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Sample records for microscopic maldi-tof ms

  1. MALDI-TOF MS in Prenatal Genomics

    PubMed Central

    Zhong, Xiao Yan; Holzgreve, Wolfgang

    2009-01-01

    Summary Prenatal diagnosis aims either to provide the reassurance to the couples at risk of having an affected child by timely appropriate therapy or to give the parents a chance to decide the fate of the unborn babies with health problems. Invasive prenatal diagnosis (IPD) is accurate, however, carrying a risk of miscarriage. Non-invasive prenatal diagnosis (NIPD) has been developed based on the existing of fetal genetic materials in maternal circulation; however, a minority fetal DNA in majority maternal background DNA hinders the detections of fetal traits. Different protocols and assays, such as homogenous MassEXTEND (hME), single allele base extension reaction (SABER), precise measuring copy number variation of each allele, and quantitative methylation and expression analysis using the high-throughput sensitive matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), allow NIPD for single gene disorders, fetal blood group genotyping and fetal aneuploidies as well as the development of fetal gender-independent biomarkers in maternal circulation for management of pathological pregnancies. In this review, we summarise the use of MALDI-TOF MS in prenatal genomics. PMID:21049077

  2. Applications of MALDI-TOF MS in environmental microbiology.

    PubMed

    Santos, Inês C; Hildenbrand, Zacariah L; Schug, Kevin A

    2016-05-10

    Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is an emerging technique for microbial identification, characterization, and typing. The single colony method can be used for obtaining a protein fingerprint or profile unique to each microorganism. This technique has been mainly used in the clinical field, but it also has significant potential in the environmental field. The applications of MALDI-TOF MS in environmental microbiology are discussed in this review. An overview on the use of MALDI-TOF MS for environmental proteomics and metabolomics is given as well as its use for bacterial strain typing and bioremediation research. A more detailed review on the use of this technique for the identification, differentiation, and categorization of environmental microorganisms is given. Some of the parameters that can influence the results and reproducibility of MALDI-TOF MS are also discussed. PMID:27072574

  3. MALDI-TOF MS quantification of coccidiostats in poultry feeds.

    PubMed

    Wang, J; Sporns, P

    2000-07-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a relatively new technique that is having a great impact on analyses. This study is the first to demonstrate the use of linear MALDI-TOF MS to identify and quantify coccidiostats in poultry feeds. 2,5-Dihydroxybenzoic acid (DHB) was found to be the best matrix. In MALDI-TOF MS, coccidiostats form predominantly [M + Na](+) ions, with additional small amounts of [M + K](+) and [M - H + 2Na](+) ions, and no obvious fragment ions. Salinomycin and narasin were unstable in the concentrated DHB matrix solution but were stable when dried on the MALDI-TOF MS probe. A simple fast Sep-pak C18 cartridge purification procedure was developed for the MALDI-TOF MS quantification of coccidiostats in poultry feeds. The MALDI-TOF MS limit of detection for lasalocid, monensin, salinomycin, and narasin standards was 251, 22, 24, and 24 fmol, respectively. The method detection limit for salinomycin and narasin in poultry feeds was 2.4 microgram/g. PMID:10898626

  4. Identification of flea species using MALDI-TOF/MS.

    PubMed

    Yssouf, Amina; Socolovschi, Cristina; Leulmi, Hamza; Kernif, Tahar; Bitam, Idir; Audoly, Gilles; Almeras, Lionel; Raoult, Didier; Parola, Philippe

    2014-05-01

    In the present study, a molecular proteomics (MALDI-TOF/MS) approach was used as a tool for identifying flea vectors. We measured the MS spectra from 38 flea specimens of 5 species including Ctenocephalides felis, Ctenocephalides canis, Archaeopsylla erinacei, Xenopsylla cheopis and Stenoponia tripectinata. A blind test performed with 24 specimens from species included in a library spectral database confirmed that MALDI-TOF/MS is an effective tool for discriminating flea species. Although fresh and 70% ethanol-conserved samples subjected to MALDI-TOF/MS in blind tests were correctly classified, only MS spectra of quality from fresh specimens were sufficient for accurate and significant identification. A cluster analysis highlighted that the MALDI Biotyper can be used for studying the phylogeny of fleas. PMID:24878069

  5. Rapid identification of Streptomyces isolates by MALDI-TOF MS.

    PubMed

    Loucif, Lotfi; Bendjama, Esma; Gacemi-Kirane, Djamila; Rolain, Jean-Marc

    2014-12-01

    The recent emergence of multidrug-resistant bacteria over the last decade has led to a renewal in the discovery of new antimicrobial drugs. Streptomyces members are practically unlimited sources of new antibiotics. However, the identification of Streptomyces species is difficult and time-consuming. Therefore, there is a need for alternative methods for their rapid identification. In this study, an efficient protocol of identification using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was developed and applied for the rapid identification of Streptomyces isolates from the El Kala lakes in northeastern Algeria. A collection of 48 Streptomyces isolates were used for this study. The optimized procedure allowed us to obtain specific and reproducible protein spectra for each Streptomyces isolate tested. The spectra generated were used to build a preliminary local database based on their initial 16S rRNA identification. The blind test used for the identification of 20 Streptomyces strains already available in our created database and 20 unknown Streptomyces isolates showed that all (100%) of the Streptomyces strains listed in the database were rapidly (<30min) identified with high scores of up to 2.8. Here, for the first time we showed that MALDI-TOF MS could be used as a cost-effective tool for the rapid identification of Streptomyces isolates. PMID:24862894

  6. Challenges in biomarker discovery with MALDI-TOF MS.

    PubMed

    Hajduk, Joanna; Matysiak, Jan; Kokot, Zenon J

    2016-07-01

    MALDI-TOF MS technique is commonly used in system biology and clinical studies to search for new potential markers associated with pathological conditions. Despite numerous concerns regarding a sample preparation or processing of complex data, this strategy is still recognized as a popular tool and its awareness has risen in the proteomic community over the last decade. In this review, we present comprehensive application of MALDI mass spectrometry with special focus on profiling research. We also discuss major advantages and disadvantages of universal sample preparation methods such as micro-SPE columns, immunodepletion or magnetic beads, and we show the potential of nanostructured materials in capturing low molecular weight subproteomes. Furthermore, as the general protocol considerably affects spectra quality and interpretation, an alternative solution for improved ion detection, including hydrophobic constituents, data processing and statistical analysis is being considered in up-to-date profiling pattern. In conclusion, many reports involving MALDI-TOF MS indicated highly abundant proteins as valuable indicators, and at the same time showed the inaccuracy of available methods in the detection of low abundant proteome that is the most interesting from the clinical perspective. Therefore, the analytical aspects of sample preparation methods should be standardized to provide a reproducible, low sample handling and credible procedure. PMID:27134187

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. Identification of Dermatophyte Species after Implementation of the In-House MALDI-TOF MS Database

