry-based identification of group A Streptococcus isolated from areas of the 2011 scarlet fever outbreak in china.

PubMed

Xiao, Di; You, Yuanhai; Bi, Zhenwang; Wang, Haibin; Zhang, Yongchan; Hu, Bin; Song, Yanyan; Zhang, Huifang; Kou, Zengqiang; Yan, Xiaomei; Zhang, Menghan; Jin, Lianmei; Jiang, Xihong; Su, Peng; Bi, Zhenqiang; Luo, Fengji; Zhang, Jianzhong

2013-03-01

There was a dramatic increase in scarlet fever cases in China from March to July 2011. Group A Streptococcus (GAS) is the only pathogen known to cause scarlet fever. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled to Biotyper system was used for GAS identification in 2011. A local reference database (LRD) was constructed, evaluated and used to identify GAS isolates. The 75 GAS strains used to evaluate the LRD were all identified correctly. Of the 157 suspected β-hemolytic strains isolated from 298 throat swab samples, 127 (100%) and 120 (94.5%) of the isolates were identified as GAS by the MALDI-TOF MS system and the conventional bacitracin sensitivity test method, respectively. All 202 (100%) isolates were identified at the species level by searching the LRD, while 182 (90.1%) were identified by searching the original reference database (ORD). There were statistically significant differences with a high degree of credibility at species level (χ(2)=6.052, P<0.05 between the LRD and ORD). The test turnaround time was shortened 36-48h, and the cost of each sample is one-tenth of the cost of conventional methods. Establishing a domestic database is the most effective way to improve the identification efficiency using a MALDI-TOF MS system. MALDI-TOF MS is a viable alternative to conventional methods and may aid in the diagnosis and surveillance of GAS. Copyright © 2013 Elsevier B.V. All rights reserved.

Direct identification of pathogens from positive blood cultures using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

PubMed

Rodríguez-Sánchez, B; Sánchez-Carrillo, C; Ruiz, A; Marín, M; Cercenado, E; Rodríguez-Créixems, M; Bouza, E

2014-07-01

In recent years, matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has proved a rapid and reliable method for the identification of bacteria and yeasts that have already been isolated. The objective of this study was to evaluate this technology as a routine method for the identification of microorganisms directly from blood culture bottles (BCBs), before isolation, in a large collection of samples. For this purpose, 1000 positive BCBs containing 1085 microorganisms have been analysed by conventional phenotypic methods and by MALDI-TOF MS. Discrepancies have been resolved using molecular methods: the amplification and sequencing of the 16S rRNA gene or the Superoxide Dismutase gene (sodA) for streptococcal isolates. MALDI-TOF predicted a species- or genus-level identification of 81.4% of the analysed microorganisms. The analysis by episode yielded a complete identification of 814 out of 1000 analysed episodes (81.4%). MALDI-TOF identification is available for clinicians within hours of a working shift, as oppose to 18 h later when conventional identification methods are performed. Moreover, although further improvement of sample preparation for polymicrobial BCBs is required, the identification of more than one pathogen in the same BCB provides a valuable indication of unexpected pathogens when their presence may remain undetected in Gram staining. Implementation of MALDI-TOF identification directly from the BCB provides a rapid and reliable identification of the causal pathogen within hours. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

The use of Gram stain and matrix-assisted laser desorption ionization time-of-flight mass spectrometry on positive blood culture: synergy between new and old technology.

PubMed

Fuglsang-Damgaard, David; Nielsen, Camilla Houlberg; Mandrup, Elisabeth; Fuursted, Kurt

2011-10-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is promising as an alternative to more costly and cumbersome methods for direct identifications in blood cultures. We wanted to evaluate a simplified pre-treatment method for using MALDI-TOF-MS directly on positive blood cultures using BacT/Alert blood culture system, and to test an algorithm combining the result of the initial microscopy with the result suggested by MALDI-TOF-MS. Using the recommended cut-off score of 1.7 the best results were obtained among Gram-negative rods with correct identifications in 91% of Enterobacteriaceae, 83% in aerobic/non-fermentative Gram-negative rods, whereas results were more modest among Gram-positive cocci with correct identifications in 52% of Staphylococci, 54% in Enterococci and only 20% in Streptococci. Combining the results of Gram stain with the top reports by MALDI-TOF-MS, increased the sensitivity from 91% to 93% in the score range from 1.5 to 1.7 and from 48% to 85% in the score range from 1.3 to 1.5. Thus, using this strategy and accepting a cut-off at 1.3 instead of the suggested 1.7, overall sensitivity could be increased from 88.1% to 96.3%. MALDI-TOF-MS is an efficient method for direct routine identification of bacterial isolates in blood culture, especially when combined with the result of the Gram stain. © 2011 The Authors. APMIS © 2011 APMIS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species

PubMed Central

Suttisunhakul, Vichaya; Pumpuang, Apinya; Ekchariyawat, Peeraya; Wuthiekanun, Vanaporn; Elrod, Mindy G.; Turner, Paul; Currie, Bart J.; Phetsouvanh, Rattanaphone; Dance, David A. B.; Limmathurotsakul, Direk; Peacock, Sharon J.

2017-01-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species. PMID:28384252

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

PubMed

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

2018-06-01

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

Identification of Weissella species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Lee, Meng-Rui; Tsai, Chia-Jung; Teng, Shih-Hua; Hsueh, Po-Ren

2015-01-01

Although some Weissella species play beneficial roles in food fermentation and in probiotic products, others such as Weissella confusa are emerging Gram-positive pathogens in immunocompromised hosts. Weissella species are difficult to identify by conventional biochemical methods and commercial automated systems and are easily misidentified as Lactobacillus and Leuconostoc species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly being used for bacterial identification. Little, however, is known about the effectiveness of MALDI-TOF MS in identifying clinical isolates of Weissella to the species level. In this study, we evaluated whether the MALDI-TOF MS Bruker Biotyper system could accurately identify a total of 20 W. confusa and 2 W. cibaria blood isolates that had been confirmed by 16s rRNA sequencing analysis. The MALDI-TOF Biotyper system yielded no reliable identification results based on the current reference spectra for the two species (all score values <1.7). New W. confusa spectra were created by randomly selecting 3 W. confusa isolates and external validation was performed by testing the remaining 17 W. confusa isolates using the new spectra. The new main spectra projection (MSP) yielded reliable score values of >2 for all isolates with the exception of one (score value, 1.963). Our results showed that the MSPs in the current database are not sufficient for correctly identifying W. confusa or W. cibaria. Further studies including more Weissella isolates are warranted to further validate the performance of MALDI-TOF in identifying Weissella species.

Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library.

PubMed

Becker, Pierre T; de Bel, Annelies; Martiny, Delphine; Ranque, Stéphane; Piarroux, Renaud; Cassagne, Carole; Detandt, Monique; Hendrickx, Marijke

2014-11-01

The identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) relies mainly on a robust and extensive database of reference spectra. To this end, a large in-house library containing 760 strains and representing 472 species was built and evaluated on 390 clinical isolates by comparing MALDI-TOF MS with the classical identification method based on morphological observations. The use of MALDI-TOF MS resulted in the correct identification of 95.4% of the isolates at species level, without considering LogScore values. Taking into account the Brukers' cutoff value for reliability (LogScore >1.70), 85.6% of the isolates were correctly identified. For a number of isolates, microscopic identification was limited to the genus, resulting in only 61.5% of the isolates correctly identified at species level while the correctness reached 94.6% at genus level. Using this extended in-house database, MALDI-TOF MS thus appears superior to morphology in order to obtain a robust and accurate identification of filamentous fungi. A continuous extension of the library is however necessary to further improve its reliability. Indeed, 15 isolates were still not represented while an additional three isolates were not recognized, probably because of a lack of intraspecific variability of the corresponding species in the database. © The Author 2014. 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.

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.

PubMed

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 in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method.

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

PubMed

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

2017-01-30

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

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.

PubMed

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.

Recognition of Clostridium difficile PCR-ribotypes 001, 027 and 126/078 using an extended MALDI-TOF MS system.

PubMed

Reil, M; Erhard, M; Kuijper, E J; Kist, M; Zaiss, H; Witte, W; Gruber, H; Borgmann, S

2011-11-01

During the last decade, Clostridium difficile infection (CDI) increased markedly inside as well as outside of hospitals. In association with the occurrence of new hypervirulent C. difficile strains, CDI became more important. Until now typing of C. difficile strains has been enabled by PCR-ribotyping. However, this method is restricted to specialized laboratories combined with high maintenance cost. Therefore, we tested MALDI-TOF mass spectrometry for typing of C. difficile to provide a fast method for surveillance of CDI. Using a standard set of 25 different C. difficile PCR ribotypes a database was made by different mass spectra recorded in the SARAMIS software (AnagnosTec, Zossen, Germany). The database was validated with 355 C. difficile strains belonging to 29 different PCR ribotypes collected prospectively from all submitted feces samples in 2009. The most frequent PCR ribotypes were type 001 (70%), 027 (4.8%) and 078/126 (4.7%). All three types were recognized by MALDI-TOF MS. We conclude that an extended MALDI-TOF system was capable to recognize specific markers for ribotypes 001, 027 and 078/126 allowing an effective identification of these strains.

Identification of Candida species isolated from vulvovaginitis using matrix assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Alizadeh, Majid; Kolecka, Anna; Boekhout, Teun; Zarrinfar, Hossein; Ghanbari Nahzag, Mohamad A; Badiee, Parisa; Rezaei-Matehkolaei, Ali; Fata, Abdolmajid; Dolatabadi, Somayeh; Najafzadeh, Mohammad J

2017-12-01

Vulvovaginal candidiasis (VVC) is a common problem in women. The purpose of this study was to identify Candida isolates by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) from women with vulvovaginitis that were referred to Ghaem Hospital, Mashhad, Iran. This study was conducted on 65 clinical samples isolated from women that were referred to Ghaem Hospital. All specimens were identified using phenotyping techniques, such as microscopy and culture on Sabouraud dextrose agar and corn meal agar. In addition, all isolates were processed for MALDI-TOF MS identification. Out of the 65 analyzed isolates, 61 (94%) samples were recognized by MALDI-TOF MS. However, the remaining four isolates (6%) had no reliable identification. According to the results, C. albicans (58.5%) was the most frequently isolated species, followed by C. tropicalis (16.9%), C. glabrata (7.7%), C. parapsilosis (7.7%), and guilliermondii (3.1%). As the findings indicated, MALDI TOF MS was successful in the identification of clinical Candida species. C. albicans was identified as the most common Candida species isolated from the women with VVC. Moreover, C. tropicalis was the most common species among the non- albicans Candida species.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed

Papac, D I; Hoyes, J; Tomer, K B

1994-09-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.

PubMed Central

Papac, D. I.; Hoyes, J.; Tomer, K. B.

1994-01-01

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP. PMID:7530543

Capillary electrophoresis-MALDI interface based on inkjet technology

PubMed Central

Vannatta, Michael W.; Whitmore, Colin D.; Dovichi, Norman J.

2010-01-01

An ink jet printer valve and nozzle were used to deliver matrix and sample from an electrophoresis capillary onto a MALDI plate. The system was evaluated by separation of a set of standard peptides. That separation generated up to 40,000 theoretical plates in less than three minutes. Detection limits were 500 amol using an ABI TOF-TOF instrument and 2 fmol for an ABI Q-TOF instrument. Over 70% coverage was obtained for the tryptic digest of α-lactalbumin in less than 2.5 minutes. PMID:19960472

MALDI-TOF mass spectrometry: avoidance of artifacts and analysis of caffeine-precipitated SII thearubigins from 15 commercial black teas.

PubMed

Drynan, J Warren; Clifford, Michael N; Obuchowicz, Jacek; Kuhnert, Nikolai

2012-05-09

Thearubigins are the quantitatively major phenolic compounds in black tea, accounting for some 60-70% of the solids in a typical black tea infusion. MALDI-TOF mass spectra for caffeine-precipitated SII thearubigins (SII CTRs) from 15 different commercial teas support previous conclusions that SII CTRs are polyhydroxylated oligomers (rather than polymers) of catechins and catechin gallates in redox equilibrium with their quinone counterparts. Some 4500 peaks were revealed in a mass range from m/z 500 to 2100. Polyphenols are redox-susceptible and readily generate artifacts during MALDI-TOF analysis when the matrix is also redox-susceptible. Of the nine matrices evaluated, 3',4',5'-trihydroxyacetophenone (F) provided the best compromise between signal intensity and redox artifact formation.

Evaluation of inter-day and inter-individual variability of tear peptide/protein profiles by MALDI-TOF MS analyses

PubMed Central

González, Nerea; Iloro, Ibon; Durán, Juan A.; Elortza, Félix

2012-01-01

Purpose To characterize the tear film peptidome and low molecular weight protein profiles of healthy control individuals, and to evaluate changes due to day-to-day and individual variation and tear collection methods, by using solid phase extraction coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling. Methods The tear protein profiles of six healthy volunteers were analyzed over seven days and inter-day and inter-individual variability was evaluated. The bilaterality of tear film and the effect of tear collection methods on protein profiles were also analyzed in some of these patients. MALDI-TOF MS analyses were performed on tear samples purified by using a solid phase extraction (SPE) method based on C18 functionalized magnetic beads for peptide and low molecular weight protein enrichment, focusing spectra acquisition on the 1 to 20 kDa range. Spectra were analyzed using principal component analysis (PCA) with MultiExperiment Viewer (TMeV) software. Volunteers were examined in terms of tear production status (Schirmer I test), clinical assessment of palpebral lids and meibomian glands, and a subjective OSD questionnaire before tear collection by a glass micro-capillary. Results Analysis of peptides and proteins in the 1–20 kDa range showed no significant inter-day differences in tear samples collected from six healthy individuals during seven days of monitoring, but revealed subtle intrinsic inter-individual differences. Profile analyses of tears collected from the right and left eyes confirmed tear bilaterality in four healthy patients. The addition of physiologic serum for tear sample collection did not affect the peptide and small protein profiles with respect to the number of resolved peaks, but it did reduce the signal intensity of the peaks, and increased variability. Magnetic beads were found to be a suitable method for tear film purification for the profiling study. Conclusions No significant variability in tear peptide and protein profiles below 20 kDa was found in healthy controls over a seven day period, nor in right versus left eye profiles from the same individual. Subtle inter-individual differences can be observed upon tear profiling analysis and confirm intrinsic variability between control subjects. Addition of physiologic serum for tear collection affects the proteome and peptidome in terms of peak intensities, but not in the composition of the profiles themselves. This work shows that MALDI-TOF MS coupled with C18 magnetic beads is an effective and reproducible methodology for tear profiling studies in the clinical monitoring of patients. PMID:22736947

Detection of biomarkers of pathogenic Naegleria fowleri through mass spectrometry and proteomics.

PubMed

Moura, Hercules; Izquierdo, Fernando; Woolfitt, Adrian R; Wagner, Glauber; Pinto, Tatiana; del Aguila, Carmen; Barr, John R

2015-01-01

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry MALDI-TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF-TOF instrument. MALDI-TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI-TOF MS fingerprinting is a rapid, reproducible, high-throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

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

PubMed

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.

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

PubMed Central

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

Highly efficient classification and identification of human pathogenic bacteria by MALDI-TOF MS.

PubMed

Hsieh, Sen-Yung; Tseng, Chiao-Li; Lee, Yun-Shien; Kuo, An-Jing; Sun, Chien-Feng; Lin, Yen-Hsiu; Chen, Jen-Kun

2008-02-01

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.

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

PubMed Central

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. PMID:27843740

Graphene as a Novel Matrix for the Analysis of Small Molecules by MALDI-TOF MS

PubMed Central

Dong, Xiaoli; Cheng, Jinsheng; Li, Jinghong; Wang, Yinsheng

2010-01-01

Graphene was utilized for the first time as matrix for the analysis of low-molecular weight compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Polar compounds including amino acids, polyamines, anticancer drugs and nucleosides could be successfully analyzed. Additionally, nonpolar compounds including steroids could be detected with high resolution and sensitivity. Compared with conventional matrix, graphene exhibited high desorption/ionization efficiency for nonpolar compounds. The graphene matrix functions as substrate to trap analytes, and it transfers energy to the analytes upon laser irradiation, which allowed for the analytes to be readily desorbed/ionized and interference of intrinsic matrix ions to be eliminated. The use of graphene as matrix avoided the fragmentation of analytes and provided good reproducibility and high salt tolerance, underscoring the potential application of graphene as matrix for MALDI-MS analysis of practical samples in complex sample matrices. We also demonstrated that the use of graphene as adsorbent for the solid-phase extraction of squalene could improve greatly the detection limit. This work not only opens a new field for applications of graphene, but also offers a new technique for high-speed analysis of low-molecular weight compounds in areas such as metabolism research and natural products characterization. PMID:20565059

Direct analysis and identification of pathogenic Lichtheimia species by matrix-assisted laser desorption ionization-time of flight analyzer-mediated mass spectrometry.

PubMed

Schrödl, Wieland; Heydel, Tilo; Schwartze, Volker U; Hoffmann, Kerstin; Grosse-Herrenthey, Anke; Walther, Grit; Alastruey-Izquierdo, Ana; Rodriguez-Tudela, Juan Luis; Olias, Philipp; Jacobsen, Ilse D; de Hoog, G Sybren; Voigt, Kerstin

2012-02-01

Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

Integration of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in blood culture diagnostics: a fast and effective approach.

PubMed

Klein, Sabrina; Zimmermann, Stefan; Köhler, Christine; Mischnik, Alexander; Alle, Werner; Bode, Konrad A

2012-03-01

Sepsis is a major cause of mortality in hospitalized patients worldwide, with lethality rates ranging from 30 to 70 %. Sepsis is caused by a variety of different pathogens, and rapid diagnosis is of outstanding importance, as early and adequate antimicrobial therapy correlates with positive clinical outcome. In recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has become a powerful tool in microbiological diagnostics. The direct identification of micro-organisms in a positive blood culture by MALDI-TOF MS can shorten the diagnostic procedure significantly. Therefore, the aim of the present study was to evaluate whether identification rates could be improved by using the new Sepsityper kit from Bruker Daltonics for direct isolation and identification of bacteria from positive blood cultures by MALDI-TOF MS compared with the use of conventional separator gel columns, and to integrate the MALDI-TOF MS-based identification method into the routine course of blood culture diagnostics in the setting of a microbiological laboratory at a university hospital in Germany. The identification of Gram-negative bacteria by MALDI-TOF MS was significantly better using the Sepsityper kit compared with a separator gel tube-based method (99 and 68 % correct identification, respectively). For Gram-positive bacteria, only 73 % were correctly identified by MALDI-TOF with the Sepsityper kit and 59 % with the separator gel tube assay. A major problem of both methods was the poor identification of Gram-positive grape-like clustered cocci. As differentiation of Staphylococcus aureus from coagulase-negative staphylococci is of clinical importance, a PCR was additionally established that was capable of identifying S. aureus directly from positive blood cultures, thus closing this diagnostic gap. Another benefit of the PCR approach is the possibility of directly detecting the genes responsible for meticillin resistance in staphylococci and for vancomycin resistance in enterococci, which is of high importance for early adequate treatment. Both of the described methods were finally integrated into a protocol for fast and effective identification of bacteria from positive blood cultures.

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

PubMed

Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

2008-02-01

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

Identification of proteins in renaissance paintings by proteomics.

PubMed

Tokarski, Caroline; Martin, Elisabeth; Rolando, Christian; Cren-Olivé, Cécile

2006-03-01

The presented work proposes a new methodology based on proteomics techniques to identify proteins in old art paintings. The main challenging tasks of this work were (i) to find appropriate conditions for extracting proteins from the binding media without protein hydrolysis in amino acids and (ii) to develop analytical methods adapted to the small sample quantity available. Starting from microsamples of painting models (ovalbumin-based, which is the major egg white protein, and egg-based paintings), multiple extraction solutions (HCl, HCOOH, NH3, NaOH) and conditions (ultrasonic bath, mortar and pestle, grinding resin) were evaluated. The best results were obtained using a commercial kit including a synthetic resin, mortar and pestle to grind the sample in an aqueous solution acidified with trifluoroacetic acid at 1% with additional multiple steps of ultrasonic baths. The resulting supernatant was analyzed by MALDI-TOF in linear mode to verify the efficiency of the extraction solution. An enzymatic hydrolysis step was also performed for protein identification; the peptide mixture was analyzed by nanoLC/nanoESI/Q-q-TOF MS/MS with an adapted chromatographic run for the low sample quantity. Finally, the developed methodology was successfully applied to Renaissance art painting microsamples of approximately 10 microg from Benedetto Bonfigli's triptych, The Virgin and Child, St. John the Baptist, St. Sebastian (XVth century), and Niccolo di Pietro Gerini's painting, The Virgin and Child (XIVth century), identifying, for the first time and without ambiguity, the presence of whole egg proteins (egg yolk and egg white) in a painting binder.

3M™ Molecular detection system versus MALDI-TOF mass spectrometry and molecular techniques for the identification of Escherichia coli 0157:H7, Salmonella spp. &Listeria spp.

PubMed

Loff, Marché; Mare, Louise; de Kwaadsteniet, Michele; Khan, Wesaal

2014-06-01

The aim of this study was to compare standard selective plating, conventional PCR (16S rRNA and species specific primers), MALDI-TOF MS and the 3M™ Molecular Detection System for the routine detection of the pathogens Listeria, Salmonella and Escherichia coli 0157:H7 in wastewater and river water samples. MALDI-TOF MS was able to positively identify 20/21 (95%) of the E. coli isolates obtained at genus and species level, while 16S rRNA sequencing only correctly identified 6/21 (28%) as E. coli strains. None of the presumptive positive Listeria spp. and Salmonella spp. isolates obtained by culturing on selective media were positively identified by MALDI-TOF and 16S rRNA analysis. The species-specific E. coli 0157:H7 PCR described in this present study, was not able to detect any E. coli 0157:H7 strains in the wastewater and river water samples analysed. However, E. coli strains, Listeria spp., L. monocytogenes and Salmonella spp. were detected using species specific PCR. Escherichia coli 0157:H7, Listeria spp. and Salmonella spp. were also sporadically detected throughout the sampling period in the wastewater and river water samples analysed by the 3M™ Molecular Detection System. MALDI-TOF MS, which is a simple, accurate and cost-effective detection method, efficiently identified the culturable organisms, while in the current study both species specific PCR (Listeria spp. and Salmonella spp.) and 3M™ Molecular Detection System could be utilised for the direct routine analysis of pathogens in water sources. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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.

Comparison of MALDI-TOF MS, nucleic acid hybridization and the MPT64 immunochromatographic test for the identification of M. tuberculosis and non-tuberculosis Mycobacterium species.