    PubMed Central

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

    2014-01-01

    Despite that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool in the clinical microbiology setting, few studies have till now focused on MALDI-TOF MS-based identification of dermatophytes. In this study, we analyze dermatophytes strains isolated from clinical samples by MALDI-TOF MS to supplement the reference database available in our laboratory. Twenty four dermatophytes (13 reference strains and 11 field isolated strains), identified by both conventional and molecular standard procedures, were analyzed by MALDI-TOF MS, and the spectra obtained were used to supplement the available database, limited to a few species. To verify the robustness of the implemented database, 64 clinical isolates other than those used for the implementation were identified by MALDI-TOF MS. The implementation allowed the identification of the species not included in the original database, reinforced the identification of the species already present and correctly identified those within the Trichophyton mentagrophytes complex previously classified as Trichophyton. tonsurans by MALDI-TOF MS. The dendrogram obtained by analyzing the proteic profiles of the different species of dermatophytes reflected their taxonomy, showing moreover, in some cases, a different clusterization between the spectra already present in the database and those newly added. In this study, MALDI-TOF MS proved to be a useful tool suitable for the identification of dermatophytes for diagnostic purpose. PMID:25216335

  9. Quantitative determination of Piroxicam by TLC-MALDI TOF MS.

    PubMed

    Crecelius, Anna; Clench, Malcolm R; Richards, Don S; Parr, Vic

    2004-04-01

    A quantitative thin-layer chromatography (TLC)-matrix-assisted laser desorption (MALDI) TOF mass spectrometry (MS) method for the determination of Piroxicam has been developed. Following preliminary experiments three different approaches to the incorporation of the internal standard (Tenoxicam) into the TLC plates were investigated. These were: (a) adding the internal standard to the mobile phase and pre-developing the plate, (b) coating the plate with internal standard by electrospraying prior to matrix application and finally, (c) mixing the internal standard into the matrix solution and electrospraying both. The most successful method was that where the internal standard was pre-developed over the plate. For this method linearity was observed over the range between 400 and 800ng of Piroxicam. The precision was found to be in the range of 1-9% R.S.D. from the average detected value (n = 5), dependent on the amount of analyte on the TLC plate. The proposed method was accurate with +/-2% deviation from the known amount of Piroxicam in the sample spot. PMID:15030877

  10. Ellagitannin Composition of Blackberry As Determined by HPLC-ESI-MS and MALDI-TOF-MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apache blackberries (Rubus sp.) were evaluated by HPLC-MS and MALDI-TOF-MS to identify ellagitannins present in the flesh, torus (receptacle tissue), and seeds. Most ellagitannins were only present or detectable in seed tissues. Ellagitannins identified by HPLC-MS in the seeds included pedunculagi...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. [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. PMID:25011591

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

    PubMed

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

    2015-01-01

    The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification. PMID:25882136

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

    NASA Astrophysics Data System (ADS)

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

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

  15. 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

  16. Rapid detection of carbapenemase activity: benefits and weaknesses of MALDI-TOF MS.

    PubMed

    Mirande, C; Canard, I; Buffet Croix Blanche, S; Charrier, J-P; van Belkum, A; Welker, M; Chatellier, S

    2015-11-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an identification procedure for bacteria and fungi. The MALDI-TOF MS-based analysis of resistance to β-lactam antibiotics has been applied to detect hydrolysis of carbapenems by different bacterial strains. However, the detection of enzymatic carbapenem degradation by MALDI-TOF MS lacks well-standardized protocols and several methods and models of interpretation using different calculations of ratio-of-peak intensities have been described in the literature. Here, we used faropenem and ertapenem hydrolysis as model compounds. In an attempt to propose a universal protocol, the hydrolysis was regularly monitored during 24 h using well-characterized bacterial strains producing different types of carbapenemases (KPC, IMP, NDM, VIM, and OXA-48). Variable responses and different timing for detectable hydrolysis, depending on the enzyme produced, were observed. KPC degrades its template antibiotics very quickly (15 min for some KPC producers) compared to other types of enzymes (more than 90 min for other enzymes). Prior bacterial lysis was shown to be of no interest in the modulation or optimization of the hydrolytic kinetics. The adequate detection of carbapenem hydrolysis would, therefore, require several MALDI-TOF MS readouts for the timely detection of rapid hydrolysis without missing slow hydrolysis. This enzymatic constraint limits the implementation of a standard protocol in routine microbiology laboratories. PMID:26337432

  17. 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

  18. 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. PMID:27117163

  19. 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

  20. 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. PMID:27044871

  1. Evaluation of the MALDI-TOF MS profiling for identification of newly described Aeromonas spp.

    PubMed

    Vávrová, Andrea; Balážová, Tereza; Sedláček, Ivo; Tvrzová, Ludmila; Šedo, Ondrej

    2015-09-01

    The genus Aeromonas comprises primarily aquatic bacteria and also serious human and animal pathogens with the occurrence in clinical material, drinking water, and food. Aeromonads are typical for their complex taxonomy and nomenclature and for limited possibilities of identification to the species level. According to studies describing the use of MALDI-TOF MS in diagnostics of aeromonads, this modern chemotaxonomical approach reveals quite high percentage of correctly identified isolates. We analyzed 64 Aeromonas reference strains from the set of 27 species. After extending the range of analyzed Aeromonas species by newly described ones, we proved that MALDI-TOF MS procedure accompanied by Biotyper tool is not a reliable diagnostic technique for aeromonads. We obtained quite high percentage of false-positive, incorrect, and uncertain results. The identification of newly described species is accompanied with misidentifications that were observed also in the case of pathogenic aeromonads. PMID:25520239

  2. Potential pitfalls in MALDI-TOF MS analysis of abiotically synthesized RNA oligonucleotides.

    PubMed

    Burcar, Bradley T; Cassidy, Lauren M; Moriarty, Elizabeth M; Joshi, Prakash C; Coari, Kristin M; McGown, Linda B

    2013-06-01

    Demonstration of the abiotic polymerization of ribonucleotides under conditions consistent with conditions that may have existed on the prebiotic Earth is an important goal in "RNA world" research. Recent reports of abiotic RNA polymerization with and without catalysis rely on techniques such as HPLC, gel electrophoresis, and MALDI-TOF MS to analyze the reaction products. It is essential to understand the limitations of these techniques in order to accurately interpret the results of these analyses. In particular, techniques that rely on mass for peak identification may not be able to distinguish between a single, linear RNA oligomer and stable aggregates of smaller linear and/or cyclic RNA molecules. In the case of MALDI-TOF MS, additional complications may arise from formation of salt adducts and MALDI matrix complexes. This is especially true for abiotic RNA polymerization reactions because the concentration of longer RNA chains can be quite low and RNA, as a polyelectrolyte, is highly susceptible to adduct formation and aggregation. Here we focus on MALDI-TOF MS analysis of abiotic polymerization products of imidazole-activated AMP in the presence and absence of montmorillonite clay as a catalyst. A low molecular weight oligonucleotide standard designed for use in MALDI-TOF MS and a 3'-5' polyadenosine monophosphate reference standard were also run for comparison and calibration. Clay-catalyzed reaction products of activated GMP and UMP were also examined. The results illustrate the ambiguities associated with assignment of m/z values in MALDI mass spectra and the need for accurate calibration of mass spectra and careful sample preparation to minimize the formation of adducts and other complications arising from the MALDI process. PMID:23793938

  3. Identification of Disseminated Cryptococcosis Using MALDI-TOF MS and Clinical Evaluation.

    PubMed

    Tarumoto, Norihito; Sakai, Jun; Kodana, Masahiro; Kawamura, Tohru; Ohno, Hideaki; Maesaki, Shigefumi

    2016-01-01

    Disseminated cryptococcosis is rare but can often become severe with a poor outcome. Given recent reports that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyser is useful for Cryptococcus species identification, it was applied retrospectively to past cases of disseminated cryptococcosis at our hospital over the past 10 years, and their clinical courses were reviewed. For each case, the retained Cryptococcus spp. were used for identification using both MALDI-TOF MS and genetic sequencing, as well as for drug susceptibility testing. A total of eight cases were found. Cryptococcus spp. were found in cerebrospinal fluid in 3 cases and blood in 5 cases; anti-HIV antibody was either negative or untested. MALDI-TOF MS identified Cryptococcus neoformans as the pathogen in all 8 cases, but genetic testing identified one of these as Cryptococcus curvatus. The outcome was death within 30 days in 5 of the total 8 cases and in 2 of the 3 cases in which C. neoformans was detected in the cerebrospinal fluid, despite regimens and dosages that followed IDSA Guidelines in all 3 cases. Drug susceptibility testing showed no drug resistance that would have affected the therapy. In conclusion, the outcomes were very poor in these drug-susceptible cases, despite treatment in full accordance with standard guidelines. This study confirmed the need to develop newer therapies as well as the high capability of MALDI-TOF MS for the identification of C. neoformans. Genetic testing, however, may be necessary if non-neoformans Cryptococcus is suspected. PMID:27581774

  4. Potential Pitfalls in MALDI-TOF MS Analysis of Abiotically Synthesized RNA Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Burcar, Bradley T.; Cassidy, Lauren M.; Moriarty, Elizabeth M.; Joshi, Prakash C.; Coari, Kristin M.; McGown, Linda B.

    2013-06-01

    Demonstration of the abiotic polymerization of ribonucleotides under conditions consistent with conditions that may have existed on the prebiotic Earth is an important goal in "RNA world" research. Recent reports of abiotic RNA polymerization with and without catalysis rely on techniques such as HPLC, gel electrophoresis, and MALDI-TOF MS to analyze the reaction products. It is essential to understand the limitations of these techniques in order to accurately interpret the results of these analyses. In particular, techniques that rely on mass for peak identification may not be able to distinguish between a single, linear RNA oligomer and stable aggregates of smaller linear and/or cyclic RNA molecules. In the case of MALDI-TOF MS, additional complications may arise from formation of salt adducts and MALDI matrix complexes. This is especially true for abiotic RNA polymerization reactions because the concentration of longer RNA chains can be quite low and RNA, as a polyelectrolyte, is highly susceptible to adduct formation and aggregation. Here we focus on MALDI-TOF MS analysis of abiotic polymerization products of imidazole-activated AMP in the presence and absence of montmorillonite clay as a catalyst. A low molecular weight oligonucleotide standard designed for use in MALDI-TOF MS and a 3'-5' polyadenosine monophosphate reference standard were also run for comparison and calibration. Clay-catalyzed reaction products of activated GMP and UMP were also examined. The results illustrate the ambiguities associated with assignment of m/z values in MALDI mass spectra and the need for accurate calibration of mass spectra and careful sample preparation to minimize the formation of adducts and other complications arising from the MALDI process.

  5. 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. PMID:27227555

  6. Assessment of MALDI-TOF MS as Alternative Tool for Streptococcus suis Identification.

    PubMed

    Pérez-Sancho, Marta; Vela, Ana Isabel; García-Seco, Teresa; Gottschalk, Marcelo; Domínguez, Lucas; Fernández-Garayzábal, José Francisco

    2015-01-01

    The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Streptococcus suis isolates obtained from pigs, wild animals, and humans was evaluated using a PCR-based identification assay as the gold standard. In addition, MALDI-TOF MS was compared with the commercial multi-tests Rapid ID 32 STREP system. From the 129 S. suis isolates included in the study and identified by the molecular method, only 31 isolates (24.03%) had score values ≥2.300 and 79 isolates (61.24%) gave score values between 2.299 and 2.000. After updating the currently available S. suis MALDI Biotyper database with the spectra of three additional clinical isolates of serotypes 2, 7, and 9, most isolates had statistically significant higher score values (mean score: 2.65) than those obtained using the original database (mean score: 2.182). Considering the results of the present study, we suggest using a less restrictive threshold score of ≥2.000 for reliable species identification of S. suis. According to this cut-off value, a total of 125 S. suis isolates (96.9%) were correctly identified using the updated database. These data indicate an excellent performance of MALDI-TOF MS for the identification of S. suis. PMID:26347858

  7. Assessment of MALDI-TOF MS as Alternative Tool for Streptococcus suis Identification

    PubMed Central

    Pérez-Sancho, Marta; Vela, Ana Isabel; García-Seco, Teresa; Gottschalk, Marcelo; Domínguez, Lucas; Fernández-Garayzábal, José Francisco

    2015-01-01

    The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Streptococcus suis isolates obtained from pigs, wild animals, and humans was evaluated using a PCR-based identification assay as the gold standard. In addition, MALDI-TOF MS was compared with the commercial multi-tests Rapid ID 32 STREP system. From the 129 S. suis isolates included in the study and identified by the molecular method, only 31 isolates (24.03%) had score values ≥2.300 and 79 isolates (61.24%) gave score values between 2.299 and 2.000. After updating the currently available S. suis MALDI Biotyper database with the spectra of three additional clinical isolates of serotypes 2, 7, and 9, most isolates had statistically significant higher score values (mean score: 2.65) than those obtained using the original database (mean score: 2.182). Considering the results of the present study, we suggest using a less restrictive threshold score of ≥2.000 for reliable species identification of S. suis. According to this cut-off value, a total of 125 S. suis isolates (96.9%) were correctly identified using the updated database. These data indicate an excellent performance of MALDI-TOF MS for the identification of S. suis. PMID:26347858

  8. 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. PMID:24338887

  9. Top-down proteomic identification of protein biomarkers of food-borne pathogens using MALDI-TOF-TOF-MS/MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter describes a step-by-step protocol and discussion of top-down proteomic identification of protein biomarkers of food-borne pathogens using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) and web-based software developed in the Pro...