PubMed

Şamlı, Asuman; İlki, Arzu

2016-10-01

Mycobacteria are an important cause of morbidity in humans. Rapid and accurate mycobacterial identification is important for improving patient outcomes. However, identification of Mycobacterium species is not easy, due to the slow and fastidious growth of mycobacteria. Recently, biochemical, sequencing, and probing methods have come to be used for identification. This study compared the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of M.tuberculosis and non-tuberculosis Mycobacteria (NTM) to those of nucleic acid hybridization (NAH) and the MPT64 immunochromatographic test. A total of 69 isolates from Marmara University Hospital, Microbiology Laboratory obtained between 2012 and 2013 were included in our study. All strains were grown on Lowenstein-Jensen and Middlebrook 7H9 medium. Among the 69 isolates, 56 (81%) were isolated as Mycobacterium tuberculosis complex (MTC), and 13 (19%) were isolated as NTM by the MPT64 ICT. NAH was able to identify all isolates to the species level. The isolated NTM included M. intracellulare (n:5), M. lentiflavum (n:3), M. xenopi (n:2), M. malmoense (n:1), M. abscessus (n:1), and M. avium (n:1). MALDI-TOF MS identified 88% of the mycobacterial isolates. All M. tuberculosis strains were identified correctly, but the ratio was 38.5% for NTM. Mycobacterial identification using MALDI-TOF MS takes 45 minutes and costs 3 Euro/test, whereas mycobacterial identification using NAH takes 6-7 hours and costs 30 Euro/test. In conclusion, MALDI-TOF MS has the potential to identify mycobacteria in the clinical laboratory setting by reducing identification turnaround time and laboratory costs for isolate referral.

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

PubMed

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.

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

PubMed

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.

Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Molds of the Fusarium Genus

PubMed Central

Stubbe, Dirk; De Cremer, Koen; Piérard, Denis; Normand, Anne-Cécile; Piarroux, Renaud; Detandt, Monique; Hendrickx, Marijke

2014-01-01

The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity. PMID:25411180

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

PubMed

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.

Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis.

PubMed

Soler, Laura; Labas, Valérie; Thélie, Aurore; Grasseau, Isabelle; Teixeira-Gomes, Ana-Paula; Blesbois, Elisabeth

2016-06-01

Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of Acinetobacter species: is Bruker biotyper MALDI-TOF mass spectrometry a good alternative to molecular techniques?

PubMed

Alvarez-Buylla, Adela; Culebras, Esther; Picazo, Juan J

2012-03-01

Acinetobacter spp. has become a leading cause of nosocomial infection in recent years. Phenotypic similarities between the species in the genus have made it difficult to identify them clearly using routine diagnostic methods. Consequently, more relevant species have been grouped together as Acinetobacter calcoaceticus-Acinetobacter baumannii complex (A. baumannii, A. calcoaceticus, Acinetobacter genospecies 3 and A. genospecies 13TU). However, there are other species that may also have clinical significance. The aims of this study were to establish the usefulness of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of Acinetobacter species by comparison with two molecular techniques, as well as determine the role of species other than A. baumannii play in nosocomial infections.The study sample comprised 109 clinical isolates of Acinetobacter. They were all identified using MALDI-TOF MS. Thirty-one isolates of these were also tested using comparator amplification of bla(OXA51-like) and sequencing of the rpoB gene. Different score values in MALDI-TOF MS revealed 87 A. baumannii, 19 A. genospecies 3, 1 Acinetobacter junii, 1 Acinetobacter baylyi and 1 Acinetobacter tjernbergiae. Amplification of bla(OXA-51)(-like) showed products in 85 isolates. Sequencing of the rpoB gene allowed us to identify all the 31 isolates analyzed: 16 were consistent with the results of spectrometry and 15 were not. This work showed that molecular techniques are still needed to identify the different species of clinical interest within the genus Acinetobacter. Although, MALDI-TOF MS could be useful to identify A. baumannii but not other species in the genus. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of protein spectra cluster analysis for Streptococcus spp. identification from various swine clinical samples.

PubMed

Matajira, Carlos E C; Moreno, Luisa Z; Gomes, Vasco T M; Silva, Ana Paula S; Mesquita, Renan E; Doto, Daniela S; Calderaro, Franco F; de Souza, Fernando N; Christ, Ana Paula G; Sato, Maria Inês Z; Moreno, Andrea M

2017-03-01

Traditional microbiological methods enable genus-level identification of Streptococcus spp. isolates. However, as the species of this genus show broad phenotypic variation, species-level identification or even differentiation within the genus is difficult. Herein we report the evaluation of protein spectra cluster analysis for the identification of Streptococcus species associated with disease in swine by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 250 S. suis-like isolates obtained from pigs with clinical signs of encephalitis, arthritis, pneumonia, metritis, and urinary or septicemic infection were studied. The isolates came from pigs in different Brazilian states from 2001 to 2014. The MALDI-TOF MS analysis identified 86% (215 of 250) as S. suis and 14% (35 of 250) as S. alactolyticus, S. dysgalactiae, S. gallinaceus, S. gallolyticus, S. gordonii, S. henryi, S. hyointestinalis, S. hyovaginalis, S. mitis, S. oralis, S. pluranimalium, and S. sanguinis. The MALDI-TOF MS identification was confirmed in 99.2% of the isolates by 16S rDNA sequencing, with MALDI-TOF MS misidentifying 2 S. pluranimalium as S. hyovaginalis. Isolates were also tested by a biochemical automated system that correctly identified all isolates of 8 of the 10 species in the database. Neither the isolates of the 3 species not in the database ( S. gallinaceus, S. henryi, and S. hyovaginalis) nor the isolates of 2 species that were in the database ( S. oralis and S. pluranimalium) could be identified. The topology of the protein spectra cluster analysis appears to sustain the species phylogenetic similarities, further supporting identification by MALDI-TOF MS examination as a rapid and accurate alternative to 16S rDNA sequencing.

Detection of Protein Modifications and Counterfeit Protein Pharmaceuticals Using iTRAQ and MALDI TOF/TOF Mass Spectrometry: Studies with Insulins

PubMed Central

Ye, Hongping; Hill, John; Kauffman, John; Gryniewicz, Connie; Han, Xianlin

2013-01-01

iTRAQ (isotope tags for relative and absolute quantification) reagent coupled with MALDI TOF/TOF mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies, and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, Lantus®) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including Human insulin (P28K29) and Lispro (K28P29), which only differ by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting. PMID:18489896

Proteins involved in neuronal differentiation of neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-Eun; Freilinger, Angelika; Gelpi, Ellen; Pollak, Arnold; Hengstschläger, Markus; Lubec, Gert

2007-06-01

Neuronal differentiation (ND) represents a well-defined phenomenon in biological terms but proteins involved have not been studied systematically. We therefore aimed to study ND by retinoic acid (RA) in a widely used neuroblastoma cell line by comparative proteomics. The ND was induced in the N1E-115 cell line by serum deprivation and RA treatment. Undifferentiated cells and cells undergoing serum deprivation served as controls. Protein extracts were run on 2-DE followed by MALDI-TOF or MALDI-TOF-TOF analysis. Quantification was carried out using specific software and stringent statistical analysis was performed. Tubulin beta 5, cat eye syndrome critical region protein 5 homolog, putative GTP-binding protein PTD004 homolog, and the metabolic proteins glyceraldehyde-3-phosphate dehydrogenase and transketolase were differentially regulated. Differential protein levels of cytoskeleton proteins including tubulins and metabolic proteins have been reported to be regulated by ND. Herein, specific signaling differences as reflected by putative GTP-binding protein PTD004 changes in differentiated cells are shown and a possible role for the Cat eye syndrome critical region protein 5 homolog is proposed. The protein disulfide isomerase associated 3 protein fits the already proposed findings of chaperon regulation by ND. The study forms the molecular basis for further evaluation of the functional roles of the differentially expressed proteins in ND.

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.

PubMed

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.

Analysis of Glycoproteins in Human Serum by Means of Glycospecific Magnetic Bead Separation and LC-MALDI-TOF/TOF Analysis with Automated Glycopeptide Detection

PubMed Central

Sparbier, Katrin; Asperger, Arndt; Resemann, Anja; Kessler, Irina; Koch, Sonja; Wenzel, Thomas; Stein, Günter; Vorwerg, Lars; Suckau, Detlev; Kostrzewa, Markus

2007-01-01

Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively. PMID:17916798

Identification of the ESKAPE pathogens by mass spectrometric analysis of microbial membrane glycolipids.

PubMed

Leung, Lisa M; Fondrie, William E; Doi, Yohei; Johnson, J Kristie; Strickland, Dudley K; Ernst, Robert K; Goodlett, David R

2017-07-25

Rapid diagnostics that enable identification of infectious agents improve patient outcomes, antimicrobial stewardship, and length of hospital stay. Current methods for pathogen detection in the clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein characterization, and genome sequencing. Pathogen identification from single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of high abundance proteins is gaining popularity in clinical laboratories. Here, we present a novel and complementary approach that utilizes essential microbial glycolipids as chemical fingerprints for identification of individual bacterial species. Gram-positive and negative bacterial glycolipids were extracted using a single optimized protocol. Extracts of the clinically significant ESKAPE pathogens: E nterococcus faecium, S taphylococcus aureus, K lebsiella pneumoniae, A cinetobacter baumannii, P seudomonas aeruginosa, and E nterobacter spp. were analyzed by MALDI-TOF-MS in negative ion mode to obtain glycolipid mass spectra. A library of glycolipid mass spectra from 50 microbial entries was developed that allowed bacterial speciation of the ESKAPE pathogens, as well as identification of pathogens directly from blood bottles without culture on solid medium and determination of antimicrobial peptide resistance. These results demonstrate that bacterial glycolipid mass spectra represent chemical barcodes that identify pathogens, potentially providing a useful alternative to existing diagnostics.

Lipid profiling of parkin-mutant human skin fibroblasts.

PubMed

Lobasso, Simona; Tanzarella, Paola; Vergara, Daniele; Maffia, Michele; Cocco, Tiziana; Corcelli, Angela

2017-12-01

Parkin mutations are a major cause of early-onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin-mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) and thin-layer chromatography (TLC). In parallel, we have performed direct MALDI-TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin-mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well-known component of PD. © 2017 Wiley Periodicals, Inc.

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

PubMed

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.

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

ERIC Educational Resources Information Center

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…

MALDI-TOF mass spectrometry for early identification of bacteria grown in blood culture bottles.

PubMed

Zabbe, Jean-Benoît; Zanardo, Laura; Mégraud, Francis; Bessède, Emilie

2015-08-01

This note reports an interesting way to rapidly identify bacteria grown from blood culture bottles. Chocolate agar plates were inoculated with 1 drop of the positive blood bottle medium. After a 3-hour incubation, the growth veil was submitted to MALDI-TOF mass spectrometry: 77% of the bacteria present have been correctly identified. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid and reliable species identification of wild mushrooms by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

PubMed

Sugawara, Ryota; Yamada, Sayumi; Tu, Zhihao; Sugawara, Akiko; Suzuki, Kousuke; Hoshiba, Toshihiro; Eisaka, Sadao; Yamaguchi, Akihiro

2016-08-31

Mushrooms are a favourite natural food in many countries. However, some wild species cause food poisoning, sometimes lethal, due to misidentification caused by confusing fruiting bodies similar to those of edible species. The morphological inspection of mycelia, spores and fruiting bodies have been traditionally used for the identification of mushrooms. More recently, DNA sequencing analysis has been successfully applied to mushrooms and to many other species. This study focuses on a simpler and more rapid methodology for the identification of wild mushrooms via protein profiling based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). A preliminary study using 6 commercially available cultivated mushrooms suggested that a more reproducible spectrum was obtained from a portion of the cap than from the stem of a fruiting body by the extraction of proteins with a formic acid-acetonitrile mixture (1 + 1). We used 157 wild mushroom-fruiting bodies collected in the centre of Hokkaido from June to November 2014. Sequencing analysis of a portion of the ribosomal RNA gene provided 134 identifications of mushrooms by genus or species, however 23 samples containing 10 unknown species that had lower concordance rate of the nucleotide sequences in a BLAST search (less than 97%) and 13 samples that had unidentifiable poor or mixed sequencing signals remained unknown. MALDI-TOF MS analysis yielded a reproducible spectrum (frequency of matching score ≥ 2.0 was ≥6 spectra from 12 spectra measurements) for 114 of 157 samples. Profiling scores that matched each other within the database gave correct species identification (with scores of ≥2.0) for 110 samples (96%). An in-house prepared database was constructed from 106 independent species, except for overlapping identifications. We used 48 wild mushrooms that were collected in autumn 2015 to validate the in-house database. As a result, 21 mushrooms were identified at the species level with scores ≥2.0 and 5 mushrooms at the genus level with scores ≥1.7, although the signals of 2 mushrooms were insufficient for analysis. The remaining 20 samples were recognized as "unreliable identification" with scores <1.7. Subsequent DNA analysis confirmed that the correct species or genus identifications were achieved by MALDI-TOF MS for the 26 former samples, whereas the 18 mushrooms with poorly matched scores were species that were not included in the database. Thus, the proposed MALDI-TOF MS coupled with our database could be a powerful tool for the rapid and reliable identification of mushrooms; however, continuous updating of the database is necessary to enrich it with more abundant species. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Candida species isolated from vulvovaginitis using matrix assisted laser desorption ionization-time of flight mass spectrometry

PubMed Central

Alizadeh, Majid; Kolecka, Anna; Boekhout, Teun; Zarrinfar, Hossein; Ghanbari Nahzag, Mohamad. A; Badiee, Parisa; Rezaei-Matehkolaei, Ali; Fata, Abdolmajid; Dolatabadi, Somayeh; Najafzadeh, Mohammad. J

2017-01-01

Background and Purpose: Vulvovaginal candidiasis (VVC) is a common problem in women. The purpose of this study was to identify Candida isolates by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) from women with vulvovaginitis that were referred to Ghaem Hospital, Mashhad, Iran. Materials and Methods: This study was conducted on 65 clinical samples isolated from women that were referred to Ghaem Hospital. All specimens were identified using phenotyping techniques, such as microscopy and culture on Sabouraud dextrose agar and corn meal agar. In addition, all isolates were processed for MALDI-TOF MS identification. Results: Out of the 65 analyzed isolates, 61 (94%) samples were recognized by MALDI-TOF MS. However, the remaining four isolates (6%) had no reliable identification. According to the results, C. albicans (58.5%) was the most frequently isolated species, followed by C. tropicalis (16.9%), C. glabrata (7.7%), C. parapsilosis (7.7%), and guilliermondii (3.1%). Conclusion: As the findings indicated, MALDI TOF MS was successful in the identification of clinical Candida species. C. albicans was identified as the most common Candida species isolated from the women with VVC. Moreover, C. tropicalis was the most common species among the non-albicans Candida species. PMID:29707675

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.

PubMed

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.

Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry-based blood group genotyping--the alternative approach.

PubMed

Gassner, Christoph; Meyer, Stefan; Frey, Beat M; Vollmert, Caren

2013-01-01

Although matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF MS) has previously been reported for high throughput blood group genotyping, those reports are limited to only a few blood group systems. This review describes the development of a large cooperative Swiss-German project, aiming to employ MALDI-TOF MS for the molecular detection of the blood groups Rh, Kell, Kidd, Duffy, MNSs, a comprehensive collection of low incidence antigens, as well as the platelet and granulocyte antigens HPA and HNA, representing a total of 101 blood group antigens, encoded by 170 alleles, respectively. Recent reports describe MALDI-TOF MS as a technology with short time-to-resolution, ability for high throughput, and cost-efficiency when used in genetic analysis, including forensics, pharmacogenetics, oncology and hematology. Furthermore, Kell and RhD genotyping have been performed on fetal DNA from maternal plasma with excellent results. In summary, this article introduces a new technological approach for high throughput blood group genotyping by means of MALDI-TOF MS. Although all data presented are preliminary, the observed success rates, data quality and concordance with known blood group types are highly impressive, underlining the accuracy and reliability of this cost-efficient high throughput method. Copyright © 2013 Elsevier Inc. All rights reserved.

Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for the identification of clinical filamentous fungi.

PubMed

Huang, Yanfei; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Wang, Jinglin; Lu, Xinxin

2017-07-01

Infections caused by filamentous fungi have become a health concern, and require rapid and accurate identification in order for effective treatment of the pathogens. To compare the performance of two MALDI-TOF MS systems (Bruker Microflex LT and Xiamen Microtyper) in the identification of filamentous fungal species. A total of 374 clinical filamentous fungal isolates sequentially collected in the Clinical Laboratory at the Beijing Tongren Hospital between January 2014 and December 2015 were identified by traditional phenotypic methods, Bruker Microflex LT and Xiamen Microtyper MALDI-TOF MS, respectively. The discrepancy between these methods was resolved by sequencing for definitive identification. Bruker Microflex LT and Xiamen Microtyper had similar correct species ID (98.9 vs. 99.2%), genus ID (99.7 vs. 100%), mis-ID (0.3 vs. 0%) and no ID (0 vs. 0). The rate of correct species identification by both MALDI-TOF MS (98.9 and 99.2%, respectively) was much higher compared with phenotypic approach (91.9%). Both MALDI-TOF MS systems provide accurate identification of clinical filamentous fungi compared with conventional phenotypic method, and have the potential to replace identification for routine identification of these fungi in clinical mycology laboratories. Both systems have similar performance in the identification of clinical filamentous fungi.

Technologies in the Whole-Genome Age: MALDI-TOF-Based Genotyping.

PubMed

Vogel, Nicolas; Schiebel, Katrin; Humeny, Andreas

2009-01-01

With the decipherment of the human genome, new questions have moved into the focus of today's research. One key aspect represents the discovery of DNA variations capable to influence gene transcription, RNA splicing, or regulating processes, and their link to pathology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a powerful tool for the qualitative investigation and relative quantification of variations like single nucleotide polymorphisms, DNA methylation, microsatellite instability, or loss of heterozygosity. After its introduction into proteomics, efforts were made to adopt this technique to DNA analysis. Initially intended for peptide/protein analysis, it held several difficulties for application to nucleic acids. Today, MALDI-TOF-MS has reached worldwide acceptance and application in nucleic acid research, with a wide spectrum of methods being available. One of the most versatile approaches relies on primer extension to genotype single alleles, microsatellite repeat lengths or the methylation status of a given cytosine. Optimized methods comprising intelligent primer design and proper nucleotide selection for primer extension enabled multiplexing of reactions, rendering the analysis more economic due to parallel genotyping of several alleles in a single experiment. Laboratories equipped with MALDI-TOF-MS possess a universal technical platform for the analysis of a large variety of different molecules.

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

PubMed

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.

Identification of Medically Relevant Species of Arthroconidial Yeasts by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Kolecka, Anna; Khayhan, Kantarawee; Groenewald, Marizeth; Theelen, Bart; Arabatzis, Michael; Velegraki, Aristea; Kostrzewa, Markus; Mares, Mihai; Taj-Aldeen, Saad J.

2013-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used for an extensive identification study of arthroconidial yeasts, using 85 reference strains from the CBS-KNAW yeast collection and 134 clinical isolates collected from medical centers in Qatar, Greece, and Romania. The test set included 72 strains of ascomycetous yeasts (Galactomyces, Geotrichum, Saprochaete, and Magnusiomyces spp.) and 147 strains of basidiomycetous yeasts (Trichosporon and Guehomyces spp.). With minimal preparation time, MALDI-TOF MS proved to be an excellent diagnostic tool that provided reliable identification of most (98%) of the tested strains to the species level, with good discriminatory power. The majority of strains were correctly identified at the species level with good scores (>2.0) and seven of the tested strains with log score values between 1.7 and 2.0. The MALDI-TOF MS results obtained were consistent with validated internal transcribed spacer (ITS) and/or large subunit (LSU) ribosomal DNA sequencing results. Expanding the mass spectrum database by increasing the number of reference strains for closely related species, including those of nonclinical origin, should enhance the usefulness of MALDI-TOF MS-based diagnostic analysis of these arthroconidial fungi in medical and other laboratories. PMID:23678074

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

PubMed

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.

Identification of Haemophilus influenzae Type b Isolates by Use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

PubMed

Månsson, Viktor; Resman, Fredrik; Kostrzewa, Markus; Nilson, Bo; Riesbeck, Kristian

2015-07-01

Haemophilus influenzae type b (Hib) is, in contrast to non-type b H. influenzae, associated with severe invasive disease, such as meningitis and epiglottitis, in small children. To date, accurate H. influenzae capsule typing requires PCR, a time-consuming and cumbersome method. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provides rapid bacterial diagnostics and is increasingly used in clinical microbiology laboratories. Here, MALDI-TOF MS was evaluated as a novel approach to separate Hib from other H. influenzae. PCR-verified Hib and non-Hib reference isolates were selected based on genetic and spectral characteristics. Mass spectra of reference isolates were acquired and used to generate different classification algorithms for Hib/non-Hib differentiation using both ClinProTools and the MALDI Biotyper software. A test series of mass spectra from 33 Hib and 77 non-Hib isolates, all characterized by PCR, was used to evaluate the algorithms. Several algorithms yielded good results, but the two best were a ClinProTools model based on 22 separating peaks and subtyping main spectra (MSPs) using MALDI Biotyper. The ClinProTools model had a sensitivity of 100% and a specificity of 99%, and the results were 98% reproducible using a different MALDI-TOF MS instrument. The Biotyper subtyping MSPs had a sensitivity of 97%, a specificity of 100%, and 93% reproducibility. Our results suggest that it is possible to use MALDI-TOF MS to differentiate Hib from other H. influenzae. This is a promising method for rapidly identifying Hib in unvaccinated populations and for the screening and surveillance of Hib carriage in vaccinated populations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

NASA Astrophysics Data System (ADS)

Webber, Nathaniel; He, Yi; Reilly, James P.

2014-02-01

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

Improvement of identification of Capnocytophaga canimorsus by matrix-assisted laser desorption ionization-time of flight mass spectrometry using enriched database.

PubMed

Magnette, Amandine; Huang, Te-Din; Renzi, Francesco; Bogaerts, Pierre; Cornelis, Guy R; Glupczynski, Youri

2016-01-01

Capnocytophaga canimorsus and Capnocytophaga cynodegmi can be transmitted from dogs or cats and cause serious human infections. We aimed to evaluate the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify these two Capnocytophaga species. Ninety-four C. canimorsus and 10 C. cynodegmi isolates identified by 16S rRNA gene sequencing were analyzed. Using the MALDI BioTyper database, correct identification was achieved for only 16 of 94 (17%) C. canimorsus and all 10 C. cynodegmi strains, according to the manufacturer's log score specifications. Following the establishment of a complementary homemade reference database by addition of 51 C. canimorsus and 8 C. cynodegmi mass spectra, MALDI-TOF MS provided reliable identification to the species level for 100% of the 45 blind-coded Capnocytophaga isolates tested. MALDI-TOF MS can accurately identify C. canimorsus and C. cynodegmi using an enriched database and thus constitutes a valuable diagnostic tool in the clinical laboratory. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Bruker Biotyper for identification of Penicillium marneffei, Paecilomyces species, Fusarium solani, Rhizopus species, and Pseudallescheria boydii

PubMed Central

Chen, Ying-Sheng; Liu, Yen-Hung; Teng, Shih-Hua; Liao, Chun-Hsing; Hung, Chien-Ching; Sheng, Wang-Huei; Teng, Lee-Jene; Hsueh, Po-Ren

2015-01-01

We evaluated the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the MALDI Bruker Biotyper system (microflex LT; Bruker Daltonik GmbH, Bremen, Germany), on the identification of 50 isolates of clinically encountered molds, including Penicillium marneffei (n = 28), Paecilomyces species (n = 12), Fusarium solani (n = 6), Rhizopus species (n = 3), and Pseudallescheria boydii (n = 1). The isolates were identified to species levels by sequence analysis of the internal transcribed spacer (ITS) regions using primers ITS1 and ITS4. None of the 28 genetically well characterized isolates of P. marneffei were identified as P. marneffei by MALDI-TOF MS, because P. marneffei was not present in either Bruker general library (DB 5627) or Bruker filamentous fungi library V1.0. However, the rate of accurate identification as P. marneffei (score value ≥ 2.000) was 85.7% based on newly created database from one P. marneffei strain (NTUH-3370) by MALDI Biotyper system. Sequencing analysis of these 22 non-P. marneffei isolates of molds revealed seven Paecilomyces variotii, six F. solani, four Paecilomyces lilacinus, and one each of Paecilomyces sinensis, Rhizopus arrhizus, R. oryzae, R. microspores, and P. boydii. Although all the seven P. variotii isolates, four of the six F. solani, two of the four P. lilacinus, and two of the three isolates of Rhizopus species, and the P. boydii isolate had concordant identification results between MALDI-TOF MS and sequencing analysis, the score values of these isolates were all of <1.700. This study indicated that the MALDI Bruker Biotyper is ineffective for identifying P. marneffei and other unusual molds because of the current database limitations. Therefore, it is necessary to continuously update the MALDI-TOF MS databases. PMID:26217315

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

PubMed

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.