  10. 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. PMID:22282100

  11. 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

    2016-09-01

    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. PMID:27479043

  12. A population-based study of aerococcal bacteraemia in the MALDI-TOF MS-era.

    PubMed

    Senneby, E; Göransson, L; Weiber, S; Rasmussen, M

    2016-05-01

    The purpose of this study was to determine the incidence of aerococcal bacteraemia in the MALDI-TOF MS-era, to describe the clinical presentation and to determine the MIC values of aerococci for ten antibiotics. Aerococci in blood cultures were identified through searches in the laboratory database for the years 2012-2014. MALDI-TOF MS, sequencing of the 16S rRNA gene and a PYR test were used for species identification. Patients' medical charts were systematically reviewed. Etests were used to determine MIC values. Seventy-seven patients were identified (Aerococcus urinae n = 49, Aerococcus viridans n = 14, Aerococcus sanguinicola n = 13 and Aerococcus christensenii n = 1) corresponding to incidences of 14 cases per 1,000,000 inhabitants per year (A. urinae) and 3.5 cases per 1,000,000 inhabitants per year (A. sanguinicola and A.viridans). A. urinae was in pure culture in 61 %, A. sanguinicola in 46 % and A. viridans in 36 % of the cases. The A. urinae and A. sanguinicola patients were old and many had urinary tract disorders, and a majority had a suspected urinary tract focus of the bacteraemia. Eighty percent of the A. urinae patients were men. Five A. urinae patients were diagnosed with infective endocarditis. Six patients died within 30 days. Most isolates had low MICs to penicillins and carbapenems. MALDI-TOF MS has led to an increased identification of aerococcal bacteremia. A. urinae remains the most common Aerococcus in blood cultures and in aerococcal IE. PMID:26838685

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

    PubMed

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

    2016-05-01

    Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe-IMAC column chromatography and subjected to LC-MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI-TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC-MS/MS systems was comparable to that of using alternative proteases such as Asp-N, Arg-C, chymotrypsin, Glu-C and Lys-C on just one LC-MS/MS instrument. Notably, MALDI-TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (∼20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC-MALDI MS/MS can be a useful complement to LC-nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides. PMID:26990019

  14. Determining and characterizing hapten loads for carrier proteins by MALDI-TOF MS and MALDI-TOF/RTOF MS.

    PubMed

    Marchetti-Deschmann, Martina; Stephan, Christopher; Häubl, Georg; Allmaier, Günter; Krska, Rudolf; Cvak, Barbara

    2016-07-15

    The increasing number of bioconjugates used for bioanalytical purposes and in pharmaceutical industries has led to an increasing demand for robust quality control of products derived from covalently linking small molecules to proteins. Here we report, for the first time, a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)-based method to determine the quantity and location of the hapten zearalenone (ZEN) introduced to the carrier protein conalbumin (Con). This bioconjugate is of special interest because of its application in lateral flow immunoassays commercially available for fast testing of food and feed for the presence of ZEN, a common contaminant of all major cereal grains worldwide. Mass spectrometry (MS) analysis of the intact protein turned out to be highly reproducible allowing for the determination of the average hapten load of the carrier protein. In that way an easy and fast method to screen for changes in ZEN load after bioconjugate synthesis was established. For a more detailed hapten load characterization, measurements at the peptide level were of importance. Systematic studies, implementing post-source decay (PSD) and high- and low-energy collision-induced dissociation (CID), showed characteristic fragmentation pattern for three model peptides carrying between one and three lysines (the primary target for the ZEN modification) besides other, less obvious modification sites (serine, arginine and the N-terminus). By this, indicative reporter ions (m/z 203 and 316) and neutral losses (Δm/z 373 and 317) for the ZEN modification in general, plus immonium ions (m/z 87, 142 and 159) for the lysine modification in particular were identified. Based on these findings, proteolytic peptides, tentatively assigned to be modified, were unequivocally confirmed to be affected by bioconjugation. For a protein carrying on average only 2-3 modifications per molecule 29 Lys out of 59 potential modifications sites were actually modified

  15. Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS.

    PubMed

    Ziegler, Dominik; Pothier, Joël F; Ardley, Julie; Fossou, Romain Kouakou; Pflüger, Valentin; de Meyer, Sofie; Vogel, Guido; Tonolla, Mauro; Howieson, John; Reeve, Wayne; Perret, Xavier

    2015-07-01

    Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS. PMID:25776061

  16. 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.

  17. 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. PMID:26956387

  18. CIEF and MALDI-TOF-MS methods for analyzing forms of the glycoprotein VEGF 165.

    PubMed

    Ongay, Sara; Puerta, Angel; Díez-Masa, Jose Carlos; Bergquist, Jonas; de Frutos, Mercedes

    2009-04-01

    The vascular endothelial growth factor (VEGF) is involved in different sicknesses (cardiovascular diseases, cancer, and other). Out of the many components of the VEGF family, the A splice variant with 165 amino acids (VEGF(165)) is the main component. In spite of the potential as biomarker that this protein has, information about its physico-chemical characteristics is scarce. In this study CIEF and MALDI-TOF-MS methods for intact recombinant human VEGF(165) are developed and applied to analyze this glycoprotein expressed in glycosylating (Sf 21 insect cells) and non-glycosylating (Escherichia coli) systems. Different parameters influencing the CIEF separation were studied. The developed CIEF method allowed for the separation of up to seven peaks in the VEGF(165) expressed in insect cells and up to three in VEGF(165) expressed in E. coli. The use of the presented method permits the estimation of the apparent pI of the different forms of VEGF(165) expressed in insect cells to be in a range of 6.8-8.2. The three peaks with intermediate pI values are observed in the protein expressed in both systems, insect cells and E. coli. The MALDI-TOF-MS method enabled to a rapid partial characterization of VEGF(165) based on its MS fingerprint. MALDI-MS analysis of VEGF(165) expressed in insect cells shows the presence of, at least, four forms or groups of forms of VEGF(165) as a result of the different PTMs of the protein. According to the MALDI-MS analysis, VEGF(165) expressed in E. coli was produced as a very homogeneous protein, although the results suggest the existence of some PTMs in the protein. The patterns of VEGF(165) of both origins obtained by CIEF and MALDI-MS indicate the possibility of using these analytical methods to compare samples from people with different pathophysiological conditions. This work is thus a starting point to make possible the study of the role of the various forms of VEGF(165) as biomarkers. Finally, to the best of our knowledge, this is the

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

    PubMed

    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. Graphical Abstract ᅟ. PMID:27056565

  20. 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.

    2016-04-01

    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.

  1. 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.

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

    PubMed Central

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

    2014-01-01

    Background Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid and inexpensive technique that has initiated a revolution in the clinical microbiology laboratory for identification of pathogens. The Vitek 2 anaerobe and Corynebacterium (ANC) identification card is a newly developed method for identification of corynebacteria and anaerobic species. The aim of this study was to evaluate the effectiveness of the ANC card and MALDI-TOF MS techniques for identification of clinical anaerobic isolates. Methods Five reference strains and a total of 50 anaerobic bacteria clinical isolates comprising ten different genera and 14 species were identified and analyzed by the ANC card together with Vitek 2 identification system and Vitek MS together with version 2.0 database respectively. 16S rRNA gene sequencing was used as reference method for accuracy in the identification. Results Vitek 2 ANC card and Vitek MS provided comparable results at species level for the five reference strains. Of 50 clinical strains, the Vitek MS provided identification for 46 strains (92%) to the species level, 47 (94%) to genus level, one (2%) low discrimination, two (4%) no identification and one (2%) misidentification. The Vitek 2 ANC card provided identification for 43 strains (86%) correct to the species level, 47 (94%) correct to the genus level, three (6%) low discrimination, three (6%) no identification and one (2%) misidentification. Conclusions Both Vitek MS and Vitek 2 ANC card can be used for accurate routine clinical anaerobe identification. Comparing to the Vitek 2 ANC card, Vitek MS is easier, faster and more economic for each test. The databases currently available for both systems should be updated and further developed to enhance performance. PMID:24822113

  3. False Results Caused by Solvent Impurity in Tetrahydrofuran for MALDI TOF MS Analysis of Amines

    NASA Astrophysics Data System (ADS)

    Lou, Xianwen; Leenders, Christianus M. A.; van Onzen, Arthur H. A. M.; Bovee, Ralf A. A.; van Dongen, Joost L. J.; Vekemans, Jef A. J. M.; Meijer, E. W.