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

PubMed

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.

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

PubMed

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.

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

PubMed Central

Park, Jonghoo; Blick, Robert H.

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 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. PMID:24152929

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

PubMed

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.

Direct identification of bacteria in blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new methodological approach.

PubMed

Kroumova, Vesselina; Gobbato, Elisa; Basso, Elisa; Mucedola, Luca; Giani, Tommaso; Fortina, Giacomo

2011-08-15

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently been demonstrated to be a powerful tool for the rapid identification of bacteria from growing colonies. In order to speed up the identification of bacteria, several authors have evaluated the usefulness of this MALDI-TOF MS technology for the direct and quick identification bacteria from positive blood cultures. The results obtained so far have been encouraging but have also shown some limitations, mainly related to the bacterial growth and to the presence of interference substances belonging to the blood cultures. In this paper, we present a new methodological approach that we have developed to overcome these limitations, based mainly on an enrichment of the sample into a growing medium before the extraction process, prior to mass spectrometric analysis. The proposed method shows important advantages for the identification of bacterial strains, yielding an increased identification score, which gives higher confidence in the results. Copyright © 2011 John Wiley & Sons, Ltd.

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

PubMed Central

Leyer, Caroline; Gregorowicz, Guillaume; Mougari, Faiza; Raskine, Laurent; Cambau, Emmanuelle

2017-01-01

ABSTRACT During the last decade, many investigators have studied matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for identification of mycobacteria. Diverse and contradictory results indicated that optimal level for routine testing has not been reached yet. This work aimed to assess Vitek MS through two distinct versions, Saramis v4.12 RUO and the IVD v3.0, under conditions close to routine laboratory practice. Overall, 111 mycobacterial isolates were subjected to protein extraction and same spectra were matched against both databases. The IVD v3.0 database proved to be superior to Saramis v4.12 and its identification rates remarkably increased, from 67% to 94% for isolates grown on Middlebrook 7H10 solid medium and from 62% to 91% for isolates grown on mycobacterial growth indicator tube (MGIT) liquid medium. With this new version, IVD v3.0, MALDI-TOF MS might be integrated into routine clinical diagnostics, although molecular techniques remain mandatory in some cases. PMID:28424252

Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the screening of vanA-positive Enterococcus faecium.

PubMed

Wang, Li-jun; Lu, Xin-xin; Wu, Wei; Sui, Wen-jun; Zhang, Gui

2014-01-01

In order to evaluate a rapid matrix-assisted laser desorption ionization-time of flight mass spectrometry (MAIDI-TOF MS) assay in screening vancomycin-resistant Enterococcus faecium, a total of 150 E. faecium clinical strains were studied, including 60 vancomycin-resistant E. faecium (VREF) isolates and 90 vancomycin-susceptible (VSEF) strains. Vancomycin resistance genes were detected by sequencing. E. faecium were identified by MALDI-TOF MS. A genetic algorithm model with ClinProTools software was generated using spectra of 30 VREF isolates and 30 VSEF isolates. Using this model, 90 test isolates were discriminated between VREF and VSEF. The results showed that all sixty VREF isolates carried the vanA gene. The performance of VREF detection by the genetic algorithm model of MALDI-TOF MS compared to the sequencing method was sensitivity = 80%, specificity = 90%, false positive rate =10%, false negative rate =10%, positive predictive value = 80%, negative predictive value= 90%. MALDI-TOF MS can be used as a screening test for discrimination between vanA-positive E. faecium and vanA-negative E. faecium.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

PubMed

Kim, Kyoung-Jin; Kim, Yoon-Woo; Kim, Yun-Gon; Park, Hae-Min; Jin, Jang Mi; Hwan Kim, Young; Yang, Yung-Hun; Kyu Lee, Jun; Chung, Junho; Lee, Sun-Gu; Saghatelian, Alan

2015-01-01

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers.

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory.

PubMed

Wattal, C; Oberoi, J K; Goel, N; Raveendran, R; Khanna, S

2017-05-01

The study evaluates the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) Vitek MS for identification of microorganisms in the routine clinical microbiology laboratory. From May 2013 to April 2014, microbial isolates recovered from various clinical samples were identified by Vitek MS. In case of failure to identify by Vitek MS, the isolate was identified using the Vitek 2 system (bioMerieux, France) and serotyping wherever applicable or otherwise by nucleic acid-mediated methods. All the moulds were identified by Lactophenol blue mounts, and mycobacterial isolates were identified by molecular identification systems including AccuProbe (bioMerieux, France) or GenoType Mycobacterium CM (Hain Lifescience, Germany). Out of the 12,003 isolates, the Vitek MS gave a good overall ID at the genus and or species level up to 97.7% for bacterial isolates, 92.8% for yeasts and 80% for filamentous fungi. Of the 26 mycobacteria tested, only 42.3% could be identified using the Saramis RUO (Research Use Only) database. VITEK MS could not identify 34 of the 35 yeast isolates identified as C. haemulonii by Vitek 2. Subsequently, 17 of these isolates were identified as Candida auris (not present in the Vitek MS database) by 18S rRNA sequencing. Using these strains, an in-house superspectrum of C. auris was created in the VITEK MS database. Use of MALDI-TOF MS allows a rapid identification of aerobic bacteria and yeasts in clinical practice. However, improved sample extraction protocols and database upgrades with inclusion of locally representative strains is required, especially for moulds.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and accurate identification of Pseudallescheria/Scedosporium species.

PubMed

Sitterlé, E; Giraud, S; Leto, J; Bouchara, J P; Rougeron, A; Morio, F; Dauphin, B; Angebault, C; Quesne, G; Beretti, J L; Hassouni, N; Nassif, X; Bougnoux, M E

2014-09-01

An increasing number of infections due to Pseudallescheria/Scedosporium species has been reported during the past decades, both in immunocompromised and immunocompetent patients. Additionally, these fungi are now recognized worldwide as common agents of fungal colonization of the airways in cystic fibrosis patients, which represents a risk factor for disseminated infections after lung transplantation. Currently six species are described within the Pseudallescheria/Scedosporium genus, including Scedosporium prolificans and species of the Pseudallescheria/Scedosporium apiospermum complex (i.e. S. apiospermum sensu stricto, Pseudallescheria boydii, Scedosporium aurantiacum, Pseudallescheria minutispora and Scedosporium dehoogii). Precise identification of clinical isolates at the species level is required because these species differ in their antifungal drug susceptibility patterns. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)/mass spectrometry (MS) is a powerful tool to rapidly identify moulds at the species level. We investigated the potential of this technology to discriminate Pseudallescheria/Scedosporium species. Forty-seven reference strains were used to build a reference database library. Profiles from 3-, 5- and 7-day-old cultures of each reference strain were analysed to identify species-specific discriminating profiles. The database was tested for accuracy using a set of 64 clinical or environmental isolates previously identified by multilocus sequencing. All isolates were unequivocally identified at the species level by MALDI-TOF/MS. Our results, obtained using a simple protocol, without prior protein extraction or standardization of the culture, demonstrate that MALDI-TOF/MS is a powerful tool for rapid identification of Pseudallescheria/Scedosporium species that cannot be currently identified by morphological examination in the clinical setting. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

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.

PubMed

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 (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, 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.

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

PubMed

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.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) coupled to XAD fractionation: Method to algal organic matter characterization.

PubMed

Nicolau, Rudy; Leloup, Maud; Lachassagne, Delphine; Pinault, Emilie; Feuillade-Cathalifaud, Geneviève

2015-05-01

This work is focused on the development of an analytical procedure for the improvement of the Organic Matter structure characterization, particularly the algal matter. Two fractions of algal organic matter from laboratory cultures of algae (Euglena gracilis) and cyanobacteria (Microcystis aeruginosa) were extracted with XAD resins. The fractions were studied using laser desorption ionization (LDI) and Matrix-Assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). A comparison with the natural organic matter characteristics from commercial humic acids and fulvic acids extracted from Suwannee River was performed. Results show that algal and natural organic matters have unique quasi-polymeric structures. Significant repeating patterns were identified. Different fractions extracted from organic matter with common origin had common structures. Thus, 44, 114 and 169Da peaks separation for fractions from E. gracilis organic matter and 28, 58 and 100Da for M. aeruginosa ones were clearly observed. Using the developed protocol, a structural scheme and organic matter composition were obtained. The range 600-2000Da contained more architectural composition differences than the range 100-600Da, suggesting that organic matter is composed of an assembly of common small molecules. Associated to specific monomers, particular patterns were common to all samples but assembly and resulting structure were unique for each organic matter. Thus, XAD fractionation coupled to mass spectroscopy allowed determining a specific fingerprint for each organic matter. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitation of quinapril in human plasma by matrix-assisted laser desorption ionization time-of-flight mass spectrometry with quinolone matrix additives.

PubMed

Lu, Chi-Yu; Liu, Fei-Tsui; Feng, Chia-Hsien

2011-09-15

The renin-angiotensin-aldosterone system (RAAS) is an essential body fluid maintenance system that controls pressure in the human body. The conversion of angiotensin I to angiotensin II by angiotensin-converting enzyme (ACE) is a key process in the RAAS because angiotensin II causes the vasoconstriction association with hypertension. Because of its effectiveness as an ACE blocker, quinipril is widely used for clinical treatment of hypertension and chronic congestive heart failure(.) Matrix-assisted laser desorption/ionization coupled with time-of-flight analyzer (MALDI-TOF) is a high throughput instrument for biological sample analysis. This study developed a micro-scale approach for using MALDI-TOF to detect quinapril in biological samples. A micro-liquid-liquid-extraction strategy combined with ion-pair interaction successfully extracted quinapril from aqueous layer to organic layer. Quinolones were then used as matrix additives to suppress undesired substances in plasma produce signals. Several factors affecting extraction efficiency were investigated in a biosample with a volume of only 10 μL. This method is successful to monitor quinapril in the clinical therapeutic range. The proposed method proved effective for monitoring the trace amounts of quinapril typically used for clinical therapy. The relative standard deviation (R.S.D.) and relative error (R.E.) used for evaluating within- and between-day assays of quinapril in plasma consistently remained below 15%. Copyright © 2011. Published by Elsevier B.V.

Direct Analysis and Identification of Pathogenic Lichtheimia Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Analyzer-Mediated Mass Spectrometry

PubMed Central

Schrödl, Wieland; Heydel, Tilo; Schwartze, Volker U.; Hoffmann, Kerstin; Große-Herrenthey, Anke; Walther, Grit; Alastruey-Izquierdo, Ana; Rodriguez-Tudela, Juan Luis; Olias, Philipp; Jacobsen, Ilse D.; de Hoog, G. Sybren

2012-01-01

Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time. PMID:22135259

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

NASA Astrophysics Data System (ADS)

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.

Accurate characterization of carcinogenic DNA adducts using MALDI tandem time-of-flight mass spectrometry

NASA Astrophysics Data System (ADS)

Barnes, Charles A.; Chiu, Norman H. L.

2009-01-01

Many chemical carcinogens and their in vivo activated metabolites react readily with genomic DNA, and form covalently bound carcinogen-DNA adducts. Clinically, carcinogen-DNA adducts have been linked to various cancer diseases. Among the current methods for DNA adduct analysis, mass spectroscopic method allows the direct measurement of unlabeled DNA adducts. The goal of this study is to explore the use of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to determine the identity of carcinogen-DNA adducts. Two of the known carcinogenic DNA adducts, namely N-(2'-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (dG-C8-PhIP) and N-(2'-deoxyguanosin-8yl)-4-aminobiphenyl (dG-C8-ABP), were selected as our models. In MALDI-TOF MS measurements, the small matrix ion and its cluster ions did not interfere with the measurements of both selected dG adducts. To achieve a higher accuracy for the characterization of selected dG adducts, 1 keV collision energy in MALDI-TOF/TOF MS/MS was used to measure the adducts. In comparison to other MS/MS techniques with lower collision energies, more extensive precursor ion dissociations were observed. The detection of the corresponding fragment ions allowed the identities of guanine, PhIP or ABP, and the position of adduction to be confirmed. Some of the fragment ions of dG-C8-PhIP have not been reported by other MS/MS techniques.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets.

PubMed

Prod'hom, Guy; Bizzini, Alain; Durussel, Christian; Bille, Jacques; Greub, Gilbert

2010-04-01

An ammonium chloride erythrocyte-lysing procedure was used to prepare a bacterial pellet from positive blood cultures for direct matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry analysis. Identification was obtained for 78.7% of the pellets tested. Moreover, 99% of the MALDI-TOF identifications were congruent at the species level when considering valid scores. This fast and accurate method is promising.

Rapid identification of microorganisms from positive blood cultures by testing early growth on solid media using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Gonzalez, Mark D; Weber, Carol J; Burnham, Carey-Ann D

2016-06-01

We performed a retrospective analysis of a simple modification to MALDI-TOF MS for microorganism identification to accurately improve the turnaround time (TAT) for identification of Enterobacteriaceae recovered in blood cultures. Relative to standard MALDI-TOF MS procedures, we reduced TAT from 28.3 (n=90) to 21.2h (n=107). Copyright © 2016 Elsevier Inc. All rights reserved.

The Phantom Menace for Patients with Hepatobiliary Diseases: Shewanella haliotis, Often Misidentified as Shewanella algae in Biochemical Tests and MALDI-TOF Analysis.

PubMed

Byun, Jung-Hyun; Park, Hyunwoong; Kim, Sunjoo

2017-03-24

Although Shewanella algae has been known to have weak pathogenicity, case reports on infections with this species have been steadily increasing. S. algae and S. haliotis are difficult to distinguish from each other with conventional phenotypic methods. We reviewed the microbiological and clinical features of S. algae and S. haliotis infections at our institute. Bacterial culture and identification reports from patient samples from 2010 to 2014 were reviewed to screen the cases of Shewanella infections. In addition to conventional biochemical tests, 16S rRNA gene sequence analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were performed for 19 stored bacterial isolates. Medical records were reviewed for clinical characteristics and laboratory findings. All isolates were identified as S. algae by using VITEK 2. MALDI-TOF also identified all isolates as S. algae with a 99.9 confidence value. In contrast, 16S rRNA analysis identified 10 isolates as S. algae and 9 isolates as S. haliotis. Both S. algae (60%) and S. haliotis (77%) infections were strongly associated with diseases of the hepatobiliary tract and pancreas. To distinguish between S. algae and S. haliotis, 16S rRNA gene sequence analysis seems more accurate than biochemical tests or MALDI-TOF. Patients with underlying diseases in the hepatobiliary tract and pancreas seem to be susceptible to these marine pathogens.

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

PubMed

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.

Dehydrogenation and dehalogenation of amines in MALDI-TOF MS investigated by isotopic labeling.

PubMed

Kang, Chuanqing; Zhou, Yihan; Du, Zhijun; Bian, Zheng; Wang, Jianwei; Qiu, Xuepeng; Gao, Lianxun; Sun, Yuequan

2013-12-01

Secondary and tertiary amines have been reported to form [M-H](+) that correspond to dehydrogenation in matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In this investigation, we studied the dehydrogenation of amines in MALDI-TOF MS by isotopic labeling. Aliphatic amines were labeled with deuterium on the methylene of an N-benzyl group, which resulted in the formation of [M-D](+) and [M-H](+) ions by dedeuteration and dehydrogenation, respectively. This method revealed the proton that was removed. The spectra of most tertiary amines with an N-benzyl group showed high-intensity [M-D](+) and [M-H](+) ion peaks, whereas those of secondary amines showed low-intensity ion peaks. Ratios between the peak intensities of [M-D](+) and [M-H](+) greater than 1 suggested chemoselective dehydrogenation at the N-benzyl groups. The presence of an electron donor group on the N-benzyl groups enhanced the selectivity. The dehalogenation of amines with an N-(4-halobenzyl) group was also observed alongside dehydrogenation. The amino ions from dehalogenation can undergo second dehydrogenation. These results provide the first direct evidence about the position at which dehydrogenation of an amine occurs and the first example of dehalogenation of haloaromatic compounds in MALDI-TOF MS. These results should be helpful in the structural identification and elucidation of synthetic and natural molecules. Copyright © 2013 John Wiley & Sons, Ltd.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry rapid detection of carbapenamase activity in Acinetobacter baumannii isolates.

PubMed

Abouseada, Noha; Raouf, May; El-Attar, Eman; Moez, Pacinte

2017-01-01

Carbapenamase-producing Acinetobacter baumannii are an increasing threat in hospitals and Intensive Care Units. Accurate and rapid detection of carbapenamase producers has a great impact on patient improvement and aids in implementation of infection control measures. In this study, we describe the use of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF MS) to identify carbapenamase-producing A. baumannii isolates in up to 3 h. Isolates and Methods: A total of 50 A. baumannii isolates (of which 39 were carabapenamase producers) were tested using MALDI TOF MS. Isolates were incubated for 3 h with 0.25 mg/ml up to 2 mg/ml of imipenem (IMP) at 37°C. Supernatants were analysed by MALDI TOF to analyse peaks corresponding to IMP (300 Da) and an IMP metabolite (254 Da) using UltrafleXtreme (Bruker Daltonics, Bremen, Germany). All carbapenamase-producing isolates were evidenced by the disappearance or reduction in intensity of the 300 Da peak of IPM and the appearance of a 254 Da peak of the IPM metabolite. In isolates that did not produce carbapenamase, the IPM 300 Da peak remained intact. MALDI TOF is a promising tool in the field of diagnostic microbiology that has the ability to transfer identification and antimicrobial susceptibility testing time from days to hours.

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

PubMed

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

2011-07-27

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

Humic acids as both matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and adsorbent for magnetic solid phase extraction.

PubMed

Zhao, Qin; Xu, Jing; Yin, Jia; Feng, Yu-Qi

2015-08-19

In the present study, humic acids (HAs) were applied as both a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and an adsorbent of magnetic solid phase extraction (MSPE) for the first time. As natural macromolecule compounds, HAs are inherently highly functionalized and contain laser energy absorbing-transferring aromatic structures. This special molecular structure made HAs a good candidate for use as a MALDI matrix in small molecule analysis. At the same time, due to its good adsorption ability, HAs was prepared as MSPE adsorbent via a simple co-mixing method, in which the commercially available HAs were directly mixed with Fe3O4 magnetic nanoparticles (MNPs) in a mortar and grinded evenly and completely. In this process, MNPs were physically wrapped and adhered to tiny HAs leading to the formation of magnetic HAs (MHAs). To verify the bi-function of the MHAs, Rhodamine B (RdB) was chosen as model compound. Our results show that the combination of MHAs-based MSPE and MALDI-TOF-MS can provide a rapid and sensitive method for the determination of RdB in chili oil. The whole analytical procedure could be completed within 30 min for simultaneous determination of more than 20 samples, and the limit of quantitation for RdB was found to be 0.02 μg/g. The recoveries in chili oil were in the range 73.8-81.5% with the RSDs less than 21.3% (intraday) and 20.3% (interday). The proposed strategy has potential applications for high-throughput analysis of small molecules in complex samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris

PubMed Central

Jiménez, Juan J.; Gútiez, Loreto; Cintas, Luis M.; Herranz, Carmen; Hernández, Pablo E.

2015-01-01

We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins. PMID:25821820

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

PubMed

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 < 0.001; the samples included in the calculation for which both option given result). Duration of the 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 the identification of blood cultures that may contribute as much as possible early appointment effective regimes of starting antibiotic therapy.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vendrell, Iolanda; CIBER Epidemiologia y Salud Publica; Carrascal, Montserrat

Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 +- 199, 345 +- 47, and 243 +- 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cellmore » protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 +- 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.« less

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

PubMed

Burillo, Almudena; Rodríguez-Sánchez, Belén; Ramiro, Ana; Cercenado, Emilia; Rodríguez-Créixems, Marta; Bouza, Emilio

2014-01-01

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 earlier withdrawal of unnecessary antibiotics.

A laser desorption ionization/matrix-assisted laser desorption ionization target system applicable for three distinct types of instruments (LinTOF/curved field RTOF, LinTOF/RTOF and QqRTOF) with different performance characteristics from three vendors.

PubMed

Rados, Edita; Pittenauer, Ernst; Frank, Johannes; Varmuza, Kurt; Allmaier, Günter

2018-04-30

We have developed a target system which enables the use of only one target (i.e. target preparation set) for three different laser desorption ionization (LDI)/matrix-assisted laser desorption ionization (MALDI) mass spectrometric instruments. The focus was on analysing small biomolecules with LDI for future use of the system for the study of meteorite samples (carbonaceous chondrites) using devices with different mass spectrometric performance characteristics. Three compounds were selected due to their potential presence in meteoritic chondrites: tryptophan, 2-deoxy-d-ribose and triphenylene. They were prepared (with and without MALDI matrix, i.e. MALDI and LDI) and analysed with three different mass spectrometers (LinTOF/curved field RTOF, LinTOF/RTOF and QqRTOF). The ion sources of two of the instruments were run at high vacuum, and one at intermediate pressure. Two devices used a laser wavelength of 355 nm and one a wavelength of 337 nm. The developed target system operated smoothly with all devices. Tryptophan, 2-deoxy-d-ribose and triphenylene showed similar desorption/ionization behaviour for all instruments using the LDI mode. Interestingly, protonated tryptophan could be observed only with the LinTOF/curved field RTOF device in LDI and MALDI mode, while sodiated molecules were observed with all three instruments (in both ion modes). Deprotonated tryptophan was almost completely obscured by matrix ions in the MALDI mode whereas LDI yielded abundant deprotonated molecules. The presented target system allowed successful analyses of the three compounds using instruments from different vendors with only one preparation showing different analyser performance characteristics. The elemental composition with the QqRTOF analyser and the high-energy 20 keV collision-induced dissociation fragmentation will be important in identifying unknown compounds in chondrites. © 2018 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Cost Analysis of Implementing Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Plus Real-Time Antimicrobial Stewardship Intervention for Bloodstream Infections

PubMed Central

Patel, Twisha S.; Kaakeh, Rola; Nagel, Jerod L.; Newton, Duane W.