    2013-11-01

    Tetrahydrofuran (THF) is one of the most frequently used solvents in the MALDI TOF MS analysis of synthetic compounds. However, it should be used with caution because a trace amount of 4-hydroxybutanal (HBA) might be generated and accumulated in THF during storage. Since only a tiny amount of analytes is required in MALDI MS measurements, a trace amount of HBA might have a significant effect on the MS results. It was found that HBA will quickly react with primary and secondary amino compounds, leading to false results about the sample composition with an extra series of ions with additional mass of 70 Da in between. The formation of HBA can be inhibited by butylated hydroxytoluene (BHT) antioxidant. Therefore, when THF is required as the solvent for sample preparation, it is strongly recommended to use a BHT-stabilized one, at least for the analysis of compounds with amino groups.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. GC-MS and MALDI-TOF MS profiling of sucrose esters from Nicotiana tabacum and N. rustica.

    PubMed

    Haliński, Łukasz P; Stepnowski, Piotr

    2013-01-01

    Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the first time to the analysis of the sucrose esters from the surface of Nicotiana L. leaves. The profiles obtained for the model plant N. tabacum were similar to those from the gas chromatography-flame ionization detector (GC-FID) analysis. The most reproducible results were obtained using a dihydroxybenzoic acid (DHB) matrix. The main advantage of this method is that crude plant extracts can be analysed without sample clean-up. GC-MS analysis of Aztec tobacco (N. rustica) extracts revealed the presence of three types of sucrose esters. All identified compounds had three C4-C8 acyl chains substituting the glucose moiety, while the fructose part of the molecule was substituted with 0, 1, or 2 acetyl groups. MALDI-TOF MS analysis of the sucrose ester fraction revealed the presence of compounds not eluting from a GC column. Combining the data from both GC-MS and MALDI-TOF MS experiments, we obtained a full sucrose ester profile, which is based on the molecular weight of the compounds and on the number of acyl chains in the molecule. PMID:23923618

  6. Multiplex MALDI-TOF MS detection of mitochondrial variants in Brazilian patients with hereditary optic neuropathy

    PubMed Central

    Matilde da Silva-Costa, Sueli; Balieiro, Juliane Cristina; Fernandes, Marcela Scabello Amaral; Alves, Rogério Marins; Guerra, Andrea Trevas Maciel; Marcondes, Ana Maria; Sartorato, Edi Lúcia

    2016-01-01

    Purpose Leber hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by bilateral vision loss. More than 95% of LHON cases are associated with one of the three main mtDNA mutations: G11778A, T14484C, and G3460A. The other 5% of cases are due to other rare mutations related to the disease. The aim of this study was to identify the prevalence and spectrum of LHON mtDNA mutations, including the haplogroup, in a cohort of Brazilian patients with optic neuropathy and to evaluate the usefulness of iPLEX Gold/matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology in detecting LHON mutations. Methods We analyzed a total of 101 patients; 67 had a clinical diagnosis of LHON and 34 had optic neuropathy of unknown etiology. Direct sequencing and iPLEX Gold/MALDI-TOF MS were used to screen for the most common pathogenic point mutations in LHON, together with the rare mutations G3733A, C4171A, T10663C, G14459A, C14482G, A14495G, C14568T, and C14482A. Results We identified mutations in 36 patients, of whom 83.3% carried the G11778A mutation and 16.7% carried the T14484C mutation. In individuals with mutations, the haplogroups found were L1/L2, L3, C, R, U, D, and H. Rare mutations were not detected in any of the patients analyzed. Conclusions The frequencies of the main LHON mutations were similar to those previously reported for Latin America. A different frequency was found only for the A3460G mutation. The most frequent haplogroups identified were of African origin. The iPLEX Gold/MALDI-TOF MS technology proved to be highly accurate and efficient for screening mutations and identifying the haplogroups related to LHON. The MassArray platform, combined with other techniques, enabled definitive diagnosis of LHON in 36% (36/101) of the cases studied. PMID:27582625

  7. Characterization of immunoglobulins through analysis of N-glycopeptides by MALDI-TOF MS.

    PubMed

    Komatsu, Emy; Buist, Marjorie; Roy, Rini; Gomes de Oliveira, Andrey Giovanni; Bodnar, Edward; Salama, Apolline; Soulillou, Jean-Paul; Perreault, Hélène

    2016-07-15

    The aim of this report is to emphasize the role, usefulness and power of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the analysis of glycoforms of antibodies (Abs) through their proteolytic glycopeptides. Abs are complex biomolecules in which glycans hold determinant properties and thus need to be thoroughly characterized following Ab production by recombinant methods or Ab collection from human/animal serum or tissue. In spite of the great robustness of MALDI-TOF MS in terms of tolerance to impurities, the analysis of Abs and Ab components using this technique requires extensive sample preparation involving all or some of chromatography, solid phase extraction, enzymatic modification, and chemical derivatization. This report focuses on a monoclonal Ab produced in cell culture, as well as on a polyclonal human immunoglobulin (Ig) G obtained commercially and a polyclonal porcine IgG obtained from serum. A method is first provided to separate Ab protein chain components (light chains, heavy chains) by gel electrophoresis, which is useful for instance for protein-A eluates of Igs either from cell culture or biological samples. This allows for in-gel proteolytic digestion of the protein gel band(s) of choice for further MS characterization. Also discussed is the more conventional in-solution overnight digestion method used here with each of two proteolytic enzymes, i.e. trypsin and chymotrypsin. The overnight method is in turn compared with a much faster approach, that of digesting Abs with trypsin or chymotrypsin through the action of microwave heating. For method comparison, glycopeptides are fractionated from digestion mixtures using mostly C-18 cartridges for simplicity, although this enrichment procedure is also compared with other published procedures. The advantages of MALDI tandem mass spectrometry are highlighted for glycopeptide analysis, and lastly an esterification method applied to glycopeptides is

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

    PubMed

    Ergin, Çağrı; Gök, Yaşar; Bayğu, Yasemin; Gümral, Ramazan; Özhak-Baysan, Betil; Döğen, Aylin; Öğünç, Dilara; Ilkit, Macit; Seyedmousavi, Seyedmojtaba

    2016-02-01

    The present study compared two chemical-based methods, namely, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, to understand the misidentification of Exophiala dermatitidis and Exophiala phaeomuriformis. The study utilized 44 E. dermatitidis and 26 E. phaeomuriformis strains, which were partially treated with strong acids and bases for further evaluation. MALDI-TOF MS and ATR-FTIR spectroscopy data of the two Exophiala species were compared. Data groupings were observed for the chromic acid- and nitric acid-treated species when the black yeast sources were categorized as creosoted-oak sleepers, concrete sleepers, or dishwasher isolates. The MALDI-TOF MS data for the metalloenzyme-containing regions were consistent with the ATR-FTIR spectroscopy data. These results indicated that environmental isolates might contain metals not found in human isolates and might interfere with chemical-based identification methods. Therefore, MALDI-TOF MS reference libraries should be created for clinical strains and should exclude petroleum-associated environmental isolates. PMID:26373644

  9. Magnetic metal-organic frameworks for selective enrichment and exclusion of proteins for MALDI-TOF MS analysis.

    PubMed

    Wan, Wei; Liang, Qionglin; Zhang, Xiaoqiong; Yan, Min; Ding, Mingyu

    2016-08-01

    We firstly report magnetic metal-organic frameworks for selective enrichment and exclusion of proteins for MALDI-TOF MS analysis. Fe3O4@MIL-100(Fe) nanoparticles were achieved by step-by-step assembly on poly(acrylic acid) modified Fe3O4. PMID:27350019

  10. Assessment of green cleaning effectiveness on polychrome surfaces by MALDI-TOF mass spectrometry and microscopic imaging.