2016-01-01

ABSTRACT Studies evaluating rapid diagnostic testing plus stewardship intervention have consistently demonstrated improved clinical outcomes for patients with bloodstream infections. However, the cost of implementing new rapid diagnostic testing can be significant, and such testing usually does not generate additional revenue. There are minimal data evaluating the impact of adding matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for rapid organism identification and dedicating pharmacy stewardship personnel time on the total hospital costs. A cost analysis was performed utilizing patient data generated from the hospital cost accounting system and included additional costs of MALDI-TOF equipment, supplies and personnel, and dedicated pharmacist time for blood culture review and of making interventions to antimicrobial therapy. The cost analysis was performed from a hospital perspective for 3-month blocks before and after implementation of MALDI-TOF plus stewardship intervention. A total of 480 patients with bloodstream infections were included in the analysis: 247 in the preintervention group and 233 in the intervention group. Thirty-day mortality was significantly improved in the intervention group (12% versus 21%, P < 0.01), and the mean length of stay was reduced, although the difference was not statistically significant (13.0 ± 16.5 days versus 14.2 ± 16.7 days, P = 0.44). The total hospital cost per bloodstream infection was lower in the intervention group ($42,580 versus $45,019). Intensive care unit cost per bloodstream infection accounted for the largest share of the total costs in each group and was also lower in the intervention group ($10,833 versus $13,727). Implementing MALDI-TOF plus stewardship review and intervention decreased mortality for patients with bloodstream infections. Despite the additional costs of implementing MALDI-TOF and of dedicating pharmacy stewardship personnel time to interventions, the total hospital costs decreased by $2,439 per bloodstream infection, for an approximate annual cost savings of $2.34 million. PMID:27795335

Microorganisms direct identification from blood culture by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Ferreira, L; Sánchez-Juanes, F; Porras-Guerra, I; García-García, M I; García-Sánchez, J E; González-Buitrago, J M; Muñoz-Bellido, J L

2011-04-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows a fast and reliable bacterial identification from culture plates. Direct analysis of clinical samples may increase its usefulness in samples in which a fast identification of microorganisms can guide empirical treatment, such as blood cultures (BC). Three hundred and thirty BC, reported as positive by the automated BC incubation device, were processed by conventional methods for BC processing, and by a fast method based on direct MALDI-TOF MS. Three hundred and eighteen of them yield growth on culture plates, and 12 were false positive. The MALDI-TOF MS-based method reported that no peaks were found, or the absence of a reliable identification profile, in all these false positive BC. No mixed cultures were found. Among these 318 BC, we isolated 61 Gram-negatives (GN), 239 Gram-positives (GP) and 18 fungi. Microorganism identifications in GN were coincident with conventional identification, at the species level, in 83.3% of BC and, at the genus level, in 96.6%. In GP, identifications were coincident with conventional identification in 31.8% of BC at the species level, and in 64.8% at the genus level. Fungaemia was not reliably detected by MALDI-TOF. In 18 BC positive for Candida species (eight C. albicans, nine C. parapsilosis and one C. tropicalis), no microorganisms were identified at the species level, and only one (5.6%) was detected at the genus level. The results of the present study show that this fast, MALDI-TOF MS-based method allows bacterial identification directly from presumptively positive BC in a short time (<30 min), with a high accuracy, especially when GN bacteria are involved. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

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

PubMed Central

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%) (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. PMID:26554930

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of molds of the Fusarium genus.

PubMed

Triest, David; Stubbe, Dirk; De Cremer, Koen; Piérard, Denis; Normand, Anne-Cécile; Piarroux, Renaud; Detandt, Monique; Hendrickx, Marijke

2015-02-01

The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity. Copyright © 2015, American Society for Microbiology. 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.

PubMed

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%) (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.

Advantage of MALDI-TOF-MS over biochemical-based phenotyping for microbial identification illustrated on industrial applications.

PubMed

Urwyler, S K; Glaubitz, J

2016-02-01

Fast microbial identification is becoming increasingly necessary in industry to improve microbial control and reduce biocide consumption. We compared the performances of two systems based on MALDI-TOF MS (VITEK MS and BIOTYPER) and two based on biochemical testing (BIOLOG, VITEK 2 Compact) with genetic methods for the identification of environmental bacteria. At genus level both MALDI-TOF MS-based systems showed the lowest number of false (4%) and approx. 60% correct identifications. In contrast, the biochemical-based systems assigned 25% of the genera incorrectly. The differences were even more apparent at the species level. The BIOTYPER was most conservative, where assigning a species led to the lowest percentage of species identifications (54%) but also to the least wrong assignments (4%). The other three systems showed higher levels of false assignments: 8·7, 40 and 46% respectively. The genus identification performance on four industrial products of the BIOTYPER could be increased up to 94·3% (average 88% of 167 isolates) by evolving the database in a product specific manner. Comparison of the bacterial population in the example of paints, and raw materials used therein, at different production steps demonstrated unequivocally that the contamination of the final paint product originated not from the main raw material. MALDI-TOF-MS has revolutionized speed and precision of microbial identification for clinical isolates outperforming conventional methods. In contrast, few performance studies have been published so far focusing on suitability for particularly industrial applications, geomicrobiology and environmental analytics. This study evaluates the performance of this proteomic phenotyping on such industrial isolates in comparison with biochemical-based phenotyping and genotyping. Further the study exemplifies the power of MALDI-TOF-MS to trace cost-efficiently the dominating cultivable bacterial species throughout an industrial paint production process. Vital information can be retrieved to identify the most crucial contaminating source for the final product. © 2015 The Authors published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.

Identification of Bacteria Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

ERIC Educational Resources Information Center

Kedney, Mollie G.; Strunk, Kevin B.; Giaquinto, Lisa M.; Wagner, Jennifer A.; Pollack, Sidney; Patton, Walter A.

2007-01-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS or simply MALDI) has become ubiquitous in the identification and analysis of biomacromolecules. As a technique that allows for the molecular weight determination of otherwise nonvolatile molecules, MALDI has had a profound impact in the molecular…

A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen

DTIC Science & Technology

2014-08-01

Electrochemical Oxidation of Catechol and Para - Aminophenol Esters in the Presence of Hydrolases. Bioelectrochem. Bioenerg. 1980, 7, 11–24. 26. Evans-Nguyen, K. M...platform. Analytical HPLC (a) and MALDI-TOF (b) traces of biligand capture agentwithno thermal treatment, and after 5 days of storage as a powder at...sample of biligand was stored for 5 days at 65 C under nitrogen atmosphere. Analy- tical HPLC traces (Figure 4a) andMALDI-TOF (Figure 4b) reveal

Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Caspofungin Susceptibility Testing of Candida and Aspergillus Species

PubMed Central

De Carolis, Elena; Vella, Antonietta; Florio, Ada R.; Posteraro, Patrizia; Perlin, David S.; Posteraro, Brunella

2012-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility. PMID:22535984

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for caspofungin susceptibility testing of Candida and Aspergillus species.

PubMed

De Carolis, Elena; Vella, Antonietta; Florio, Ada R; Posteraro, Patrizia; Perlin, David S; Sanguinetti, Maurizio; Posteraro, Brunella

2012-07-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility.

Identification of the Corn Pathogen Pantoea stewartii by Mass Spectrometry of Whole-Cell Extracts and Its Detection with Novel PCR Primers ▿

PubMed Central

Wensing, Annette; Zimmermann, Stefan; Geider, Klaus

2010-01-01

Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn (Zea mays). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans, Pantoea dispersa, and Pantoea ananatis. Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA. PMID:20656863

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

NASA Astrophysics Data System (ADS)

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.

High-energy Collision-induced Dissociation by MALDI TOF/TOF Causes Charge-Remote Fragmentation of Steroid Sulfates

PubMed Central

Yan, Yuetian; Ubukata, Masaaki; Cody, Robert B.; Holy, Timothy E.; Gross, Michael L.

2014-01-01

A method for structural elucidation of biomolecules dating to the 1980s utilized high-energy collisions (~10 keV, laboratory frame) that induced charge-remote fragmentations (CRF), a class of fragmentations particularly informative for lipids, steroids, surfactants, and peptides. Unfortunately, the capability for high-energy activation has largely disappeared with the demise of magnetic sector instruments. With the latest designs of tandem time-of-flight mass spectrometers (TOF/TOF), however, this capability is now being restored to coincide with the renewed interest in metabolites and lipids including steroid-sulfates and other steroid metabolites. For these metabolites, structure determinations are required at concentration levels below that appropriate for NMR. To meet this need, we explored CRF with TOF/TOF mass spectrometry for two groups of steroid sulfates, 3-sulfates and 21-sulfates. We demonstrated that the current generation of MALDI TOF/TOF instruments can generate charge-remote-fragmentations for these materials. The resulting collision-induced dissociation (CID) spectra are useful for positional isomer differentiation and very often allow the complete structure determination of the steroid. We also propose a new nomenclature that directly indicates the cleavage sites on the steroid ring with carbon numbers. PMID:24781458

High-energy collision-induced dissociation by MALDI TOF/TOF causes charge-remote fragmentation of steroid sulfates.

PubMed

Yan, Yuetian; Ubukata, Masaaki; Cody, Robert B; Holy, Timothy E; Gross, Michael L

2014-08-01

A method for structural elucidation of biomolecules dating to the 1980s utilized high-energy collisions (~10 keV, laboratory frame) that induced charge-remote fragmentations (CRF), a class of fragmentations particularly informative for lipids, steroids, surfactants, and peptides. Unfortunately, the capability for high-energy activation has largely disappeared with the demise of magnetic sector instruments. With the latest designs of tandem time-of-flight mass spectrometers (TOF/TOF), however, this capability is now being restored to coincide with the renewed interest in metabolites and lipids, including steroid-sulfates and other steroid metabolites. For these metabolites, structure determinations are required at concentration levels below that appropriate for NMR. To meet this need, we explored CRF with TOF/TOF mass spectrometry for two groups of steroid sulfates, 3-sulfates and 21-sulfates. We demonstrated that the current generation of MALDI TOF/TOF instruments can generate charge-remote fragmentations for these materials. The resulting collision-induced dissociation (CID) spectra are useful for positional isomer differentiation and very often allow the complete structure determination of the steroid. We also propose a new nomenclature that directly indicates the cleavage sites on the steroid ring with carbon numbers.

MALDI-TOF mass spectrometry as a tool for differentiation of Bradyrhizobium species: application to the identification of Lupinus nodulating strains.

PubMed

Sánchez-Juanes, Fernando; Ferreira, Laura; Alonso de la Vega, Pablo; Valverde, Angel; Barrios, Milagros León; Rivas, Raúl; Mateos, Pedro F; Martínez-Molina, Eustoquio; González-Buitrago, José Manuel; Trujillo, Martha E; Velázquez, Encarna

2013-12-01

Genus Bradyrhizobium includes slow growing bacteria able to nodulate different legumes as well as species isolated from plant tumours. The slow growth presented by the members of this genus and the phylogenetic closeness of most of its species difficults their identification. In the present work we applied for the first time Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to the analysis of Bradyrhizobium species after the extension of MALDI Biotyper 2.0 database with the currently valid species of this genus. With this methodology it was possible to identify strains belonging to phylogenetically closely related species of genus Bradyrhizobium allowing the discrimination among species with rrs gene identities higher than 99%. The application of MALDI-TOF MS to strains isolated from nodules of different Lupinus species in diverse geographical locations allowed their correct identification when comparing with the results of rrs gene and ITS analyses. The nodulation of Lupinus gredensis, an endemic species of the west of Spain, by B. canariense supports the European origin of this species. Copyright © 2013. Published by Elsevier GmbH.

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

PubMed Central

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

Fast detection of Piscirickettsia salmonis in Salmo salar serum through MALDI-TOF-MS profiling.

PubMed

Olate, Verónica R; Nachtigall, Fabiane M; Santos, Leonardo S; Soto, Alex; Araya, Macarena; Oyanedel, Sandra; Díaz, Verónica; Marchant, Vanessa; Rios-Momberg, Mauricio

2016-03-01

Piscirickettsia salmonis is a pathogenic bacteria known as the aetiological agent of the salmonid rickettsial syndrome and causes a high mortality in farmed salmonid fishes. Detection of P. salmonis in farmed fishes is based mainly on molecular biology and immunohistochemistry techniques. These techniques are in most of the cases expensive and time consuming. In the search of new alternatives to detect the presence of P. salmonis in salmonid fishes, this work proposed the use of MALDI-TOF-MS to compare serum protein profiles from Salmo salar fish, including experimentally infected and non-infected fishes using principal component analysis (PCA). Samples were obtained from a controlled bioassay where S. salar was challenged with P. salmonis in a cohabitation model and classified according to the presence or absence of the bacteria by real time PCR analysis. MALDI spectra of the fish serum samples showed differences in its serum protein composition. These differences were corroborated with PCA analysis. The results demonstrated that the use of both MALDI-TOF-MS and PCA represents a useful tool to discriminate the fish status through the analysis of salmonid serum samples. Copyright © 2016 John Wiley & Sons, Ltd.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry for comprehensive indexing of East African ixodid tick species.

PubMed

Rothen, Julian; Githaka, Naftaly; Kanduma, Esther G; Olds, Cassandra; Pflüger, Valentin; Mwaura, Stephen; Bishop, Richard P; Daubenberger, Claudia

2016-03-15

The tick population of Africa includes several important genera belonging to the family Ixodidae. Many of these ticks are vectors of protozoan and rickettsial pathogens including Theileria parva that causes East Coast fever, a debilitating cattle disease endemic to eastern, central and southern Africa. Effective surveillance of tick-borne pathogens depends on accurate identification and mapping of their tick vectors. A simple and reproducible technique for rapid and reliable differentiation of large numbers of closely related field-collected ticks, which are often difficult and tedious to discriminate purely by morphology, will be an essential component of this strategy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is increasingly becoming a useful tool in arthropod identification and has the potential to overcome the limitations of classical morphology-based species identification. In this study, we applied MALDI-TOF MS to a collection of laboratory and field ticks found in Eastern Africa. The objective was to determine the utility of this proteomic tool for reliable species identification of closely related afrotropical ticks. A total of 398 ixodid ticks from laboratory maintained colonies, extracted from the hides of animals or systematically collected from vegetation in Kenya, Sudan and Zimbabwe were analyzed in the present investigation. The cytochrome c oxidase I (COI) genes from 33 specimens were sequenced to confirm the tentatively assigned specimen taxa identity on the basis of morphological analyses. Subsequently, the legs of ticks were homogenized and analyzed by MALDI-TOF MS. A collection of reference mass spectra, based on the mass profiles of four individual ticks per species, was developed and deposited in the spectral database SARAMIS™. The ability of these superspectra (SSp.) to identify and reliably validate a set of ticks was demonstrated using the remaining individual 333 ticks. Ultimately, ten different tick species within the genera Amblyomma, Hyalomma, Rhipicephalus and Rhipicephalus (Boophilus) based on molecular COI typing and morphology were included into the study analysis. The robustness of the 12 distinct SSp. developed here proved to be very high, with 319 out of 333 ticks used for validation identified correctly at species level. Moreover, these novel SSp. allowed for diagnostic specificity of 99.7 %. The failure of species identification for 14 ticks was directly linked to low quality mass spectra, most likely due to poor specimen quality that was received in the laboratory before sample preparation. Our results are consistent with earlier studies demonstrating the potential of MALDI-TOF MS as a reliable tool for differentiating ticks originating from the field, especially females that are difficult to identify after blood feeding. This work provides further evidence of the utility of MALDI-TOF MS to identify morphologically and genetically highly similar tick species and indicates the potential of this tool for large-scale monitoring of tick populations, species distributions and host preferences.

Bacterial rapid identification with matrix assisted laser desorption/ionization time-of-flight mass spectrometry: development of an 'in-house method' and comparison with Bruker Sepsityper(®) kit.

PubMed

Frédéric Ric, S; Antoine, M; Bodson, A; Lissoir, B

2015-10-01

The objective of this study was to compare an in-house matrix-assisted laser desorption ionization with time of flight (MALDI-TOF) method and a commercial MALDI-TOF kit (Sepsityper(®) kit) for direct bacterial identification in positive blood cultures. We also evaluated the time saved and the cost associated with the rapid identification techniques. We used the BACTEC(®) automated system for detecting positive blood cultures. Direct identification using Sepsityper kit and the in-house method were compared with conventional identification by MALDI-TOF using pure bacterial culture on the solid phase. We also evaluated different cut-off scores for rapid bacterial identification. In total, 127 positive blood vials were selected. The rate of rapid identification with the MALDI Sepsityper kit was 25.2% with the standard cut-off and 33.9% with the enlarged cut-off, while the results for the in-house method were 44.1 and 61.4%, respectively. Error rates with the enlarged cut-off were 6.98 (n = 3) and 2.56% (n = 2) for Sepsityper and the in-house method, respectively. Identification rates were higher for gram-negative bacteria. Direct bacterial identification succeeded in supplying rapid identification of the causative organism in cases of sepsis. The time taken to obtain a result was nearly 24  hours shorter for the direct bacterial identification methods than for conventional MALDI-TOF on solid phase culture. Compared with the Sepsityper kit, the in-house method offered better results and fewer errors, was more cost-effective and easier to use.

Direct screening of herbal blends for new synthetic cannabinoids by MALDI-TOF MS.

PubMed

Gottardo, Rossella; Chiarini, Anna; Dal Prà, Ilaria; Seri, Catia; Rimondo, Claudia; Serpelloni, Giovanni; Armato, Ubaldo; Tagliaro, Franco

2012-01-01

Since 2004, a number of herbal blends containing different synthetic compounds mimicking the pharmacological activity of cannabinoids and displaying a high toxicological potential have appeared in the market. Their availability is mainly based on the so-called "e-commerce", being sold as legal alternatives to cannabis and cannabis derivatives. Although highly selective, sensitive, accurate, and quantitative methods based on GC-MS and LC-MS are available, they lack simplicity, rapidity, versatility and throughput, which are required for product monitoring. In this context, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers a simple and rapid operation with high throughput. Thus, the aim of the present work was to develop a MALDI-TOF MS method for the rapid qualitative direct analysis of herbal blend preparations for synthetic cannabinoids to be used as front screening of confiscated clandestine preparations. The sample preparation was limited to herbal blend leaves finely grinding in a mortar and loading onto the MALDI plate followed by addition of 2 µl of the matrix/surfactant mixture [α-cyano-4-hydroxy-cinnamic acid/cetyltrimethylammonium bromide (CTAB)]. After drying, the sample plate was introduced into the ion source for analysis. MALDI-TOF conditions were as follows: mass spectra were analyzed in the range m/z 150-550 by averaging the data from 50 laser shots and using an accelerating voltage of 20 kV. The described method was successfully applied to the screening of 31 commercial herbal blends, previously analyzed by GC-MS. Among the samples analyzed, 21 contained synthetic cannabinoids (namely JWH-018, JWH-073, JWH-081, JWH-250, JWH-210, JWH-019, and AM-694). All the results were in agreement with GC-MS, which was used as the reference technique. Copyright © 2012 John Wiley & Sons, Ltd.

Chemical analysis of pharmaceuticals and explosives in fingermarks using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry.

PubMed

Kaplan-Sandquist, Kimberly; LeBeau, Marc A; Miller, Mark L

2014-02-01

Chemical analysis of latent fingermarks, "touch chemistry," has the potential of providing intelligence or forensically relevant information. Matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS) was used as an analytical platform for obtaining mass spectra and chemical images of target drugs and explosives in fingermark residues following conventional fingerprint development methods and MALDI matrix processing. There were two main purposes of this research: (1) develop effective laboratory methods for detecting drugs and explosives in fingermark residues and (2) determine the feasibility of detecting drugs and explosives after casual contact with pills, powders, and residues. Further, synthetic latent print reference pads were evaluated as mimics of natural fingermark residue to determine if the pads could be used for method development and quality control. The results suggest that artificial amino acid and sebaceous oil residue pads are not suitable to adequately simulate natural fingermark chemistry for MALDI/TOF MS analysis. However, the pads were useful for designing experiments and setting instrumental parameters. Based on the natural fingermark residue experiments, handling whole or broken pills did not transfer sufficient quantities of drugs to allow for definitive detection. Transferring drugs or explosives in the form of powders and residues was successful for preparing analytes for detection after contact with fingers and deposition of fingermark residue. One downfall to handling powders was that the analyte particles were easily spread beyond the original fingermark during development. Analyte particles were confined in the original fingermark when using transfer residues. The MALDI/TOF MS was able to detect procaine, pseudoephedrine, TNT, and RDX from contact residue under laboratory conditions with the integration of conventional fingerprint development methods and MALDI matrix. MALDI/TOF MS is a nondestructive technique which provides chemical information in both the mass spectra and chemical images. Published by Elsevier Ireland Ltd.

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.

PubMed

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.

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

PubMed

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 healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples. Copyright 2009 Elsevier B.V. All rights reserved.

A nano-patterned self assembled monolayer (SAM) rutile titania cancer chip for rapid, low cost, highly sensitive, direct cancer analysis in MALDI-MS.

PubMed

Manikandan, M; Gopal, Judy; Hasan, Nazim; Wu, Hui-Fen

2014-12-01

We developed a cancer chip by nano-patterning a highly sensitive SAM titanium surface capable of capturing and sensing concentrations as low as 10 cancer cells/mL from the environment by Matrix Assisted Laser Desorption and Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). The current approach evades any form of pretreatment and sample preparation processes; it is time saving and does not require the (expensive) conventional MALDI target plate. The home made aluminium (Al) target holder cost, on which we loaded the cancer chips for MALDI-TOF MS analysis, is about 60 USD. While the conventional stainless steel MALDI target plate is more than 700 USD. The SAM surface was an effective platform leading to on-chip direct MALDI-MS detection of cancer cells. We compared the functionality of this chip with the unmodified titanium surfaces and thermally oxidized (TO) titanium surfaces. The lowest detectable concentration of the TO chip was 10(3) cells/mL, while the lowest detectable concentration of the control or unmodified titanium chips was 10(6) cells/mL. Compared to the control surface, the SAM cancer chip showed 100,000 times of enhanced sensitivity and compared with the TO chip, 1000 times of increased sensitivity. The high sensitivity of the SAM surfaces is attributed to the presence of the rutile SAM, surface roughness and surface wettability as confirmed by AFM, XRD, contact angle microscope and FE-SEM. This study opens a new avenue for the potent application of the SAM cancer chip for direct cancer diagnosis by MALDI-TOF MS in the near future. Copyright © 2014. Published by Elsevier B.V.

Analysis of the endogenous peptide profile of milk: identification of 248 mainly casein-derived peptides.

PubMed

Baum, Florian; Fedorova, Maria; Ebner, Jennifer; Hoffmann, Ralf; Pischetsrieder, Monika

2013-12-06

Milk is an excellent source of bioactive peptides. However, the composition of the native milk peptidome has only been partially elucidated. The present study applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) directly or after prefractionation of the milk peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) or OFFGEL fractionation for the comprehensive analysis of the peptide profile of raw milk. The peptide sequences were determined by MALDI-TOF/TOF or nano-ultra-performance liquid chromatography-nanoelectrospray ionization-LTQ-Orbitrap-MS. Direct MALDI-TOF-MS analysis led to the assignment of 57 peptides. Prefractionation by both complementary methods led to the assignment of another 191 peptides. Most peptides originate from α(S1)-casein, followed by β-casein, and α(S2)-casein. κ-Casein and whey proteins seem to play only a minor role as peptide precursors. The formation of many, but not all, peptides could be explained by the activity of the endogenous peptidases, plasmin or cathepsin D, B, and G. Database searches revealed the presence of 22 peptides with established physiological function, including those with angiotensin-converting-enzyme (ACE) inhibitory, immunomodulating, or antimicrobial activity.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

PubMed Central

Zhang, Lin; Vranckx, Katleen; Janssens, Koen; Sandrin, Todd R.

2015-01-01

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level. PMID:25590854

Use of MALDI-TOF mass spectrometry and a custom database to characterize bacteria indigenous to a unique cave environment (Kartchner Caverns, AZ, USA).

PubMed

Zhang, Lin; Vranckx, Katleen; Janssens, Koen; Sandrin, Todd R

2015-01-02

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Analysis of benzylpenicillin in milk using MALDI-TOF mass spectrometry with top-down synthesized TiO2 nanowires as the solid matrix.

PubMed

Kim, Jo-Il; Park, Jong-Min; Noh, Joo-Yoon; Hwang, Seong-Ju; Kang, Min-Jung; Pyun, Jae-Chul

2016-01-01

In this work, the wet-corrosion process for the synthesis of titanium oxide (TiO2) nanowires in the anatase phase was optimized as the solid matrix in MALDI-TOF mass spectrometry, and the solid matrix of the TiO2 nanowires was applied to the detection of antibiotics in a daily milk sample. The influence of the alkali concentration and the heat treatment temperature on the crystal structure of the TiO2 nanowires was investigated. The ionization activity of the TiO2 nanowires was estimated for each synthetic condition using amino acids as model analytes with low molecular weights. For the detection of antibiotics in milk, benzylpenicillin was spiked in daily milk samples, and MALDI-TOF mass spectrometry with the TiO2 nanowires was demonstrated to detect the benzylpenicillin at the cut-off concentration of the EU directive. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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.

A Sol-gel Integrated Dual-readout Microarray Platform for Quantification and Identification of Prostate-specific Antigen.

PubMed

Lee, SangWook; Lee, Jong Hyun; Kwon, Hyuck Gi; Laurell, Thomas; Jeong, Ok Chan; Kim, Soyoun

2018-01-01

Here, we report a sol-gel integrated affinity microarray for on-chip matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that enables capture and identification of prostate?specific antigen (PSA) in samples. An anti-PSA antibody (H117) was mixed with a sol?gel, and the mixture was spotted onto a porous silicon (pSi) surface without additional surface modifications. The antibody easily penetrates the sol-gel macropore fluidic network structure, making possible high affinities. To assess the capture affinity of the platform, we performed a direct assay using fluorescein isothiocyanate-labeled PSA. Pure PSA was subjected to on-chip MALDI-TOF-MS analysis, yielding three clear mass peptide peaks (m/z = 1272, 1407, and 1872). The sol-gel microarray platform enables dual readout of PSA both fluorometric and MALDI-TOF MS analysis in biological samples. Here we report a useful method for a means for discovery of biomarkers in complex body fluids.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applied to virus identification

PubMed Central

Calderaro, Adriana; Arcangeletti, Maria-Cristina; Rodighiero, Isabella; Buttrini, Mirko; Gorrini, Chiara; Motta, Federica; Germini, Diego; Medici, Maria-Cristina; Chezzi, Carlo; De Conto, Flora

2014-01-01

Virus detection and/or identification traditionally rely on methods based on cell culture, electron microscopy and antigen or nucleic acid detection. These techniques are good, but often expensive and/or time-consuming; furthermore, they not always lead to virus identification at the species and/or type level. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was tested as an innovative tool to identify human polioviruses and to identify specific viral protein biomarkers in infected cells. The results revealed MALDI-TOF MS to be an effective and inexpensive tool for the identification of the three poliovirus serotypes. The method was firstly applied to Sabin reference strains, and then to isolates from different clinical samples, highlighting its value as a time-saving, sensitive and specific technique when compared to the gold standard neutralization assay and casting new light on its possible application to virus detection and/or identification. PMID:25354905

N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation.

PubMed

Balog, Crina I A; Stavenhagen, Kathrin; Fung, Wesley L J; Koeleman, Carolien A; McDonnell, Liam A; Verhoeven, Aswin; Mesker, Wilma E; Tollenaar, Rob A E M; Deelder, André M; Wuhrer, Manfred

2012-09-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ~1 million cases and an annual mortality rate of ~655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers.

N-glycosylation of Colorectal Cancer Tissues

PubMed Central

Balog, Crina I. A.; Stavenhagen, Kathrin; Fung, Wesley L. J.; Koeleman, Carolien A.; McDonnell, Liam A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

2012-01-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ∼1 million cases and an annual mortality rate of ∼655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers. PMID:22573871

Illustrating the Concepts of Isotopes and Mass Spectrometry in Introductory Courses: A MALDI-TOF Mass Spectrometry Laboratory Experiment

ERIC Educational Resources Information Center

Dopke, Nancy Carter; Lovett, Timothy Neal

2007-01-01

Mass spectrometry is a widely used and versatile tool for scientists in many different fields. Soft ionization techniques such as matrix-assisted laser desorption/ionization (MALDI) allow for the analysis of biomolecules, polymers, and clusters. This article describes a MALDI mass spectrometry experiment designed for students in introductory…

Profiling N-glycans of the egg jelly coat of the sea urchin Paracentrotus lividus by MALDI-TOF mass spectrometry and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectrometry systems.

PubMed

Şahar, Umut; Deveci, Remziye

2017-05-01

Sea urchin eggs are surrounded by a carbohydrate-rich layer, termed the jelly coat, that consists of polysaccharides and glycoproteins. In the present study, we describe two mass spectrometric strategies to characterize the N-glycosylation of the Paracentrotus lividus egg jelly coat, which has an alecithal-type extracellular matrix like mammalian eggs. Egg jelly was isolated, lyophilized, and dialyzed, followed by peptide N-glycosidase F (PNGase-F) treatment to release N-glycans from their protein chain. These N-glycans were then derivatized by permethylation reaction, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and capillary liquid chromatography electrospray ionization-ion trap tandem mass spectroscopy (CapLC ESI-Ion trap-MS/MS). N-glycans in the egg jelly coat glycoproteins were indicated by sodiated molecules at m/z 1579.8, 1783.9, 1988.0, 2192.0, and 2397.1 for permethylated oligosaccharides on MALDI-TOF MS. Fragmentation and structural characterization of these oligosaccharides were performed by ESI-Ion trap MS/MS. Then, MALDI-TOF-MS and ESI-Ion trap-MS/MS spectra were interpreted using the GlycoWorkbench software suite, a tool for building, displaying, and profiling glycan masses, to identify the original oligosaccharide structures. The oligosaccharides of the isolated egg jelly coat were mainly of the high mannose type. © 2017 Wiley Periodicals, Inc.

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.

PubMed

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.

Detection of Staphylococcus aureus Delta-Toxin Production by Whole-Cell MALDI-TOF Mass Spectrometry

PubMed Central

Gagnaire, Julie; Dauwalder, Olivier; Boisset, Sandrine; Khau, David; Freydière, Anne-Marie; Ader, Florence; Bes, Michèle; Lina, Gerard; Tristan, Anne; Reverdy, Marie-Elisabeth; Marchand, Adrienne; Geissmann, Thomas; Benito, Yvonne; Durand, Géraldine; Charrier, Jean-Philippe; Etienne, Jerome; Welker, Martin; Van Belkum, Alex; Vandenesch, François

2012-01-01

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. PMID:22792394

Identification of coryneform Actinomyces neuii by MALDI-TOF MS: 5 case reports and review of literature.

PubMed

De Vreese, K; Verhaegen, J

2013-01-01

We describe five cases of Actinomyces neuii, isolated from different clinical specimens over a period of five months (from June to October 2011), followed by a review of literature on infections with this micro-organism. All Actinomyces neuii strains were cultured or subcultured on horse blood agar. Identification took place using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Identification was confirmed by conventional biochemical tests and API Coryne test strips (BioMérieux SA). Susceptibility testing was performed on Mueller-Hinton agar supplemented with horse blood, using E-tests (BioMérieux SA). The minimal inhibitory concentrations were determined after 24 and 48 hours of incubation in a 5% CO2 environment. Isolation of this micro-organism was associated with abscesses in two patients and chronic osteomyelitis in one patient. The remaining two patients had positive blood cultures which grew Actinomyces neuii, either as contamination or as catheter-related infection. All Actinomyces neuii identifications were obtained by MALDI-TOF MS and were confirmed by conventional biochemical and API Coryne tests. Identification of one isolate was also confirmed by 16S rRNA sequencing. All strains were susceptible to penicillin. One strain showed heteroresistance for macrolides and lincosamides. Minimal inhibitory concentrations were more reliable and easier to read after 48 hours of incubation, as compared to 24 hours. MALDI-TOF MS analysis allows rapid and reliable identification of Actinomyces neuii, even at subspecies level.

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

PubMed Central

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. PMID:27442502

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

NASA Astrophysics Data System (ADS)

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.

MALDI-TOF MS analysis of bovine and zoonotic Trichophyton verrucosum isolates reveals a distinct peak and cluster formation of a subgroup with Trichophyton benhamiae.

PubMed

Bartosch, Theresa; Heydel, Tilo; Uhrlaß, Silke; Nenoff, Pietro; Müller, Hendrik; Baums, Christoph Georg; Schrödl, Wieland

2018-07-01

The zoophilic dermatophyte Trichophyton verrucosum is the most important causative agent of bovine dermatophytosis. Additionally, it causes profound and poorly healing skin infections in humans indicating the high zoonotic potential. The objective of this study was to establish differentiation of T. verrucosum from other dermatophytes by mass spectrometry and to identify distinct features of the mass spectra. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successful for identification of this pathogen only after extension of the database of the manufacturer with spectra from T. verrucosum strains, which were identified as such by sequencing of the internal transcribed spacer (ITS) region. MALDI-TOF MS analysis was conducted with 46 field isolates from cattle, two live vaccine strains, and 10 isolates from humans identified as T. verrucosum by sequence analysis of the ITS region. The results suggest a very good agreement of both methods. Comparison with the mass spectra of 68 strains of other keratinophilic fungi revealed that most T. verrucosum wild-type isolates showed a characteristic peak at 7950-7954 m/z, which was missing in the spectra of other keratinophilic fungi and the live vaccine strains. The spectra of T. verrucosum were most similar to the spectra of T. benhamiae, an emerging zoophilic dermatophyte. In summary, MALDI-TOF MS is a powerful and reliable tool to identify T. verrucosum.

Superbasic alkyl-substituted bisphosphazene proton sponges: a new class of deprotonating matrices for negative ion matrix-assisted ionization/laser desorption mass spectrometry of low molecular weight hardly ionizable analytes.

PubMed

Calvano, C D; Cataldi, T R I; Kögel, J F; Monopoli, A; Palmisano, F; Sundermeyer, J

2016-07-30

Here hardly ionizable and low molecular weight compounds are detected in negative ion mode by using novel superbasic proton sponges based on 1,8-bisphosphazenylnaphthalene (PN) as MALDI matrices. Among the selected proton sponges, 1,8-bis(trispyrrolidinophosphazenyl)naphthalene (TPPN) has shown the best behaviour as matrix since it allows the direct detection of intact cholesterol without derivatization also in real challenging samples. Very weakly acidic compounds such as sterols, steroids, fatty alcohols and saccharides were detected in reflectron negative ion mode by a MALDI TOF/TOF system equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm) with typical mass accuracy of 10 ppm. MS/MS experiments were performed by using ambient air as the collision gas. Contrary to traditional MALDI matrices, superbasic proton sponges allowed the easy deprotonation of an alcohol functional group without a previous chemical derivatization step. Experimental evidence indicates that analyte deprotonation is achieved in the condensed phase, i.e. PN superbasic proton sponges operate according to a recently proposed model named matrix assisted ionization/laser desorption (MAILD). A detection limit of 3 pmol/spot of cholesterol (model compound) with a signal-to-noise ratio ≥ 10 was typically obtained. For the first time, the usefulness of novel superbasic proton sponges is demonstrated for MALDI detection of hardly ionizable compounds such as sterols, steroids, fatty alcohols and saccharides. The leading candidate TPPN has been successfully applied for negative ion MAILD-MS analysis of cholesterol, fatty acids and phospholipids in egg yolk and brain tissue extracts. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of prohibitin 1 as a potential prognostic biomarker in human pancreatic carcinoma using modified aqueous two-phase partition system combined with 2D-MALDI-TOF-TOF-MS/MS.

PubMed

Zhong, Ning; Cui, Yazhou; Zhou, Xiaoyan; Li, Tianliang; Han, Jinxiang

2015-02-01

Membrane proteins are an important source of potential targets for anticancer drugs or biomarkers for early diagnosis. In this study, we used a modified aqueous two-phase partition system combined with two-dimensional (2D) matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS, 2D-MALDI-TOF-TOF-MS/MS) analysis to isolate and identify membrane proteins in PANC-1 pancreatic cancer cells. Using this method, we identified 55 proteins, of which 31 (56.4 %) were membrane proteins, which, according to gene ontology annotation, are associated with various cellular processes including cell signal transduction, differentiation, and apoptosis. Immunohistochemical analysis showed that the expression level of one of the identified mitochondria membrane proteins, prohibitin 1 (PHB1), is correlated with pancreatic carcinoma differentiation; PHB1 is expressed at a higher level in normal pancreatic tissue than in well-differentiated carcinoma tissue. Further studies showed that PHB1 plays a proapoptotic role in human pancreatic cancer cells, which suggests that PHB1 has antitumorigenic properties. In conclusion, we have provided a modified method for isolating and identifying membrane proteins and demonstrated that PHB1 may be a promising biomarker for early diagnosis and therapy of pancreatic (and potentially other) cancers.

Facile MALDI-MS analysis of neutral glycans in NaOH-doped matrixes: microwave-assisted deglycosylation and one-step purification with diamond nanoparticles.

PubMed

Tzeng, Yan-Kai; Chang, Cheng-Chun; Huang, Chien-Ning; Wu, Chih-Che; Han, Chau-Chung; Chang, Huan-Cheng

2008-09-01

A streamlined protocol has been developed to accelerate, simplify, and enhance matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) of neutral underivatized glycans released from glycoproteins. It involved microwave-assisted enzymatic digestion and release of glycans, followed by rapid removal of proteins and peptides with carboxylated/oxidized diamond nanoparticles, and finally treating the analytes with NaOH before mixing them with acidic matrix (such as 2,5-dihydroxybenzoic acid) to suppress the formation of both peptide and potassiated oligosaccharide ions in MS analysis. The advantages of this protocol were demonstrated with MALDI-TOF-MS of N-linked glycans released from ovalbumin and ribonuclease B.

Understanding Coatings that Protect Plasmonic Structures for Materials Characterization and Detection and Identification of Chemical, Biological and Explosive Agents

DTIC Science & Technology

2013-04-11

in the top monomolecular layer of a blend film using mass spectrometry. This technique we call Surface Layer-Matrix Assisted Laser Desorption...C., Foster, M.D. “Probing Surface Concentration of Cyclic/linear Blend Films Using Surface MALDI-TOF Mass Spectrometry,” Dept. of Polymer Science...Isotopically Labeled Species in a Polymer Blend Using Tip Enhanced Raman Spectroscopy, ACS Macro Letters (11 2012) TOTAL: 2 Books Number of Manuscripts

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detection and identification of albumin phosphylation by organophosphorus pesticides and G- and V-type nerve agents.

PubMed

John, Harald; Breyer, Felicitas; Thumfart, Jörg Oliver; Höchstetter, Hans; Thiermann, Horst

2010-11-01

Toxic organophosphorus compounds (OPC), e.g., pesticides and nerve agents (NA), are known to phosphylate distinct endogenous proteins in vivo and in vitro. OPC adducts of butyrylcholinesterase and albumin are considered to be valuable biomarkers for retrospective verification of OPC exposure. Therefore, we have detected and identified novel adducts of human serum albumin (HSA) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Pure albumin and plasma were incubated with numerous pesticides and NA of the V- and G-type in different molar ratios. Samples were prepared either by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel enzymatic cleavage using endoproteinase Glu-C (Glu-C) or by combining highly albumin-selective affinity extraction with ultrafiltration followed by reduction, carbamidomethylation, and enzymatic cleavage (Glu-C) prior to MALDI-TOF MS analysis. Characteristic mass shifts for phosphylation revealed tyrosine adducts at Y(411) (Y(401)KFQNALLVRY(411)TKKVPQVSTPTLVE(425)), Y(148) and Y(150) (I(142)ARRHPY(148)FY(150)APE(153), single and double labeled), and Y(161) (L(154)LFFAKRY(161)KAAFTE(167)) produced by original NA (tabun, sarin, soman, cyclosarin, VX, Chinese VX, and Russian VX) as well as by chlorpyrifos-oxon, diisopropyl fluorophosphate (DFP), paraoxon-ethyl (POE), and profenofos. MALDI-MS/MS of the single-labeled I(142)-E(153) peptide demonstrated that Y(150) was phosphylated with preference to Y(148). Aged albumin adducts were not detected. The procedure described was reproducible and feasible for detection of adducts at the most reactive Y(411)-residue (S/N ≥ 3) when at least 1% of total albumin was labeled. This was achieved by incubating plasma with molar HSA/OPC ratios ranging from approximately 1:0.03 (all G-type NA, DFP, and POE) to 1:3 (V-type NA, profenofos). Relative signal intensity of the Y(411) adduct correlated well with the spotted relative molar amount underlining the usefulness for quantitative adduct determination. In conclusion, the current analytical design exhibits potential as a verification tool for high-dose exposure.

Effect of manuka honey on the expression of universal stress protein A in meticillin-resistant Staphylococcus aureus.

PubMed

Jenkins, Rowena; Burton, Neil; Cooper, Rose

2011-04-01

Staphylococcus aureus is an important pathogen that can cause many problems, from impetigo to endocarditis. With its continued resistance to multiple antibiotics, S. aureus remains a serious health threat. Honey has been used to eradicate meticillin-resistant S. aureus (MRSA) strains from wounds, but its mode of action is not yet understood. Proteomics provides a potent group of techniques that can be used to analyse differences in protein expression between untreated bacterial cells and those treated with inhibitory concentrations of manuka honey. In this study, two-dimensional (2D) electrophoresis was combined with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to determine the identities of proteins whose levels of expression were changed at least two-fold following treatment with manuka honey. Protein extracts were obtained from cells grown in tryptone soy broth (with or without manuka honey) by mechanical disruption and were separated on 2D polyacrylamide gels. A protein was isolated in gels prepared from untreated cell extract that was absent from gels made using honey-treated cell extract. Using MALDI-TOF MS, the protein was identified as universal stress protein A (UspA). Downregulation of this protein was confirmed by real-time polymerase chain reaction (PCR), which showed a 16-fold downregulation in honey-treated cells compared with untreated samples. This protein is involved in the stress stamina response and its downregulation could help to explain the inhibition of MRSA by manuka honey. Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation.

PubMed

Vasu, Prasanna; Savary, Brett J; Cameron, Randall G

2012-07-15

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6-9) and moderate thermostability (optimum ca. 70°C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure. Copyright © 2012 Elsevier Ltd. All rights reserved.

Detection of Escherichia coli sequence type 131 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: implications for infection control policies?

PubMed

Lafolie, J; Sauget, M; Cabrolier, N; Hocquet, D; Bertrand, X

2015-07-01

Sequence type 131 (ST131) is a predominant lineage among extraintestinal pathogenic Escherichia coli. It plays a major role in the worldwide dissemination of extended-spectrum β-lactamase (ESBL)-producing E. coli. The ST131 pandemic is mainly the result of clonal expansion of the single well-adapted subclone H30-Rx, which is acquired in hospitals more frequently than other ESBL-producing E. coli clones. To develop a rapid method using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify ST131 for infection control purposes. Peak biomarkers of ST131 were identified from the mass spectrum profiles of 109 E. coli isolates (including 50 ST131 isolates). The models accurately identified ST131 isolates from mass spectrum profiles obtained with and without protein extraction. The rapid identification of ST131 isolates with MALDI-TOF MS can be easily implemented in the laboratory, and could help to target infection control measures in patients carrying multi-drug-resistant E. coli that are more likely to spread. Copyright © 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Lipidomic fingerprint of almonds (Prunus dulcis L. cv Nonpareil) using TiO₂ nanoparticle based matrix solid-phase dispersion and MALDI-TOF/MS and its potential in geographical origin verification.

PubMed

Shen, Qing; Dong, Wei; Yang, Mei; Li, Linqiu; Cheung, Hon-Yeung; Zhang, Zhifeng

2013-08-14

A matrix solid-phase dispersion (MSPD) procedure with titanium dioxide (TiO2) nanoparticles (NP) as sorbent was developed for the selective extraction of phospholipids from almond samples, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) was employed for analysis. A remarkable increase in the signals of phospholipid accompanied by a decrease in those of triacylglycerols and diacylglycerols was observed in the relevant mass spectra. The proposed method was applied to five batches of almonds originating from four geographical areas, whereas principal component analysis (PCA) was utilized to normalize the relative amounts of the identified phospholipid species. The results indicated that the lipidomic fingerprint of almonds was successfully established by the negative ion mode spectrum, and the ratio of m/z 833.6 to 835.6 as well as m/z 821.6 could be introduced as potential markers for the differentiation of the tested almonds with different geographical origins. The whole method is of great promise for selective separation of phospholipids from nonphospholipids, especially the glycerides, and superior in fast screening and characterization of phospholipids in almond samples.

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

PubMed

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.

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media.

PubMed

Leyer, Caroline; Gregorowicz, Guillaume; Mougari, Faiza; Raskine, Laurent; Cambau, Emmanuelle; de Briel, Dominique

2017-07-01

During the last decade, many investigators have studied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification of mycobacteria. Diverse and contradictory results indicated that optimal level for routine testing has not been reached yet. This work aimed to assess Vitek MS through two distinct versions, Saramis v4.12 RUO and the IVD v3.0, under conditions close to routine laboratory practice. Overall, 111 mycobacterial isolates were subjected to protein extraction and same spectra were matched against both databases. The IVD v3.0 database proved to be superior to Saramis v4.12 and its identification rates remarkably increased, from 67% to 94% for isolates grown on Middlebrook 7H10 solid medium and from 62% to 91% for isolates grown on mycobacterial growth indicator tube (MGIT) liquid medium. With this new version, IVD v3.0, MALDI-TOF MS might be integrated into routine clinical diagnostics, although molecular techniques remain mandatory in some cases. Copyright © 2017 American Society for Microbiology.

[Separation and identification of bovine lactoferricin by high performance liquid chromatography-matrix-assisted laser desorption/ionization time of flight/ time of flight mass spectrometry].

PubMed

An, Meichen; Liu, Ning

2010-02-01

A high performance liquid chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (HPLC-MALDI-TOF/TOF MS) method was developed for the separation and identification of bovine lactoferricin (LfcinB). Bovine lactoferrin was hydrolyzed by pepsin and then separated by ion exchange chromatography and reversed-phase liquid chromatography (RP-LC). The antibacterial activities of the fractions from RP-LC separation were determined and the protein concentration of the fraction with the highest activity was measured, whose sequence was indentified by MALDI-TOF/TOF MS. The relative molecular mass of LfcinB was 3 124.89 and the protein concentration was 18.20 microg/mL. The method of producing LfcinB proposed in this study has fast speed, high accuracy and high resolution.

Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis.

PubMed

Baldwin, M A; Medzihradszky, K F; Lock, C M; Fisher, B; Settineri, T A; Burlingame, A L

2001-04-15

The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.