    PubMed

    Hrdlickova Kuckova, Stepanka; Crhova Krizkova, Michaela; Pereira, Catarina Luísa Cortes; Hynek, Radovan; Lavrova, Olga; Busani, Tito; Branco, Luis Cobra; Sandu, Irina Crina Anca

    2014-08-01

    This article proposes an innovative methodology which employs nondestructive techniques to assess the effectiveness of new formulations based on ionic liquids, as alternative solvents for enzymes (proteases), for the removal of proteinaceous materials from painted surfaces during restoration treatments. Ionic liquids (ILs), also known as "designer" solvents, because of their peculiar properties which can be adjusted by selecting different cation-anion combinations, are potentially green solvents due totheir low vapour pressure. In this study, two ionic liquids were selected: IL1 (1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4 ])) and IL2 (1-ethyl-3-methylimidazolium ethylsulphate ([EMIM][EtSO4 ])). New formulations were prepared with these ILs and two different proteases (E): one acid (E1-pepsin) and one alkaline (E2-obtained from Aspergillus sojae). These formulations were tested on tempera and oil mock-up samples, prepared in accordance with historically documented recipes, and covered with two different types of protein-based varnishes (egg white and isinglass-fish glue). A noninvasive multiscale imaging methodology was applied before and after the treatment to evaluate the cleaning's effectiveness. Different microscopic techniques-optical microscopy (OM) with visible and fluorescent light, scanning electron microscopy (SEM) and atomic force microscopy (AFM)-together with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) were applied on areas cleaned with the new formulations (IL + E) and reference areas cleaned only with the commercial enzyme formulations (gels). MALDI-TOF proved particularly very useful for comparing the diversity and abundance of peptides released by using different enzymatic systems. Microsc. Res. Tech. 77:574-585, 2014. © 2014 Wiley Periodicals, Inc. PMID:24825619

  11. Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome.

    PubMed

    Angelini, Roberto; Lobasso, Simona; Gorgoglione, Ruggiero; Bowron, Ann; Steward, Colin G; Corcelli, Angela

    2015-09-01

    Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of card-iolipin (CL) species in mitochondria. Diagnosis of the disease is often compromised by lack of rapid and widely available diagnostic laboratory tests. The present study describes a new method for BTHS screening based on MALDI-TOF/MS analysis of leukocyte lipids. This generates a "CL fingerprint" and allows quick and simple assay of the relative levels of CL and monolysocardiolipin species in leukocyte total lipid profiles. To validate the method, we used vector algebra to analyze the difference in lipid composition between controls (24 healthy donors) and patients (8 boys affected by BTHS) in the high-mass phospholipid range. The method of lipid analysis described represents an important additional tool for the diagnosis of BTHS and potentially enables therapeutic monitoring of drug targets, which have been shown to ameliorate abnormal CL profiles in cells. PMID:26144817

  12. Detection of Ricin Intoxication in Mice Using Serum Peptide Profiling by MALDI-TOF/MS

    PubMed Central

    Zhao, Siyan; Liu, Wen-Sen; Wang, Meng; Li, Jiping; Sun, Yucheng; Li, Nan; Hou, Feng; Wan, Jia-Yu; Li, Zhongyi; Qian, Jun; Liu, Linna

    2012-01-01

    Ricin toxin has been regarded as one of the most potent poisons in the plant kingdom, and there is no effective therapeutic countermeasure or licensed vaccine against it. Consequently, early detection of ricin intoxication is necessary. In this study, we took mice as test subjects, and used the technique of Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) and ClinProt™ microparticle beads to set up an effective detection model with an accuracy of almost 100%. Eighty-two peaks in the mass range 1000–10,000 m/z were detected by ClinProTools software, and five different peaks with m/z of 4982.49, 1333.25, 1537.86, 4285.05 and 2738.88 had the greatest contribution to the accuracy and sensitivity of this model. They may therefore provide biomarkers for ricin intoxication. PMID:23202975

  13. A MALDI-TOF MS study of oligomeric poly(m-phenyleneisophthalamide).

    PubMed

    Gies, Anthony P; Nonidez, William K; Ellison, Sparkle T; Ji, Haining; Mays, Jimmy W

    2005-02-01

    MALDI-TOF MS was used to study the end-group distribution of a series of poly(m-phenyleneisophthalamide) oligomers which were synthesized using various mole percent ratios of diamine to diacid chloride (90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, and 10:90) to clarify results obtained in previous work published in this journal. Oligomers synthesized with excess diamine or excess diacid chloride were found to contain abundances of amine or carboxylate end groups, respectively, as expected. Oligomers synthesized with equal molar ratios of reactants produced cyclic species which were also found in a previous publication as an oligomer in commercially produced, high molecular mass Nomex. PMID:15679344

  14. Rapid Detection of K1 Hypervirulent Klebsiella pneumoniae by MALDI-TOF MS

    PubMed Central

    Huang, Yonglu; Li, Jiaping; Gu, Danxia; Fang, Ying; Chan, Edward W.; Chen, Sheng; Zhang, Rong

    2015-01-01

    Hypervirulent strains of Klebsiella pneumoniae (hvKP) are genetic variants of K. pneumoniae which can cause life-threatening community-acquired infection in healthy individuals. Currently, methods for efficient differentiation between classic K. pneumoniae (cKP) and hvKP strains are not available, often causing delay in diagnosis and treatment of hvKP infections. To address this issue, we devised a Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) approach for rapid identification of K1 hvKP strains. Four standard algorithms, genetic algorithm (GA), support vector machine (SVM), supervised neural network (SNN), and quick classifier (QC), were tested for their power to differentiate between K1 and non-K1 strains, among which SVM was the most reliable algorithm. Analysis of the receiver operating characteristic curves of the interest peaks generated by the SVM model was found to confer highly accurate detection sensitivity and specificity, consistently producing distinguishable profiles for K1 hvKP and non-K1 strains. Of the 43 K. pneumoniae modeling strains tested by this approach, all were correctly identified as K1 hvKP and non-K1 capsule type. Of the 20 non-K1 and 17 K1 hvKP validation isolates, the accuracy of K1 hvKP and non-K1 identification was 94.1 and 90.0%, respectively, according to the SVM model. In summary, the MALDI-TOF MS approach can be applied alongside the conventional genotyping techniques to provide rapid and accurate diagnosis, and hence prompt treatment of infections caused by hvKP. PMID:26733976