Development of an improved rapid BACpro® protocol and a method for direct identification from blood-culture-positive bottles using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Yonezawa, Takatoshi; Watari, Tomohisa; Ashizawa, Kazuho; Hanada, Daisuke; Yanagiya, Takako; Watanabe, Naoki; Terada, Takashi; Tomoda, Yutaka; Fujii, Satoshi

2018-05-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been incorporated into pathogenic bacterial identification methods and has improved their rapidity. Various methods have been reported to directly identify bacteria with MALDI-TOF MS by pretreating culture medium in blood culture bottles. Rapid BACpro® (Nittobo Medical Co., Ltd.) is a pretreatment kit for effective collection of bacteria with cationic copolymers. However, the Rapid BACpro® pretreatment kit is adapted only for MALDI Biotyper (Bruker Daltonics K.K.), and there has been a desire to expand its use to VITEK MS (VMS; bioMerieux SA). We improved the protocol and made it possible to analyze with VMS. The culture medium bacteria collection method was changed to a method with centrifugation after hemolysis using saponin; the cationic copolymer concentration was changed to 30% of the original concentration; the sequence with which reagents were added was changed; and a change was made to an ethanol/formic acid extraction method. The improved protocol enhanced the identification performance. When VMS was used, the identification rate was 100% with control samples. With clinical samples, the identification agreement rate with the cell smear method was 96.3%. The improved protocol is effective in blood culture rapid identification, being both simpler and having an improved identification performance compared with the original. Copyright © 2018 Elsevier B.V. All rights reserved.

A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen

DTIC Science & Technology

2013-09-24

Oxidation of Catechol and Para - Aminophenol Esters in the Presence of Hydrolases. Bioelectrochem. Bioenerg. 1980, 7, 11–24. 26. Evans-Nguyen, K. M.; Tao, S...Analytical HPLC (a) and MALDI-TOF (b) traces of biligand capture agentwithno thermal treatment, and after 5 days of storage as a powder at 65 Cunder...biligand was stored for 5 days at 65 C under nitrogen atmosphere. Analy- tical HPLC traces (Figure 4a) andMALDI-TOF (Figure 4b) reveal that the chemical

Conventional Matrices Loaded Onto a Graphene Layer Enhances MALDI-TOF/TOF Signal: Its Application to Improve Detection of Phosphorylated Peptides

NASA Astrophysics Data System (ADS)

Rodríguez, Carlos E.; Palacios, Javier; Fajardo, Ignacio; Urdiales, José Luis; Le Guével, Xavier; Lozano, José; Sánchez-Jiménez, Francisca

2016-02-01

This is the first study where graphene is used as a MALDI adjuvant in combination with the traditional matrix α-cyano-4-hydroxycinnamic acid (CHCA) to improve the signal intensity of peptide samples. Use of this amended matrix not only leads to increased signals but also to a higher number of peaks detected in complex samples. Additionally, the use of graphene has a stabilizing effect that can also be exploited to improve the detection of easily cleavable molecules.

An in-house assay is superior to Sepsityper for direct matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification of yeast species in blood cultures.

PubMed

Bidart, Marie; Bonnet, Isabelle; Hennebique, Aurélie; Kherraf, Zine Eddine; Pelloux, Hervé; Berger, François; Cornet, Muriel; Bailly, Sébastien; Maubon, Danièle

2015-05-01

We developed an in-house assay for the direct identification, by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, of yeasts in blood culture. Sixty-one representative strains from 12 species were analyzed in spiked blood cultures. Our assay accurately identified 95 of 107 (88.8%) positive blood cultures and outperformed the commercial Sepsityper kit (81.7% identification). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Use with Positive Blood Cultures: Methodology, Performance, and Optimization.

PubMed

Faron, Matthew L; Buchan, Blake W; Ledeboer, Nathan A

2017-12-01

Early initiation of effective antibiotics for septic patients is essential for patient survival. Matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized clinical microbiology for isolate identification and has the possibility to impact how blood culture testing is performed. This review discusses the various uses of MALDI-TOF MS for the identification and susceptibility testing of positive blood cultures, the performance of these methods, and the outcomes involved with its implementation. Copyright © 2017 American Society for Microbiology.

Development of a direct in-matrix extraction (DIME) protocol for MALDI-TOF-MS detection of glycated phospholipids in heat-treated food samples.

PubMed

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

2014-09-01

In foodstuffs, one of the main factors inducing modifications in phospholipids (PLs) structure is the heat treatment. Among PLs, only phosphatidylethanolamines and phosphatidylserines, due to their free amino group, can be involved in Maillard reaction and can form adducts with reducing sugars, besides other by-products called advanced glycation end-products. To date, glycated lipid products are less characterized in comparison to proteins. The aim of this work was to develop a novel, rapid and sensitive extraction protocol for the detection and characterization of modified PLs (glycated and oxidized) by means of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). At first, to investigate the formation of glycated and/or short chain by-products in different classes of PLs, representative standards were heated with or without sugar (lactose or glucose) and subjected to traditional lipid extraction methods as Bligh and Dyer and to the novel direct in matrix extraction (DIME) using 1,8-bis(dimethylamino)naphthalene as preconcentrating matrix. MALDI-MS analysis in negative ion mode allowed detecting glycation and oxidation products both on fatty acid and glucose moieties. Then, the procedure was successfully applied to different heat-treated and powdered samples (milk powders, pasteurized milk, ultra-high-temperature milk and soy flour) for the detection of modified PLs in complex foods. The currently developed DIME protocol could be a powerful tool for understanding lipid glycation also in biological samples. Copyright © 2014 John Wiley & Sons, Ltd.

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials.

PubMed

Heinz, Andrea; Ruttkies, Christoph K H; Jahreis, Günther; Schräder, Christoph U; Wichapong, Kanin; Sippl, Wolfgang; Keeley, Fred W; Neubert, Reinhard H H; Schmelzer, Christian E H

2013-04-01

Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown. Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry. MS(2) data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations. The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials. The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin. Copyright © 2013 Elsevier B.V. All rights reserved.

Proteomics of the Autographa californica Nucleopolyhedrovirus Budded Virions ▿

PubMed Central

Wang, RanRan; Deng, Fei; Hou, Dianhai; Zhao, Yong; Guo, Lin; Wang, Hualin; Hu, Zhihong

2010-01-01

Baculoviruses produce two progeny phenotypes during their replication cycles. The occlusion-derived virus (ODV) is responsible for initiating primary infection in the larval midgut, and the budded virus (BV) phenotype is responsible for the secondary infection. The proteomics of several baculovirus ODVs have been revealed, but so far, no extensive analysis of BV-associated proteins has been conducted. In this study, the protein composition of the BV of Autographa californica nucleopolyhedrovirus (AcMNPV), the type species of baculoviruses, was analyzed by various mass spectrometry (MS) techniques, including liquid chromatography-triple quadrupole linear ion trap (LC-Qtrap), liquid chromatography-quadrupole time of flight (LC-Q-TOF), and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). SDS-PAGE and MALDI-TOF analyses showed that the three most abundant proteins of the AcMNPV BV were GP64, VP39, and P6.9. A total of 34 viral proteins associated with the AcMNPV BV were identified by the indicated methods. Thirteen of these proteins, PP31, AC58/59, AC66, IAP-2, AC73, AC74, AC114, AC124, chitinase, polyhedron envelope protein (PEP), AC132, ODV-E18, and ODV-E56, were identified for the first time to be BV-associated proteins. Western blot analyses showed that ODV-E18 and ODV-E25, which were previously thought to be ODV-specific proteins, were also present in the envelop fraction of BV. In addition, 11 cellular proteins were found to be associated with the AcMNPV BV by both LC-Qtrap and LC-Q-TOF analyses. Interestingly, seven of these proteins were also identified in other enveloped viruses, suggesting that many enveloped viruses may commonly utilize certain conserved cellular pathways. PMID:20444894

MALDI-TOF Mass Spectrometry Is a Fast and Reliable Platform for Identification and Ecological Studies of Species from Family Rhizobiaceae

PubMed Central

Ferreira, Laura; Sánchez-Juanes, Fernando; García-Fraile, Paula; Rivas, Raúl; Mateos, Pedro F.; Martínez-Molina, Eustoquio; González-Buitrago, José Manuel; Velázquez, Encarna

2011-01-01

Family Rhizobiaceae includes fast growing bacteria currently arranged into three genera, Rhizobium, Ensifer and Shinella, that contain pathogenic, symbiotic and saprophytic species. The identification of these species is not possible on the basis of physiological or biochemical traits and should be based on sequencing of several genes. Therefore alternative methods are necessary for rapid and reliable identification of members from family Rhizobiaceae. In this work we evaluated the suitability of Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for this purpose. Firstly, we evaluated the capability of this methodology to differentiate among species of family Rhizobiaceae including those closely related and then we extended the database of MALDI Biotyper 2.0 including the type strains of 56 species from genera Rhizobium, Ensifer and Shinella. Secondly, we evaluated the identification potential of this methodology by using several strains isolated from different sources previously identified on the basis of their rrs, recA and atpD gene sequences. The 100% of these strains were correctly identified showing that MALDI-TOF MS is an excellent tool for identification of fast growing rhizobia applicable to large populations of isolates in ecological and taxonomic studies. PMID:21655291

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

PubMed

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

2008-02-13

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

Applications of copolymer for rapid identification of bacteria in blood culture broths using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Ashizawa, Kazuho; Murata, Syota; Terada, Takashi; Ito, Daisuke; Bunya, Masaru; Watanabe, Koji; Teruuchi, Yoko; Tsuchida, Sachio; Satoh, Mamoru; Nishimura, Motoi; Matsushita, Kazuyuki; Sugama, Yuji; Nomura, Fumio

2017-08-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify pathogens in blood culture samples. However, sample pretreatment is needed for direct identification of microbes in blood culture bottles. Conventional protocols are complex and time-consuming. Therefore, in this study, we developed a method for collecting bacteria using polyallylamine-polystyrene copolymer for application in wastewater treatment technology. Using representative bacterial species Escherichia coli and Staphylococcus capitis, we found that polyallylamine-polystyrene can form visible aggregates with bacteria, which can be identified using MALDI-TOF MS. The processing time of our protocol was as short as 15min. Hemoglobin interference in MALDI spectra analysis was significantly decreased in our method compared with the conventional method. In a preliminary experiment, we evaluated the use of our protocol to identify clinical isolates from blood culture bottles. MALDI-TOF MS-based identification of 17 strains from five bacterial species (E. coli, Klebsiella pneumoniae, Enterococcus faecalis, S. aureus, and S. capitis) collected by our protocol was satisfactory. Prospective large-scale studies are needed to further evaluate the clinical application of this novel and simple method of collecting bacteria in blood culture bottles. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis.

PubMed

Wang, Jun; Chen, Wen Feng; Li, Qing X

2012-02-24

The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4-99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9-99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification. Copyright © 2011 Elsevier B.V. All rights reserved.

Cost Analysis of Implementing Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Plus Real-Time Antimicrobial Stewardship Intervention for Bloodstream Infections.

PubMed

Patel, Twisha S; Kaakeh, Rola; Nagel, Jerod L; Newton, Duane W; Stevenson, James G

2017-01-01

Studies evaluating rapid diagnostic testing plus stewardship intervention have consistently demonstrated improved clinical outcomes for patients with bloodstream infections. However, the cost of implementing new rapid diagnostic testing can be significant, and such testing usually does not generate additional revenue. There are minimal data evaluating the impact of adding matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid organism identification and dedicating pharmacy stewardship personnel time on the total hospital costs. A cost analysis was performed utilizing patient data generated from the hospital cost accounting system and included additional costs of MALDI-TOF equipment, supplies and personnel, and dedicated pharmacist time for blood culture review and of making interventions to antimicrobial therapy. The cost analysis was performed from a hospital perspective for 3-month blocks before and after implementation of MALDI-TOF plus stewardship intervention. A total of 480 patients with bloodstream infections were included in the analysis: 247 in the preintervention group and 233 in the intervention group. Thirty-day mortality was significantly improved in the intervention group (12% versus 21%, P < 0.01), and the mean length of stay was reduced, although the difference was not statistically significant (13.0 ± 16.5 days versus 14.2 ± 16.7 days, P = 0.44). The total hospital cost per bloodstream infection was lower in the intervention group ($42,580 versus $45,019). Intensive care unit cost per bloodstream infection accounted for the largest share of the total costs in each group and was also lower in the intervention group ($10,833 versus $13,727). Implementing MALDI-TOF plus stewardship review and intervention decreased mortality for patients with bloodstream infections. Despite the additional costs of implementing MALDI-TOF and of dedicating pharmacy stewardship personnel time to interventions, the total hospital costs decreased by $2,439 per bloodstream infection, for an approximate annual cost savings of $2.34 million. Copyright © 2016 American Society for Microbiology.

Evaluation of MALDI-TOF mass spectrometry for identification of environmental yeasts and development of supplementary database.

PubMed

Agustini, Bruna Carla; Silva, Luciano Paulino; Bloch, Carlos; Bonfim, Tania M B; da Silva, Gildo Almeida

2014-06-01

Yeast identification using traditional methods which employ morphological, physiological, and biochemical characteristics can be considered a hard task as it requires experienced microbiologists and a rigorous control in culture conditions that could implicate in different outcomes. Considering clinical or industrial applications, the fast and accurate identification of microorganisms is a crescent demand. Hence, molecular biology approaches has been extensively used and, more recently, protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved to be an even more efficient tool for taxonomic purposes. Nonetheless, concerning to mass spectrometry, data available for the differentiation of yeast species for industrial purpose is limited and reference databases commercially available comprise almost exclusively clinical microorganisms. In this context, studies focusing on environmental isolates are required to extend the existing databases. The development of a supplementary database and the assessment of a commercial database for taxonomic identifications of environmental yeast are the aims of this study. We challenge MALDI-TOF MS to create protein profiles for 845 yeast strains isolated from grape must and 67.7 % of the strains were successfully identified according to previously available manufacturer database. The remaining 32.3 % strains were not identified due to the absence of a reference spectrum. After matching the correct taxon for these strains by using molecular biology approaches, the spectra concerning the missing species were added in a supplementary database. This new library was able to accurately predict unidentified species at first instance by MALDI-TOF MS, proving it is a powerful tool for the identification of environmental yeasts.

Evaluation of MALDI-TOF-MS for the Identification of Yeast Isolates Causing Bloodstream Infection.

PubMed

Turhan, Ozge; Ozhak-Baysan, Betil; Zaragoza, Oscar; Er, Halil; Sarıtas, Zubeyde Eres; Ongut, Gozde; Ogunc, Dilara; Colak, Dilek; Cuenca-Estrella, Manuel

2017-04-01

Infections due to Candida species are major causes of morbidity and mortality in humans, causing a diverse spectrum of clinical disease ranging from superficial and mucosal infections to invasive disease. Several authors have demonstrated that mortality is closely linked to both timing of therapy and/or source control. The rapid identification of pathogenic species is helpful to start timely and effective antifungal therapy. The aim of this study was to assess the performance of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system for the correct and rapid identification of yeast isolates causing bloodstream infection. Between January 2014 and January 2015, a total of 117 yeast like organisms isolated from blood culture samples of 117 episodes from 102 patients who had blood stream infections were included in the study. The isolates were identified by MALDI-TOF MS. The results were compared with those obtained by the standard mycological methods and/or sequence analysis. One hundred and seventeen yeast isolates including 115 Candida spp and two non-Candida yeasts were analysed. The Biotyper correctly identified 115 (98.3%) isolates to the genus level and 102 (87.2%) isolates to the species level using the manufacturer's recommended cutoff scores. The Bruker Biotyper is a rapid, easy, inexpensive, and highly reliable system for the identification of yeast isolates. Early identification with MALDI-TOF MS would save time for determination of antifungal susceptibility and proper treatment strategy. The expansion of the database of the library by addition of less common species will improve the performance of the system.

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

PubMed Central

Celandroni, Francesco; Salvetti, Sara; Gueye, Sokhna Aissatou; Mazzantini, Diletta; Lupetti, Antonella; Senesi, Sonia; Ghelardi, Emilia

2016-01-01

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance. PMID:27031639

Direct bacterial identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry: A systematic review and meta-analysis.

PubMed

Ruiz-Aragón, Jesús; Ballestero-Téllez, Mónica; Gutiérrez-Gutiérrez, Belén; de Cueto, Marina; Rodríguez-Baño, Jesús; Pascual, Álvaro

2017-10-27

The rapid identification of bacteraemia-causing pathogens could assist clinicians in the timely prescription of targeted therapy, thereby reducing the morbidity and mortality of this infection. In recent years, numerous techniques that rapidly and directly identify positive blood cultures have been marketed, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) being one of the most commonly used. The aim of this systematic review and meta-analysis was to evaluate the accuracy of MALDI-TOF (Bruker ® ) for the direct identification of positive blood culture bottles. A meta-analysis was performed to summarize the results of the 32 studies evaluated. The overall quality of the studies was moderate. For Gram-positive bacteria, overall rates of correct identification of the species ranged from 0.17 to 0.98, with a cumulative rate (random-effects model) of 0.72 (95% CI: 0.64-0.80). For Gram-negative bacteria, correct identification rates ranged from 0.66 to 1.00, with a cumulative effect of 0.92 (95% CI: 0.88-0.95). For Enterobacteriaceae, the rate was 0.96 (95% CI: 0.94-0.97). MALDI-TOF mass spectrometry shows high accuracy for the correct identification of Gram-negative bacteria, particularly Enterobacteriaceae, directly from positive blood culture bottles, and moderate accuracy for the identification of Gram-positive bacteria (low for some species). Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

[Evaluation of mass spectrometry for the identification of clinically interesting yeasts].

PubMed

Galán, Fátima; García-Agudo, Lidia; Guerrero, Inmaculada; Marín, Pilar; García-Tapia, Ana; García-Martos, Pedro; Rodríguez-Iglesias, Manuel

2015-01-01

Identification of yeasts is based on morphological, biochemical and nutritional characteristics, and using molecular methods. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, a new method for the identification of microorganisms, has demonstrated to be very useful. The aim of this study is to evaluate this new method in the identification of yeasts. A total of 600 strains of yeasts isolated from clinical specimens belonging to 9 genera and 43 species were tested. Identification was made by sequencing of the ITS regions of ribosomal DNA, assimilation of carbon compounds (ID 32C), and mass spectrometry on a Microflex spectrometer (Bruker Daltonics GmbH, Germany). A total of 569 strains (94.8%) were identified to species level by ID 32C, and 580 (96.7%) by MALDI-TOF. Concordance between both methods was observed for 553 strains (92.2%), with 100% in clinically relevant species: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and almost 100% in C. krusei. MALDI-TOF identified species requiring molecular methods: Candida dubliniensis, C. nivariensis, C. metapsilosis and C. orthopsilosis. Some irregularities were observed in the identification of arthroconidia yeast and basidiomycetes. MALDI-TOF is a rapid, effective and economic method, which enables the identification of most clinically important yeasts and the differentiation of closely related species. It would be desirable to include more species in its database to expand its performance. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter.

PubMed

Zaluga, Joanna; Heylen, Kim; Van Hoorde, Koenraad; Hoste, Bart; Van Vaerenbergh, Johan; Maes, Martine; De Vos, Paul

2011-09-01

The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens. Copyright © 2011 Elsevier GmbH. All rights reserved.

Comprehensive MALDI-TOF biotyping of the non-redundant Harvard Pseudomonas aeruginosa PA14 transposon insertion mutant library.

PubMed

Oumeraci, Tonio; Jensen, Vanessa; Talbot, Steven R; Hofmann, Winfried; Kostrzewa, Markus; Schlegelberger, Brigitte; von Neuhoff, Nils; Häussler, Susanne

2015-01-01

Pseudomonas aeruginosa is a gram-negative bacterium that is ubiquitously present in the aerobic biosphere. As an antibiotic-resistant facultative pathogen, it is a major cause of hospital-acquired infections. Its rapid and accurate identification is crucial in clinical and therapeutic environments. In a large-scale MALDI-TOF mass spectrometry-based screen of the Harvard transposon insertion mutant library of P. aeruginosa strain PA14, intact-cell proteome profile spectra of 5547 PA14 transposon mutants exhibiting a plethora of different phenotypes were acquired and analyzed. Of all P. aeruginosa PA14 mutant profiles 99.7% were correctly identified as P. aeruginosa with the Biotyper software on the species level. On the strain level, 99.99% of the profiles were mapped to five different individual P. aeruginosa Biotyper database entries. A principal component analysis-based approach was used to determine the most important discriminatory mass features between these Biotyper groups. Although technical replicas were consistently categorized to specific Biotyper groups in 94.2% of the mutant profiles, biological replicas were not, indicating that the distinct proteotypes are affected by growth conditions. The PA14 mutant profile collection presented here constitutes the largest coherent P. aeruginosa MALDI-TOF spectral dataset publicly available today. Transposon insertions in thousands of different P. aeruginosa genes did not affect species identification from MALDI-TOF mass spectra, clearly demonstrating the robustness of the approach. However, the assignment of the individual spectra to sub-groups proved to be non-consistent in biological replicas, indicating that the differentiation between biotyper groups in this nosocomial pathogen is unassured.

Novel monolithic enzymatic microreactor based on single-enzyme nanoparticles for highly efficient proteolysis and its application in multidimensional liquid chromatography.

PubMed

Gao, Mingxia; Zhang, Peng; Hong, Guangfeng; Guan, Xia; Yan, Guoquan; Deng, Chunhui; Zhang, Xiangmin

2009-10-30

In this work, a novel and facile monolithic enzymatic microreactor was prepared in the fused-silica capillary via a two-step procedure including surface acryloylation and in situ aqueous polymerization/immobilization to encapsulate a single enzyme, and its application to fast protein digestion through a direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis was demonstrated. At first, vinyl groups on the protein surface were generated by a mild acryloylation with N-acryloxysuccinimide in alkali buffer. Then, acryloylated enzyme was encapsulated into polyacrylates by free-radical copolymerization with acrylamide as the monomer, N,N'-methylenebisacrylamide as the cross-linker, and N,N,N',N'-tetramethylethylenediamine/ammonium persulfate as the initiator. Finally, polymers were immobilized onto the activated inner wall of capillaries via the reaction of vinyl groups. Capability of the enzyme-immobilized monolithic microreactor was demonstrated by myoglobin and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF-MS with sequence coverage of 94% and 29% observed. This microreactor was also applied to the analysis of fractions through two-dimensional separation of weak anion exchange/reversed-phase liquid chromatography of human liver extract. After a database search, 16 unique peptides corresponding to 3 proteins were identified when two RPLC fractions of human liver extract were digested by the microreactor. This opens a route for its future application in top-down proteomic analysis.

Magazines in waiting areas of hospital: a forgotten microbial reservoir?

PubMed

Adé, Mathias; Burger, Sandrine; Cuntzmann, Anaelle; Exinger, Julien; Meunier, Olivier

2017-12-01

The hospital environment is a potential source of microbial contamination. Thus, the magazines in hospital's waiting rooms are handled by patients and visitors whose health and hygiene conditions can vary widely. In this context, we had measured the microbial load on the surface of magazines. Fifteen magazines from 5 waiting rooms of hospital are sampled by agar prints at the areas taken in hand. The agar plates are incubated at 30̊C for 72h. The colonies are counted and identified by MALDI-TOF mass spectrometry (Vitek ® -MS). The extraction efficiency of bacteria by the agar print method on the magazines is calculated. All the samples highlight a varied bacterial flora: 32CFU/agar in mean. Isolated bacteria come principally from the skin flora (>60%), but we also isolate potentially pathogenic micro-organisme like S. aureus, E. faecalis, A. viridans and Aspergillus sp. as well as oropharyngeal flora bacteria like A. iwolfii and M. osloensis and fecal like B. stercoris. Some species rarely described in hospital are also isolated such as P. yeei or K. sedentarius. The extraction efficiency of the sampling method on a magazine is 36%. Our study, which is the first to be interested in the bacterial contamination of magazines in hospital, could make them consider as microbial reservoir to be controlled, especially for the most fragile patients. New bacterial identification techniques as the MALDI-TOF allow to reveal the presence of rarely described and often underestimated species.

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

PubMed

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.

Characterization and comparison of Fumonisin B(1)-protein conjugates by six methods.

PubMed

Wang, Ying; He, Cheng-Hua; Zheng, Hao; Zhang, Hai-Bin

2012-01-01

In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten (Fumonisin B(1), FB(1)) was coupled to ovalbumin (OVA) and bovine serum albumin (BSA), respectively by a short cross-linker reagent (glutaraldehyde, GA). To develop a technique for detecting the conjugation, the hapten-protein conjugates (FB(1)-OVA and FB(1)-BSA) were characterized thoroughly by ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. The molecular weights of FB(1)-BSA and FB(1)-OVA were 74,355.301 Da and 48,009.212 Da, respectively determined by the method of MALDI-TOF-MS. The molecular coupling ratios were 11 and 5 in FB(1)-BSA and FB(1)-OVA, respectively. In this experiment, MALDI-TOF-MS was selected as the most efficient method to evaluate the cross-linking effect and calculate the molecular coupling ratio.

Rapid Screening of Ergot Alkaloids in Sclerotia by MALDI-TOF Mass Spectrometry.

PubMed

Sivagnanam, Kumaran; Komatsu, Emy; Patrick, Susan; Rampitsch, Christoph; Perreault, Hélène; Gräfenhan, Tom

2016-07-01

Ergot is a common disease of wheat and other cereal grains that is predominantly caused by Claviceps purpurea in the field, often affecting crop yield in addition to the environment. Infected grain can be contaminated with dark sclerotia, which contain fungal metabolites such as ergot alkaloids. The occurrence of ergot alkaloids in cereal grain is a major health concern for humans and livestock. Effective and rapid screening of these mycotoxins is crucial for producers, processors, and consumers of cereal-based food and feed grain. Established methods of ergot alkaloid screening based on LC-MS or GC-MS require laborious processes. A novel method using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS was developed to identify four ergot alkaloids. Using dihydroxybenzoic acid as the matrix, ergosine, ergocornine, ergocryptine, and ergocristine were readily detected in individual sclerotia of C. purpurea. The accuracy of the identified ergot alkaloids was further confirmed by tandem MS analysis. MALDI-TOF MS is suitable for high-throughput screening of ergot alkaloids because it permits rapid and accurate identification, simple sample preparation, and no derivatization or chromatographic separation.

Novel approach for differentiating Shigella species and Escherichia coli by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Khot, Prasanna D; Fisher, Mark A

2013-11-01

Shigella species are so closely related to Escherichia coli that routine matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) cannot reliably differentiate them. Biochemical and serological methods are typically used to distinguish these species; however, "inactive" isolates of E. coli are biochemically very similar to Shigella species and thus pose a greater diagnostic challenge. We used ClinProTools (Bruker Daltonics) software to discover MALDI-TOF MS biomarker peaks and to generate classification models based on the genetic algorithm to differentiate between Shigella species and E. coli. Sixty-six Shigella spp. and 72 E. coli isolates were used to generate and test classification models, and the optimal models contained 15 biomarker peaks for genus-level classification and 12 peaks for species-level classification. We were able to identify 90% of E. coli and Shigella clinical isolates correctly to the species level. Only 3% of tested isolates were misidentified. This novel MALDI-TOF MS approach allows laboratories to streamline the identification of E. coli and Shigella species.

Semi-quantitative MALDI-TOF for antimicrobial susceptibility testing in Staphylococcus aureus.

PubMed

Maxson, Tucker; Taylor-Howell, Cheryl L; Minogue, Timothy D

2017-01-01

Antibiotic resistant bacterial infections are a significant problem in the healthcare setting, in many cases requiring the rapid administration of appropriate and effective antibiotic therapy. Diagnostic assays capable of quickly and accurately determining the pathogen resistance profile are therefore crucial to initiate or modify care. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a standard method for species identification in many clinical microbiology laboratories and is well positioned to be applied towards antimicrobial susceptibility testing. One recently reported approach utilizes semi-quantitative MALDI-TOF MS for growth rate analysis to provide a resistance profile independent of resistance mechanism. This method was previously successfully applied to Gram-negative pathogens and mycobacteria; here, we evaluated this method with the Gram-positive pathogen Staphylococcus aureus. Specifically, we used 35 strains of S. aureus and four antibiotics to optimize and test the assay, resulting in an overall accuracy rate of 95%. Application of the optimized assay also successfully determined susceptibility from mock blood cultures, allowing both species identification and resistance determination for all four antibiotics within 3 hours of blood culture positivity.

Successful Identification of Clinical Dermatophyte and Neoscytalidium Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Alshawa, Kinda; Beretti, Jean-Luc; Lacroix, Claire; Feuilhade, Martine; Dauphin, Brunhilde; Quesne, Gilles; Hassouni, Noura; Nassif, Xavier

2012-01-01

Dermatophytes are keratinolytic fungi responsible for a wide variety of diseases of glabrous skin, nails, and hair. Their identification, currently based on morphological criteria, is hindered by intraspecies morphological variability and the atypical morphology of some clinical isolates. The aim of this study was to evaluate matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a routine tool for identifying dermatophyte and Neoscytalidium species, both of which cause dermatomycoses. We first developed a spectral database of 12 different species of common and unusual dermatophytes and two molds responsible for dermatomycoses (Neoscytalidium dimidiatum and N. dimidiatum var. hyalinum). We then prospectively tested the performance of the database on 381 clinical dermatophyte and Neoscytalidium isolates. Correct identification of the species was obtained for 331/360 dermatophytes (91.9%) and 18/21 Neoscytalidium isolates (85.7%). The results of MALDI-TOF MS and standard identification disagreed for only 2 isolates. These results suggest that MALDI-TOF MS could be a useful tool for routine and fast identification of dermatophytes and Neoscytalidium spp. in clinical mycology laboratories. PMID:22535981

Porous extraction paddle: a solid phase extraction technique for studying the urine metabolome

PubMed Central

Shao, Gang; MacNeil, Michael; Yao, Yuanyuan; Giese, Roger W.

2016-01-01

RATIONALE A method was needed to accomplish solid phase extraction of a large urine volume in a convenient way where resources are limited, towards a goal of metabolome and xenobiotic exposome analysis at another, distant location. METHODS A porous extraction paddle (PEP) was set up, comprising a porous nylon bag containing extraction particles that is flattened and immobilized between two stainless steel meshes. Stirring the PEP after attachment to a shaft of a motor mounted on the lid of the jar containing the urine accomplishes extraction. The bag contained a mixture of nonpolar and partly nonpolar particles to extract a diversity of corresponding compounds. RESULTS Elution of a urine-exposed, water-washed PEP with aqueous methanol containing triethylammonium acetate (conditions intended to give a complete elution), followed by MALDI-TOF/TOF-MS, demonstrated that a diversity of compounds had been extracted ranging from uric acid to peptides. CONCLUSION The PEP allows the user to extract a large liquid sample in a jar simply by turning on a motor. The technique will be helpful in conducting metabolomics and xenobiotic exposome studies of urine, encouraging the extraction of large volumes to set up a convenient repository sample (e.g. 2 g of exposed adsorbent in a cryovial) for shipment and re-analysis in various ways in the future, including scaled-up isolation of unknown chemicals for identification. PMID:27624170

Porous extraction paddle: a solid phase extraction technique for studying the urine metabolome.

PubMed

Shao, Gang; MacNeil, Michael; Yao, Yuanyuan; Giese, Roger W

2016-09-14

A method was needed to accomplish solid phase extraction of a large urine volume in a convenient way where resources are limited, towards a goal of metabolome and xenobiotic exposome analysis at another, distant location. A porous extraction paddle (PEP) was set up, comprising a porous nylon bag containing extraction particles that is flattened and immobilized between two stainless steel meshes. Stirring the PEP after attachment to a shaft of a motor mounted on the lid of the jar containing the urine accomplishes extraction. The bag contained a mixture of nonpolar and partly nonpolar particles to extract a diversity of corresponding compounds. Elution of a urine-exposed, water-washed PEP with aqueous methanol containing triethylammonium acetate (conditions intended to give a complete elution), followed by MALDI-TOF/TOF-MS, demonstrated that a diversity of compounds had been extracted ranging from uric acid to peptides. The PEP allows the user to extract a large liquid sample in a jar simply by turning on a motor. The technique will be helpful in conducting metabolomics and xenobiotic exposome studies of urine, encouraging the extraction of large volumes to set up a convenient repository sample (e.g. 2 g of exposed adsorbent in a cryovial) for shipment and re-analysis in various ways in the future, including scaled-up isolation of unknown chemicals for identification. This article is protected by copyright. All rights reserved.

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

PubMed

Krásný, Lukáš; Rohlová, Eva; Růžičková, Helena; Santrůček, Jiří; Hynek, Radovan; Hochel, Igor

2014-03-01

Intact cell MALDI-TOF mass spectrometry is a rapid tool for the identification and classification of microorganisms, now widely used even in clinical laboratories. However, its distinctive power is not sufficient for some closely-related species. The genus Cronobacter, formerly known as Enterobacter sakazakii, contains such species. In this work, a new method for the differentiation of five Cronobacter species is presented involving the tryptic digestion of cytoplasmatic proteins followed by MALDI mass spectrometry analysis. A database was developed for use in Bruker Biotyper software including 52 reference spectra and tested on a set of 45 samples with an overall accuracy of about 80%. The possibility of measurement automation and the short time and low cost requirements of this method compared to those of biochemical tests or PCR methods make it a supplementary option to intact cell MALDI, providing additional information about the differentiation of problematic species. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

Lou, Xianwen; de Waal, Bas F M; Milroy, Lech-Gustav; van Dongen, Joost L J

2015-05-01

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 suppression effect (ASE). In our method, analytes are deposited on top of the surface of matrix preloaded on the MALDI plate. To prevent embedding of analyte into the matrix crystals, the sample solution were prepared without matrix and efforts were taken not to re-dissolve the preloaded matrix. The results with model mixtures of peptides, synthetic polymers and lipids show that detection of analyte ions, which were completely suppressed using the conventional dried-droplet method, could be effectively recovered by using our method. Our findings suggest that the incorporation of analytes in the matrix crystals has an important contributory effect on ASE. By reducing ASE, our method should be useful for the direct MALDI MS analysis of multicomponent mixtures. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of soils - Part II: Determination of oligosaccharides in soils by MALDI-time-of-flight mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schuerch, S.; Howald, M.; Schlunegger, U.P.

1995-12-31

Polysaccharides are the most abundant organic compounds in nature. Decomposition of plant and animal residues leads to a high polysaccharide content in soils. The decomposition of carbohydrates and subsequent mineralization of the products are part of the cycle of life on earth. In extracts of soils collected in the Valle Onsernone (Ticino, Switzerland), oligosaccharides of different size and structure have been identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The selected soils show identical climatic parameters and pedological factors, whereas the age of fallow land is the only varying factor. Identification and structure elucidation of the oligosaccharides is performedmore » by substrate-specific enzymatic hydrolysis. Moreover the appearance and the distribution of the oligosaccharides is correlated to soil genesis.« less

Species Identification of Clinical Prevotella Isolates by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Soetens, Oriane; De Bel, Annelies; Echahidi, Fedoua; Vancutsem, Ellen; Vandoorslaer, Kristof; Piérard, Denis

2012-01-01

The performance of matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS) for species identification of Prevotella was evaluated and compared with 16S rRNA gene sequencing. Using a Bruker database, 62.7% of the 102 clinical isolates were identified to the species level and 73.5% to the genus level. Extension of the commercial database improved these figures to, respectively, 83.3% and 89.2%. MALDI-TOF MS identification of Prevotella is reliable but needs a more extensive database. PMID:22301022

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

NASA Astrophysics Data System (ADS)

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

2015-09-01

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

Unlocking the proteomic information encoded in MALDI-TOF-MS data used for microbial identification and characterization

USDA-ARS?s Scientific Manuscript database

Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)is increasingly utilized as a rapid technique to identify microorganisms including pathogenic bacteria. However, little attention has been paid to the significant proteomic information encoded in ...

Use of MALDI-TOF mass spectrometry to analyze the molecular profile of Pseudomonas aeruginosa biofilms grown on glass and plastic surfaces.

PubMed

Pereira, Flávio D E S; Bonatto, Cínthia C; Lopes, Cláudio A P; Pereira, Alex L; Silva, Luciano P

2015-09-01

Biofilms are microbial sessile communities attached to surfaces that are known for causing many medical problems. A bacterial biofilm of clinical relevance is formed by the gram-negative bacteria Pseudomonas aeruginosa. During the formation of a biofilm, the initial adhesion of the cells is of crucial importance, and the characteristics of the contact surface have great influence on this step. In the present study, we aimed to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling as a new methodology to monitor P. aeruginosa biofilm development. Biofilms were grown within polypropylene tubes containing a glass slide, and were harvested after 3, 5, 7, 9, or 12 days of inoculation. Planktonic cells were obtained separately by centrifugation as control. Two independent MALDI-TOF experiments were performed, one by collecting biofilms from both the glass slide and the polypropylene tube internal surface, and the other by acquiring biofilms from these surfaces separately. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to evaluate the morphological progression of the biofilm. The molecular results showed that MALDI profiling is able not only to distinguish between different biofilm stages, but it is also appropriate to indicate when the biofilm cells are released at the dispersion stage, which occurred first on polypropylene surface. Finally, the present study pointed out that MALDI profiling may emerge as a promising tool for the clinical diagnostic and prognostic workup of biofilms formation and control. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of sixteen peptides reflecting heat and/or storage induced processes by profiling of commercial milk samples.

PubMed

Ebner, Jennifer; Baum, Florian; Pischetsrieder, Monika

2016-09-16

Peptide profiles of different drinking milk samples were examined to study how the peptide fingerprint of milk reflects processing conditions. The combination of a simple and fast method for peptide extraction using stage tips and MALDI-TOF-MS enabled the fast and easy generation and relative quantification of peptide fingerprints for high-temperature short-time (HTST), extended shelf life (ESL) and ultra-high temperature (UHT) milk of the same dairies. The relative quantity of 16 peptides changed as a function of increasing heat load. Additional heating experiments showed that among those, the intensity of peptide β-casein 196-209 (m/z 1460.9Da) was most heavily influenced by heat treatment indicating a putative marker peptide for milk processing conditions. Storage experiments with HTST- and UHT milk revealed that the differences between different types of milk samples were not only caused by the heating process. Relevant was also the proteolytic activity of enzymes during storage, which were differently influenced by the heat treatment. These results indicate that the peptide profile may be suitable to monitor processing as well as storage conditions of milk. In the present study, peptide profiling of different types of milk was carried out by MALDI-TOF-MS after stage-tip extraction and relative quantification using an internal reference peptide. Although MALDI-TOF-MS covers only part of the peptidome, the method is easy and quick and is, therefore, suited for routine analysis to address several aspects of food authenticity. Using this method, 16 native peptides were detected in milk that could be modulated by different industrial processes. Subsequent heating and storage experiments with pasteurized and UHT milk confirmed that these peptides are indeed related to the production or storage conditions of the respective products. Furthermore, the heating experiments revealed one peptide, namely the β-casein-derived sequence β-casein 196-209, which underwent particularly sensitive modulation by heat treatment. The present results indicate that the modulated peptides, and especially β-casein 196-209, may be suitable markers to monitor processing parameters for industrial milk production. Furthermore, the model experiments suggest mechanisms leading to the formation or degradation of peptides, which help to evaluate putative marker peptides. Copyright © 2016 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jian, E-mail: lujian@ujs.edu.cn; Institute of Life Sciences, Jiangsu University, Zhenjiang 212013; Zhou, Zhongping

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 linemore » 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.« less

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

PubMed

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, p<0.001; saponin vs. centrifugation, p=0.003). These results suggest that the saponin method is superior to the centrifugation method and comparable to the Sepsityper method in the accuracy of rapid bacterial identification directly 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.

Rapid Identification of Intact Staphylococcal Bacteriophages Using Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

PubMed

Štveráková, Dana; Šedo, Ondrej; Benešík, Martin; Zdráhal, Zbyněk; Doškař, Jiří; Pantůček, Roman

2018-04-04

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae , Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus , as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus -specific bacteriophages.

Rapid Identification of Intact Staphylococcal Bacteriophages Using Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

PubMed Central

Štveráková, Dana; Šedo, Ondrej; Benešík, Martin; Zdráhal, Zbyněk; Doškař, Jiří

2018-01-01

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages. PMID:29617332

Comparison of Heat Inactivation and Cell Disruption Protocols for Identification of Mycobacteria from Solid Culture Media by Use of Vitek Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Machen, Alexandra; Kobayashi, Miwako; Connelly, Mary Robin

2013-01-01

Two novel protocols for inactivation and extraction were developed and used to identify 107 Mycobacterium clinical isolates, including Mycobacterium tuberculosis complex, from solid cultures using Vitek matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The protocol using heat inactivation with sonication and cell disruption with glass beads resulted in 82.2% and 88.8% species and genus level identifications, respectively. PMID:24068013

Impact of rapid microbial identification directly from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on patient management.

PubMed

Martiny, D; Debaugnies, F; Gateff, D; Gérard, M; Aoun, M; Martin, C; Konopnicki, D; Loizidou, A; Georgala, A; Hainaut, M; Chantrenne, M; Dediste, A; Vandenberg, O; Van Praet, S

2013-12-01

For septic patients, delaying the initiation of antimicrobial therapy or choosing an inappropriate antibiotic can considerably worsen their prognosis. This study evaluated the impact of rapid microbial identification (RMI) from positive blood cultures on the management of patients with suspected sepsis. During a 6-month period, RMI by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed for all new episodes of bacteraemia. For each patient, the infectious disease specialist was contacted and questioned about his therapeutic decisions made based on the Gram staining and the RMI. This information was collected to evaluate the number of RMIs that led to a therapeutic change or to a modification of the patient's general management (e.g. fast removal of infected catheters). During the study period, 277 new episodes of bacteraemia were recorded. In 71.12% of the cases, MALDI-TOF MS resulted in a successful RMI (197/277). For adult and paediatric patients, 13.38% (21/157) and 2.50% (1/40) of the RMIs, respectively, resulted in modification of the treatment regimen, according to the survey. In many other cases, the MALDI-TOF MS was a helpful tool for infectious disease specialists because it confirmed suspected cases of contamination, especially in the paediatric population (15/40 RMIs, 37.50%), or suggested complementary diagnostic testing. This study emphasizes the benefits of RMI from positive blood cultures. Although the use of this technique represents an extra cost for the laboratory, RMI using MALDI-TOF MS has been implemented in our daily practice. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment.

PubMed

Rodrigues, Naiara Miranda Bento; Bronzato, Greiciane França; Santiago, Gabrielli Stefaninni; Botelho, Larissa Alvarenga Batista; Moreira, Beatriz Meurer; Coelho, Irene da Silva; Souza, Miliane Moreira Soares de; Coelho, Shana de Mattos de Oliveira

Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n=47) and fecal samples (n=94) from cows, and samples from water (n=23) and milk lines (n=19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Nilsson, C L; Puchades, M; Westman, A; Blennow, K; Davidsson, P

1999-01-01

Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

NASA Astrophysics Data System (ADS)

Prentice, Boone M.; Chumbley, Chad W.; Caprioli, Richard M.

2017-01-01

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

A First Insight into Escherichia coli ST131 High-Risk Clone Among Extended-Spectrum Beta-Lactamase-Producing Urine Isolates in Istanbul with the Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry and Real-Time PCR.

PubMed

Aktaş, Elif; Otlu, Barış; Erdemir, Duygu; Ekici, Hatice; Bulut, Emin

2017-12-01

We aim to investigate, as a first insight, the presence and rates of high-risk Escherichia coli ST131 clone in Istanbul and evaluate antimicrobial resistance and CTX-M-15 production of ST131 and non-ST131 isolates. The use of MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry) to detect E. coli ST131 clone is also evaluated. A total of 203 extended-spectrum beta-lactamase (ESBL)-producing urinary isolates from a training hospital in Istanbul were investigated. Detection of E. coli ST131 was done by MALDI-TOF MS and real-time PCR melting curve analysis. The presence of CTX-M and CTX-M-15 beta-lactamases was investigated by PCR and sequence analysis. Of the 203 isolates, 81 (39.9%) and 75 (36.9%) isolates were identified as ST131 clone by PCR and MALDI-TOF MS, respectively. Resistance to ciprofloxacin was significantly higher among ST131 isolates. A total of 169 (83.5%) isolates produced CTX-M beta-lactamase, of which 72 (43%) were CTX-M-15. The production of CTX-M and CTX-M-15 were significantly higher among ST131 isolates. We have demonstrated, for the first time, high rates of ST131 clone among ESBL-producing E. coli isolates in Istanbul, a region with high rates of resistance to third-generation cephalosporins and fluoroquinolones. Further investigation of this high-risk clone and its contribution to high antimicrobial resistance in Turkey is essential. MALDI-TOF MS is a useful tool for detection of high-risk clones and associated resistance patterns, simultaneous to bacterial identification.

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.

PubMed

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.

Evidence of genotypic diversity among Candida auris isolates by multilocus sequence typing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and amplified fragment length polymorphism.

PubMed

Prakash, A; Sharma, C; Singh, A; Kumar Singh, P; Kumar, A; Hagen, F; Govender, N P; Colombo, A L; Meis, J F; Chowdhary, A

2016-03-01

Candida auris is a multidrug-resistant nosocomial bloodstream pathogen that has been reported from Asian countries and South Africa. Herein, we studied the population structure and genetic relatedness among 104 global C. auris isolates from India, South Africa and Brazil using multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RPB1, RPB2 and internal transcribed spacer (ITS) and D1/D2 regions of the ribosomal DNA were sequenced for MLST. Further, genetic variation and proteomic assessment was carried out using AFLP and MALDI-TOF MS, respectively. Both MLST and AFLP typing clearly demarcated two major clusters comprising Indian and Brazilian isolates. However, the South African isolates were randomly distributed, suggesting different genotypes. MALDI-TOF MS spectral profiling also revealed evidence of geographical clustering but did not correlate fully with the genotyping methods. Notably, overall the population structure of C. auris showed evidence of geographical clustering by all the three techniques analysed. Antifungal susceptibility testing by the CLSI microbroth dilution method revealed that fluconazole had limited activity against 87% of isolates (MIC90, 64 mg/L). Also, MIC90 of AMB was 4 mg/L. Candida auris is emerging as an important yeast pathogen globally and requires reproducible laboratory methods for identification and typing. Evaluation of MALDI-TOF MS as a typing method for this yeast is warranted. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.