  15. MALDI-TOF MS portrait of emetic and non-emetic Bacillus cereus group members.

    PubMed

    Fiedoruk, Krzysztof; Daniluk, Tamara; Fiodor, Angelika; Drewicka, Ewa; Buczynska, Katarzyna; Leszczynska, Katarzyna; Bideshi, Dennis Ken; Swiecicka, Izabela

    2016-08-01

    The number of foodborne intoxications caused by emetic Bacillus cereus isolates has increased significantly. As such, rapid and reliable methods to identify emetic strains appear to be clinically relevant. In this study, intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to differentiate emetic and non-emetic bacilli. The phyloproteomic clustering of 34 B. cereus emetic and 88 non-emetic isolates classified as B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus mycoides, showed (i) a clear separation of both groups at a similarity level of 43%, and (ii) a high relatedness among the emetic isolates (similarity of 78%). Specifically, 83 mass peak classes were recognized in the spectral window range between m/z 4000 and 12 000 that were tentatively assigned to 41 protein variants based on a bioinformatic approach. Mass variation between the emetic and the non-emetic subsets was recorded for 27 of them, including ten ribosomal subunit proteins, for which inter-strain polymorphism was confirmed by gene sequencing. Additional peaks were assigned to other proteins such as small acid soluble proteins, cold shock proteins and hypothetical proteins, e.g., carbohydrate kinase. Moreover, the results were supported by in silico analysis of the biomarkers in 259 members of B. cereus group, including Bacillus anthracis, based on their whole-genome sequences. In conclusion, the proteomic profiling by MALDI-TOF MS is a promising and rapid method for pre-screening B. cereus to identify medically relevant isolates and for epidemiologic purposes. PMID:27196540

  16. Differentiation of Bacillus pumilus and Bacillus safensis Using MALDI-TOF-MS

    PubMed Central

    Branquinho, Raquel; Sousa, Clara; Lopes, João; Pintado, Manuela E.; Peixe, Luísa V.; Osório, Hugo

    2014-01-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification. PMID:25314655

  17. 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. PMID:22967923

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

    PubMed

    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

  19. 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

  20. Use of MALDI-TOF MS for Identification of Nontuberculous Mycobacterium Species Isolated from Clinical Specimens

    PubMed Central

    Mediavilla-Gradolph, María Concepción; De Toro-Peinado, Inmaculada; Bermúdez-Ruiz, María Pilar; García-Martínez, María de los Ángeles; Ortega-Torres, María; Montiel Quezel-Guerraz, Natalia; Palop-Borrás, Begoña

    2015-01-01

    The aim of this study was to compare the results obtained for identification by MALDI-TOF of nontuberculous mycobacteria (NTM) isolated in clinical samples with those obtained by GenoType Mycobacterium CM/AS (common mycobacteria/additional species). A total of 66 Mycobacterium isolates from various clinical specimens (mainly respiratory) were tested in this study. They were identified using MALDI-TOF Bruker from strains isolated in Lowenstein, following the recommended protocol of heat inactivation and extraction, and were simultaneously analyzed through hybridization by GenoType Mycobacterium from liquid culture MGIT. Our results showed that identification by MALDI-TOF was correct in 98.4% (65/66) of NTM isolated in our clinical practice (M. avium, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum, M. mucogenicum, M. kansasii, and M. scrofulaceum). MALDI-TOF was found to be an accurate, rapid, and cost-effective system for identification of mycobacteria species. PMID:26106617

  1. 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. PMID:26259660

  2. Quantitation of Alpha-Glucosidase Activity Using Fluorinated Carbohydrate Array and MALDI-TOF-MS.

    PubMed

    Yang, Hyojik; Chan, Allen L; LaVallo, Vincent; Cheng, Quan

    2016-02-01

    Quantitation of alpha-glucosidase (α-GD) activity is of significance to diagnosis of many diseases including Pompe disease and type II diabetes. We report here a new method to determine α-GD activity using matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) in combination with carbohydrate microarray and affinity surface chemistry. Carbohydrate probes are synthesized for capture of the enzymatic reaction products and the adducts are loaded onto a fluorinated gold surface to generate an array, which is followed by characterization by MALDI-TOF-MS. The ratio of intensities is used to determine the level of activity of several enzymes. In addition, half maximal inhibitory concentration (IC50) of acarbose and epigallocatechin gallate are also determined using this approach, and the results agree well with the reported values. This method is advantageous as compared to conventional colorimetric techniques that typically suffer matrix interference problems from samples. The use of the polyfluorinated surface has effectively suppressed the interference. PMID:26760440

  3. Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS.

    PubMed

    Geyer, H; Schmitt, S; Wuhrer, M; Geyer, R

    1999-01-15

    Exoglycosidase digestion combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been demonstrated to be an effective method for the structural characterization of glycoconjugates and oligosaccharides in picomolar amounts. A sample preparation method is described, in which 6-aza-2-thiothymine (ATT) in water is used as matrix and enzymes are dialyzed before use against a low concentration of volatile buffer such as ammonium acetate. Under these conditions, a series of sequential on-target exoglycosidase treatments was carried out in one single analyte spot in the presence of ATT matrix. Subsequent mass spectrometric analysis of the resulting products yielded information on both the completeness of the reaction and structural features of the glycoconjugates such as monosaccharide sequence, branching pattern, and anomeric configurations of the corresponding glycosidic linkages. The results show that all exoglycosidases used retain their activity in the presence of ATT matrix. Hence, structural analysis of carbohydrates or mixtures thereof can be performed very fast, without intermediate desalting steps or sample splitting. This approach is illustrated by the analysis of underivatized glycans, oligosaccharide derivatives, glycopeptides, and glycolipids. Depending on the analyte, amounts of sample required could be limited to a few picomoles. PMID:9949734

  4. 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.

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

    PubMed

    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. Graphical Abstract ᅟ. PMID:26797827

  6. MALDI-TOF MS to monitor the kinetics of phospholipase A2-digestion of oxidized phospholipids.

    PubMed

    Schröter, Jenny; Süß, Rosmarie; Schiller, Jürgen

    2016-07-15

    Free fatty acids (FFA) are released through phospholipase A2 (PLA2), which cleaves the fatty acyl residue at the sn-2 position of phospholipids (PL). During inflammatory diseases, reactive oxygen species (such as HOCl) lead to the formation of oxidatively modified PL (e.g., chlorohydrin generation). It is still widely unknown to which extent the oxidation of PL influences their digestibility by PLA2. Additionally, investigations on the impact of the position of the unsaturated fatty acyl residue (sn-1 versus sn-2 position) and modifications of the headgroup (for instance phosphatidylcholine (PC) versus phosphatidylethanolamine (PE)) are also lacking. Therefore, the aim of this study is the investigation of these aspects using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to elucidate the PL/lysophospholipid (LPL) ratios as measures of the PLA2 digestibility. We will show that oxidative modifications of PL by HOCl have a considerable impact on the PLA2 digestibility, i.e., oxidation of the unsaturated fatty acyl residues leads to a reduced digestibility of both PC and PE. Besides, it will be shown that MALDI MS is a convenient and reliable tool to investigate the related changes. PMID:26721598

  7. Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS.