PubMed

Clerc, O; Prod'hom, G; Senn, L; Jaton, K; Zanetti, G; Calandra, T; Greub, G

2014-04-01

Effective empirical treatment is of paramount importance to improve the outcome of patients with Staphylococcus aureus bacteraemia. We aimed to evaluate a PCR-based rapid diagnosis of methicillin resistance (GeneXpert MRSA) after early detection of S. aureus bacteraemia using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients with a first episode of S. aureus bacteraemia identified using MALDI-TOF MS were randomized in a prospective interventional open study between October 2010 and August 2012. In the control group, antibiotic susceptibility testing was performed after MALDI-TOF MS identification on blood culture pellets. In the intervention group, a GeneXpert MRSA was performed after S. aureus identification. The primary outcome was the performance of GeneXpert MRSA directly on blood cultures. We then assessed the impact of early diagnosis of methicillin resistance on the empirical treatment. In all, 197 episodes of S. aureus bacteraemia were included in the study, of which 106 were included in the intervention group. Median time from MALDI-TOF MS identification to GeneXpert MRSA result was 97 min (range 25-250). Detection of methicillin resistance using GeneXpert MRSA had a sensitivity of 99% and a specificity of 100%. There was less unnecessary coverage of MRSA in the intervention group (17.1% versus 29.2%, p 0.09). GeneXpert MRSA was highly reliable in diagnosing methicillin resistance when performed directly on positive blood cultures. This could help to avoid unnecessary prescriptions of anti-MRSA agents and promote the introduction of earlier adequate coverage in unsuspected cases. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Angiotensin-I is Largely Converted to Angiotensin-(1-7) and Angiotensin-(2-10) by Isolated Rat Glomeruli

PubMed Central

JC, Velez; KJ, Ryan; CE, Harbeson; AM, Bland; MN, Budisavljevic; JM, Arthur; WR, Fitzgibbon; JR, Raymond; MG, Janech

2009-01-01

Intraglomerular renin-angiotensin system (RAS) enzyme activities have been examined previously using glomerular lysates and immune-based assays. However, preparation of glomerular extracts compromises the integrity of their anatomic architecture. In addition, antibody-based assays focus on angiotensin (ANG)-II detection, ignoring the generation of other ANG-I-derived metabolites, some of which may cross-react with ANG-II. Therefore, our aim was to examine the metabolism of ANG-I in freshly isolated intact glomeruli using MALDI-TOF mass spectrometry (MS) as an analytical method. Glomeruli from male Sprague-Dawley rats were isolated by sieving and incubated in Krebs buffer in the presence of 1 μM ANG-I for 15 - 90 minutes, with or without various peptidase inhibitors. Peptide sequences were confirmed by MALDI-TOF MS/MS or linear-trap-quadrupole MS. Peaks were quantified using customized valine-13C.15N-labeled peptides as standards. The most prominent peaks resulting from ANG-I cleavage were 899 and 1181 m/z, corresponding to ANG-1-7 and ANG-2-10, respectively. Smaller peaks for ANG-II, ANG-1-9 and ANG-3-10 also were detected. The disappearance of ANG-I was significantly reduced during inhibition of aminopeptidase-A or neprilysin. In contrast, captopril did not alter ANG-I degradation. Furthermore, during simultaneous inhibition of aminopeptidase-A and neprilysin, the disappearance of ANG-I was markedly attenuated compared to all other conditions. These results suggest that there is prominent intraglomerular conversion of ANG-I to ANG-2-10 and ANG-1-7, mediated by aminopeptidase-A and neprilysin, respectively. Formation of these alternative ANG peptides may be critical to counterbalance the local actions of ANG-II. Enhancement of these enzymatic activities may constitute potential therapeutic targets for ANG-II mediated glomerular diseases. PMID:19289651

[Proteome analysis on interaction between Anoectochilus roxburghii and Mycorrhizal fungus].

PubMed

Gao, Chuan; Guo, Shun-Xing; Zhang, Jing; Chen, Juan; Zhang, Li-Chun

2012-12-01

To study the mechanism of plant growing promoted by Mycorrhizal fungus through the difference of proteomes. The differential proteomes between uninoculated and inoculated endophytic fungi, Epulorhiza sp. on Anoectochilus roxburghii were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrum. Twenty-seven protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Twenty-two candidate proteins were identified by database comparisons. The function of these proteins mostly involved in signal transduction, metabolic regulation, as well as photosynthesis and substance metabolism. The results indicate that the regulator control system of plant is influenced by fungi action, and the positive regulation improves substance metabolism and photosynthesis, which results in strong plant and higher resistance. It is also deduced that silent genes may exist in endosymbiosis plants.

Matrix assisted laser desorption ionization time-of-flight mass spectrometry: Velocity measurements using orthogonal and axial injection and applications to characterization of wheat proteins

NASA Astrophysics Data System (ADS)

Dworschak, Ragnar G.

Orthogonal-injection was introduced to allow continuous ion sources to be coupled to time-of-flight mass spectrometers, but also demonstrated promising features for pulsed sources such as MALDI. We tested the feasibility of using a simple implementation orthogonal injection TOF with a MALDI source without collisional cooling. The experiment demonstrated that high resolution is achievable in principle in such an instrument, but only with impractical sacrifices in intensity. Subsequent work in this laboratory has demonstrated that the addition of collisional cooling makes orthogonal MALDI not only feasible, but advantageous in several respects. The instrument used for the above feasibility test was well-suited for measurements of initial velocity distributions in MALDI, avoiding problems of field penetration and questions of timescale of the plume expansion that seemed to produce rather conflicting results in axial TOF measurements. Average initial velocities of peptides and proteins above about 1000 daltons were found to be largely mass independent around 800 m/s, plus or minus about 15% depending on the matrix used. This result is slightly higher, but still quite consistent with earlier measurements using axial TOF with the field-free method (˜750 m/s), but a factor of two higher than the first reports using the delayed-extraction method. The experiments also showed that in contrast to the average velocity, the width of the velocity distribution increases significantly with increasing mass. The matrix velocity measurements confirm earlier experiments that show the benzoic acid derivatives have generally higher velocities than the cinnamic acid derivatives. Measurements of the velocity component in the direction back toward the laser with different sample orientations suggest that the surface orientation is the main determiner of the plume direction. On the other hand, preliminary measurements using the field-free method in the axial TOF geometry show higher velocities of matrix and analyte ions for more normal laser incidence, and for single crystals parallel to the sample surface compared to polycrystalline surfaces, suggesting the orientation of the crystal face with respect to the incident laser direction plays a role in the desorption process. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyse the protein composition in several common and durum wheat varieties. Mass spectra were obtained directly from crude and partially purified wheat gliadin and reduced glutenin subunit fractions. Mass spectra of the gliadins and the low molecular weight glutenin subunits show a complex pattern of proteins in the 30--40 kDa range. The observed gliadin patterns showed some promise for variety identification. The mass spectra of the high molecular weight glutenin subunits are much simpler and the complete high molecular weight subunit profile can be determined directly from a single mass spectrum. This may prove particularly useful in wheat breeding programs for rapid identification of lines containing subunits associated with superior quality.

Corynebacterium tuberculostearicum: a Potentially Misidentified and Multiresistant Corynebacterium Species Isolated from Clinical Specimens

PubMed Central

Hinić, V.; Lang, C.; Weisser, M.; Straub, C.; Frei, R.

2012-01-01

Corynebacterium tuberculostearicum is a lipophilic corynebacterium validly characterized in 2004. We provide clinical information on 18 patients from whom this organism was isolated. The majority of the patients were hospitalized and had a history of prolonged treatment with broad-spectrum antimicrobials. In 7 (38.9%) of the 18 cases, the isolates were found to be clinically relevant. The present report also includes detailed data on the biochemical and molecular identification of C. tuberculostearicum, as well as its identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Our data demonstrate that routine biochemical tests do not provide reliable identification of C. tuberculostearicum. MALDI-TOF MS represents a helpful tool for the identification of this species, since all of the strains matched C. tuberculostearicum as the first choice and 58.3% (7/12) of the strains processed with the full extraction protocol generated scores of >2.000. Nevertheless, partial 16S rRNA gene sequencing still represents the gold standard for the identification of this species. Due to the challenging identification of C. tuberculostearicum, we presume that this organism is often misidentified and its clinical relevance is underestimated. The antimicrobial susceptibility profile of C. tuberculostearicum presented here reveals that 14 (87.5%) of the 16 strains analyzed exhibited multidrug resistance. PMID:22593594

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

PubMed Central

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

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. PMID:26391984

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

PubMed

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

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 (1)H 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.

Fundamental studies of MALDI with an orthogonal TOF mass spectrometer

NASA Astrophysics Data System (ADS)

Qiao, Hui

The interaction between the matrix and analyte molecules are studied with a high resolution MALDI imaging technique in an orthogonal-injection time of flight (TOF) mass spectrometer. The analyte incorporation and distribution patterns have been clearly demonstrated. Purified protein analytes were found to be homogeneously incorporated in large single crystals of DHB and sinapinic acid matrices, with no evidence for preferred crystal faces. Segregation of some species was observed and appeared to correlate with analyte hydrophobicity, and to a lesser extent analyte mass or mobility. Similar segregation phenomena were observed with confocal laser scanning microscopy of the same analytes labeled with fluorescent dyes in 2,5-DHB single crystals. The above investigations may shed some light on optimizing sample preparation with different matrices. The influence of incident laser parameters on sensitivity in MALDI has been investigated using orthogonal-injection TOF instruments. A qualitative comparison was first made between the beam profiles obtained with a N 2 laser and a Nd:YAG laser using 2-m long optical fibers. The N 2 laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N2 laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. Thus, the optimum sensitivity is achieved at high fluence. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. The results have particular relevance for imaging MALDI, where sensitivity and spatial resolution are important figures of merit. Finally the detection properties of the MCP detector were studied with a hybrid MCP and CuBe venetian blind converter detector. The measurements show that the detection efficiency of the MCP drops with the increasing of ion mass and the decreasing of the ion energy. For transferrin (79,500 Da), the relative detection efficiency of the MCP is about 40% at 10.6 keV and it decreases to about 5% at 4.6 keV. The secondary electron emission coefficient of the MCP shows a linear dependence on mass and a power law dependence on velocity (˜3.2). No clear velocity threshold is observed for secondary electron emission.

[Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts].

PubMed

Paco, Karen; Ponce-Soto, Luis Alberto; Lopez-Ilasaca, Marco; Aguilar, José L

2016-01-01

To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

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

PubMed

Justesen, Ulrik Stenz; Holm, Anette; Knudsen, Elisa; Andersen, Line Bisgaard; Jensen, Thøger Gorm; Kemp, Michael; Skov, Marianne Nielsine; Gahrn-Hansen, Bente; Møller, Jens Kjølseth

2011-12-01

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

Data on the identity and myristoylation state of recombinant, purified hippocalcin.

PubMed

Krishnan, Anuradha; Viviano, Jeffrey; Morozov, Yaroslav; Venkataraman, Venkat

2016-09-01

In this data article we report on the purity and post translation modification of bacterially expressed and purified recombinant hippocalcin (HPCA): a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. MALDI-TOF in source decay (ISD) analysis was used to identify both the myristoylated or non-myristoylated forms of the protein. MALDI-TOF ISD data on the identity of the protein, amino acid sequence and myristoylation efficiency are provided. This data relates to the article "Single-Column Purification of the Tag-free, Recombinant Form of the Neuronal Calcium Sensor Protein, Hippocalcin Expressed in Eschericia coli" [1].

[Studies of bacterial typing with MALDI-TOF].

PubMed

Culebras, Esther; Alvarez-Buylla, Adela; Jose Artacho Reinoso, M; Antonio Lepe, Jose

2016-06-01

MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry has emerged as a potential tool for microbial characterization and identification in many microbiology departments. The technology is rapid, sensitive, and relatively inexpensive in terms of both the labour and costs involved. This review provides an overview on its utility for strain typing and epidemiological studies and explains the methodological approaches that can be used both for the performance of the technique and for the analysis of results. Finally, the review summarizes studies on the characterization of distinct bacterial species. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of degradation products after treatment of methylene blue aqueous solution with three-dimensionally integrated microsolution plasma

NASA Astrophysics Data System (ADS)

Shirafuji, Tatsuru; Nomura, Ayano; Hayashi, Yui; Tanaka, Kenji; Goto, Motonobu

2016-01-01

Methylene blue can be degraded in three-dimensionally integrated microsolution plasma. The degradation products have been analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry to understand the degradation mechanisms. The results of MALDI TOF mass spectrometry have shown that sulfoxide is formed at the first stage of the oxidation. Then, partial oxidation proceeds on the methyl groups left on the sulfoxide. The sulfoxide is subsequently separated to two benzene derivatives. Finally, weak functional groups are removed from the benzene derivatives.

Comparative evaluation of matrix-assisted laser desorption ionisation-time of flight mass spectrometry and conventional phenotypic-based methods for identification of clinically important yeasts in a UK-based medical microbiology laboratory.

PubMed

Fatania, Nita; Fraser, Mark; Savage, Mike; Hart, Jason; Abdolrasouli, Alireza

2015-12-01

Performance of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was compared in a side-by side-analysis with conventional phenotypic methods currently in use in our laboratory for identification of yeasts in a routine diagnostic setting. A diverse collection of 200 clinically important yeasts (19 species, five genera) were identified by both methods using standard protocols. Discordant or unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene. MALDI-TOF and conventional methods were in agreement for 182 isolates (91%) with correct identification to species level. Eighteen discordant results (9%) were due to rarely encountered species, hence the difficulty in their identification using traditional phenotypic methods. MALDI-TOF MS enabled rapid, reliable and accurate identification of clinically important yeasts in a routine diagnostic microbiology laboratory. Isolates with rare, unusual or low probability identifications should be confirmed using robust molecular methods. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Rapid Identification of Bacteria in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry▿

PubMed Central

Stevenson, Lindsay G.; Drake, Steven K.; Murray, Patrick R.

2010-01-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid, accurate method for identifying bacteria and fungi recovered on agar culture media. We report herein a method for the direct identification of bacteria in positive blood culture broths by MALDI-TOF mass spectrometry. A total of 212 positive cultures were examined, representing 32 genera and 60 species or groups. The identification of bacterial isolates by MALDI-TOF mass spectrometry was compared with biochemical testing, and discrepancies were resolved by gene sequencing. No identification (spectral score of <1.7) was obtained for 42 (19.8%) of the isolates, due most commonly to insufficient numbers of bacteria in the blood culture broth. Of the bacteria with a spectral score of ≥1.7, 162 (95.3%) of 170 isolates were correctly identified. All 8 isolates of Streptococcus mitis were misidentified as being Streptococcus pneumoniae isolates. This method provides a rapid, accurate, definitive identification of bacteria within 1 h of detection in positive blood cultures with the caveat that the identification of S. pneumoniae would have to be confirmed by an alternative test. PMID:19955282

Differentiation in MALDI-TOF MS and FTIR spectra between two pathovars of Xanthomonas oryzae

NASA Astrophysics Data System (ADS)

Ge, Mengyu; Li, Bin; Wang, Li; Tao, Zhongyun; Mao, Shengfeng; Wang, Yangli; Xie, Guanlin; Sun, Guochang

2014-12-01

Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains are closely related phenotypically and genetically, which make it difficult to differentiate between the two pathovars based on phenotypic and DNA-based methods. In this study, a fast and accurate method was developed based on the differences in MALDI-TOF MS and FTIR spectra between the two pathovars. MALDI-TOF MS analysis revealed that 9 and 10 peaks are specific to Xoo and Xoc, respectively, which can be used as biomarkers to identify and differentiate the two closely related pathovars. Furthermore, FTIR analysis showed that there is a significant difference in both the band frequencies and absorption intensity of various functional groups between the two pathovars. In particular, the 6 peaks at 3433, 2867, 1273, 1065, 983 and 951 cm-1 were specific to the Xoo strains, while one peak at 1572 cm-1 was specific to the Xoc strains. Overall, this study gives the first attempt to identify and differentiate the two pathovars of X. oryzae based on mass and FTIR spectra, which will be helpful for the early detection and prevention of the two rice diseases caused by both X. oryzae pathovars.

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in Alzheimer's disease.

PubMed

Grela, Agatha; Turek, Agata; Piekoszewski, Wojciech

2012-02-11

Alzheimer's disease is becoming an increasing problem in our aging society. According to our knowledge, so far, no effective pharmacotherapy to cure the cause of the disease has been developed. Therefore, early diagnosis is needed, which will result in implementation of a drug therapy aimed at decreasing and/or inhibiting disease development. Mass spectrometry techniques (MS) have a wide range of applications in proteomics and the search for biomarkers of neurodegenerative disorders, opening new possibilities in diagnostics. Identification of proteins in body fluids (like cerebrospinal fluid or blood) is possible due to MS spectra analysis. The detected changes in protein concentrations are connected with pathological states in an organism and, therefore, can be regarded as biomarkers. Developing procedures for proteome analysis might result in fast diagnosis, as well as creating better suited pharmaceuticals. This paper reviews the search of biomarkers in cerebrospinal fluid and blood. Later on, the use of matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in proteomics, focusing on blood-related biomarkers, is discussed. The aim of the work is also to highlight the advantages and disadvantages of MALDI-TOF-based analyses.

Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Stevenson, Lindsay G; Drake, Steven K; Murray, Patrick R

2010-02-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid, accurate method for identifying bacteria and fungi recovered on agar culture media. We report herein a method for the direct identification of bacteria in positive blood culture broths by MALDI-TOF mass spectrometry. A total of 212 positive cultures were examined, representing 32 genera and 60 species or groups. The identification of bacterial isolates by MALDI-TOF mass spectrometry was compared with biochemical testing, and discrepancies were resolved by gene sequencing. No identification (spectral score of < 1.7) was obtained for 42 (19.8%) of the isolates, due most commonly to insufficient numbers of bacteria in the blood culture broth. Of the bacteria with a spectral score of > or = 1.7, 162 (95.3%) of 170 isolates were correctly identified. All 8 isolates of Streptococcus mitis were misidentified as being Streptococcus pneumoniae isolates. This method provides a rapid, accurate, definitive identification of bacteria within 1 h of detection in positive blood cultures with the caveat that the identification of S. pneumoniae would have to be confirmed by an alternative test.

Microbiology neutralization of zearalenone using Lactococcus lactis and Bifidobacterium sp.

PubMed

Król, A; Pomastowski, P; Rafińska, K; Railean-Plugaru, V; Walczak, J; Buszewski, B

2018-01-01

The aim of the study was to neutralize zearalenone by lactic acid bacteria (LAB) such as Lactococcus lactis and Bifidobacterium sp. and investigate the mechanism of zearalenone (ZEA) binding. Neutralization of ZEA by LAB was confirmed by identification of binding kinetics and spectroscopic studies such as Fourier transform infrared spectroscopy (FT-IR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The obtained results showed that the kinetic process of zearalenone binding to L. lactis is not homogeneous but is expressed with an initial rapid stage with about 90% of ZEA biosorption and with a much slower second step. In case of Bifidobacterium sp., the neutralization process is homogeneous; the main stage can be described with about 88% of ZEA biosorption. MALDI-TOF-MS measurements and FTIR analysis confirmed the uptake of zearalenone molecules by bacterial species. Moreover, the assessment of dead and live lactic acid bacteria cells after zearalenone treatment was performed using fluorescence microscopy. Graphical abstract Microbiology neutralization of zearalenone using Lactococcus lactis and Bifidobacterium sp. was confirmed by identification of binding kinetics and spectroscopic studies such as FT-IR spectroscopy and MALDI-TOF-MS spectrometry. The mechanism of ZEA binding was also investigated.

Short communication: Identification of subclinical cow mastitis pathogens in milk by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Barreiro, J R; Ferreira, C R; Sanvido, G B; Kostrzewa, M; Maier, T; Wegemann, B; Böttcher, V; Eberlin, M N; dos Santos, M V

2010-12-01

Subclinical mastitis is a common and easily disseminated disease in dairy herds. Its routine diagnosis via bacterial culture and biochemical identification is a difficult and time-consuming process. In this work, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows bacterial identification with high confidence and speed (1 d for bacterial growth and analysis). With the use of MALDI-TOF MS, 33 bacterial culture isolates from milk of different dairy cows from several farms were analyzed, and the results were compared with those obtained by classical biochemical methods. This proof-of-concept case demonstrates the reliability of MALDI-TOF MS bacterial identification, and its increased selectivity as illustrated by the additional identification of coagulase-negative Staphylococcus species and mixed bacterial cultures. Matrix-assisted laser desorption-ionization mass spectrometry considerably accelerates the diagnosis of mastitis pathogens, especially in cases of subclinical mastitis. More immediate and efficient animal management strategies for mastitis and milk quality control in the dairy industry can therefore be applied. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

High Throughput Detection of Tetracycline Residues in Milk Using Graphene or Graphene Oxide as MALDI-TOF MS Matrix

NASA Astrophysics Data System (ADS)

Liu, Junyan; Liu, Yang; Gao, Mingxia; Zhang, Xiangmin

2012-08-01

In this work, a new pre-analysis method for tetracyclines (TCs) detection from the milk samples was established. As a good accomplishment for the existing accurate quantification strategies for TCs detection, the new pre-analysis method was demonstrated to be simple, sensitive, fast, cost effective, and high throughput, which would do a great favor to the routine quality pre-analysis of TCs from milk samples. Graphene or graphene oxide was utilized, for the first time, as a duel-platform to enrich and detect the TCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All together, four TCs were chosen as models: tetracycline, oxytetracycline, demeclocycline, and chlortetracycline. Due to the excellent electronic, thermal, and mechanical properties, graphene and graphene oxide were successfully applied as matrices for MALDI-TOF MS with free background inference in low mass range. Meanwhile, graphene or graphene oxide has a large surface area and strong interaction force with the analytes. By taking the advantage of these features, TCs were effectively enriched with the limit of detection (LOD) as low as 2 nM.

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

PubMed

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 (<500 Da). In this work, a novel 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.

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

USDA-ARS?s Scientific Manuscript database

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

MALDI-based intact spore mass spectrometry of downy and powdery mildews.

PubMed

Chalupová, Jana; Sedlářová, Michaela; Helmel, Michaela; Rehulka, Pavel; Marchetti-Deschmann, Martina; Allmaier, Günter; Sebela, Marek

2012-08-01

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI-TOF MS-based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer-based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10(9) spores per ml. The best peptide/protein profiles (in terms of signal-to-noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright © 2012 John Wiley & Sons, Ltd.

Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation.

PubMed

Schmitt, Bryan H; Cunningham, Scott A; Dailey, Aaron L; Gustafson, Daniel R; Patel, Robin

2013-03-01

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria.

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

PubMed

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.

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

PubMed Central

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. PMID:28555175

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

PubMed

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

2018-01-01

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

MALDI-typing of infectious algae of the genus Prototheca using SOM portraits.

PubMed

Wirth, Henry; von Bergen, Martin; Murugaiyan, Jayaseelan; Rösler, Uwe; Stokowy, Tomasz; Binder, Hans

2012-01-01

MALDI-typing has become a frequently used approach for the identification of microorganisms and recently also of invertebrates. Similarity-comparisons are usually based on single-spectral data. We apply self-organizing maps (SOM) to portray the MS-spectral data with individual resolution and to improve the typing of Prototheca algae by using meta-spectra representing prototypes of groups of similar-behaving single spectra. The MALDI-TOF peaklists of more than 300 algae extracts referring to five Prototheca species were transformed into colored mosaic images serving as molecular portraits of the individual samples. The portraits visualize the algae-specific distribution of high- and low-amplitude peaks in two dimensions. Species-specific pattern of MS intensities were readily discernable in terms of unique single spots of high amplitude MS-peaks which collect characteristic fingerprint spectra. The spot patterns allow the visual identification of groups of samples referring to different species, genotypes or isolates. The use of meta-peaks instead of single-peaks reduces the dimension of the data and leads to an increased discriminating power in downstream analysis. We expect that our SOM portray method improves MS-based classifications and feature selection in upcoming applications of MALDI-typing based species identifications especially of closely related species. Copyright © 2011. Published by Elsevier B.V.