    PubMed

    Lee, Jeonghoon; Musyimi, Harrison K; Soper, Steven A; Murray, Kermit K

    2008-07-01

    An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 microm wide, 100 microm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 microL/min, producing a reaction time of approximately 24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, bovine serum albumin (BSA), myoglobin, and phosphorylase b, respectively. PMID:18479934

  8. Efficient Analysis of Non-Polar Environmental Contaminants by MALDI-TOF MS with Graphene as Matrix

    NASA Astrophysics Data System (ADS)

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

    2011-07-01

    In this Application Note, we describe, for the first time, the rapid analysis of hydrophobic compounds present in environmental contaminants, which includes polycyclic aromatic hydrocarbons (PAHs) and estrogen, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the use of graphene as matrix. MALDI-TOF MS with conventional matrix has limitations in analyzing low-polarity compounds owing to their difficulty in ionization. We demonstrate that compared with conventional matrix, graphene displays higher desorption/ionization efficiencies for PAHs, and no fragment ions are observed. The method also holds potential in quantitative analysis. In addition, the ionization signal increases with the increasing number of benzene rings in the PAHs, suggesting that graphene binds to PAHs via π-π stacking interactions. Furthermore, graphene as adsorbent for solid-phase extraction of coronene from river water sample displays good performance with a detection limit of 10-7 M. This work provides a novel and convenient method for analyzing low-polarity environmental contaminants by MALDI-TOF MS.

  9. The intact muscle lipid composition of bulls: an investigation by MALDI-TOF MS and 31P NMR.

    PubMed

    Dannenberger, Dirk; Süss, Rosmarie; Teuber, Kristin; Fuchs, Beate; Nuernberg, Karin; Schiller, Jürgen

    2010-02-01

    The analysis of beef lipids is normally based on chromatographic techniques and/or gas chromatography in combination with mass spectrometry (GC/MS). Modern techniques of soft-ionization MS were so far scarcely used to investigate the intact lipids in muscle tissues of beef. The objective of the study was to investigate whether matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry and (31)P nuclear magnetic resonance (NMR) spectroscopy are useful tools to study the intact lipid composition of beef. For the MALDI-TOF MS and (31)P NMR investigations muscle samples were selected from a feeding experiment with German Simmental bulls fed different diets. Beside the triacylglycerols (TAGs), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylinositol (PI) species the MALDI-TOF mass spectra of total muscle lipids gave also intense signals of cardiolipin (CL) species. The application of different matrix compounds, 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), leads to completely different mass spectra: 9-AA is particularly useful for the detection of (polar) phospholipids, whereas apolar lipids, such as cholesterol and triacylglycerols, are exclusively detected if DHB is used. Finally, the quality of the negative ion mass spectra is much higher if 9-AA is used. PMID:19900429

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

    PubMed

    Cerezo, María; Cerný, Viktor; Carracedo, Angel; Salas, Antonio

    2009-11-01

    Analysis of the mitochondrial DNA variation in populations is commonly carried out in many fields of biomedical research. We propose the analysis of mitochondrial DNA coding region SNP (mtSNP) variation to a high level of phylogenetic resolution based on MALDI-TOF MS. The African phylogeny has been chosen to test the applicability of the technique but any other part of the worldwide phylogeny (or any other mtSNP panel) could be equally suitable for MALDI-TOF MS genotyping. SNP selection thus aimed to fully cover all the mtSNPs defining major and minor branches of the known African tree, including, macro-haplogroup L, and haplogroups M1, and U6. A total of 230 mtSNPs were finally selected. We used tests samples collected from two different African locations, namely, Mozambique and Chad Basin. Different internal genotyping controls and other indirect approaches (e.g. phylogenetic checking coupled with automatic sequencing) were used in order to evaluate the reproducibility of the technique, which resulted to be 100% using samples previously subjected to whole genome amplification. The advantages of the MALDI-TOF MS are also discussed in comparison with other popular methods such as minisequencing, highlighting its high-throughput nature, which is particularly suitable for case-control medical studies, forensic databasing or population and anthropological studies. PMID:19862743

  11. Fractional Factorial Design of MALDI-TOF-MS Sample Preparations for the Optimized Detection of Phospholipids and Acylglycerols.

    PubMed

    AlMasoud, Najla; Correa, Elon; Trivedi, Drupad K; Goodacre, Royston

    2016-06-21

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has successfully been used for the analysis of high molecular weight compounds, such as proteins and nucleic acids. By contrast, analysis of low molecular weight compounds with this technique has been less successful due to interference from matrix peaks which have a similar mass to the target analyte(s). Recently, a variety of modified matrices and matrix additives have been used to overcome these limitations. An increased interest in lipid analysis arose from the feasibility of correlating these components with many diseases, e.g. atherosclerosis and metabolic dysfunctions. Lipids have a wide range of chemical properties making their analysis difficult with traditional methods. MALDI-TOF-MS shows excellent potential for sensitive and rapid analysis of lipids, and therefore this study focuses on computational-analytical optimization of the analysis of five lipids (4 phospholipids and 1 acylglycerol) in complex mixtures using MALDI-TOF-MS with fractional factorial design (FFD) and Pareto optimality. Five different experimental factors were investigated using FFD which reduced the number of experiments performed by identifying 720 key experiments from a total of 8064 possible analyses. Factors investigated included the following: matrices, matrix preparations, matrix additives, additive concentrations, and deposition methods. This led to a significant reduction in time and cost of sample analysis with near optimal conditions. We discovered that the key factors used to produce high quality spectra were the matrix and use of appropriate matrix additives. PMID:27228355

  12. Biotyping of Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates from France and Algeria Using MALDI-TOF MS

    PubMed Central

    Berrazeg, Meryem; Diene, Seydina M.; Drissi, Mourad; Kempf, Marie; Richet, Hervé; Landraud, Luce; Rolain, Jean-Marc

    2013-01-01

    Background Klebsiella pneumoniae is one of the most important pathogens responsible for nosocomial outbreaks worldwide. Epidemiological analyses are useful in determining the extent of an outbreak and in elucidating the sources and the spread of infections. The aim of this study was to investigate the epidemiological spread of K. pneumoniae strains using a MALDI-TOF MS approach. Methods Five hundred and thirty-five strains of K. pneumoniae were collected between January 2008 and March 2011 from hospitals in France and Algeria and were identified using MALDI-TOF. Antibiotic resistance patterns were investigated. Clinical and epidemiological data were recorded in an Excel file, including clustering obtained from the MSP dendrogram, and were analyzed using PASW Statistics software. Results Antibiotic susceptibility and phenotypic tests of the 535 isolates showed the presence of six resistance profiles distributed unequally between the two countries. The MSP dendrogram revealed five distinct clusters according to an arbitrary cut-off at the distance level of 500. Data mining analysis of the five clusters showed that K. pneumoniae strains isolated in Algerian hospitals were significantly associated with respiratory infections and the ESBL phenotype, whereas those from French hospitals were significantly associated with urinary tract infections and the wild-type phenotype. Conclusions MALDI-TOF was found to be a promising tool to identify and differentiate between K. pneumoniae strains according to their phenotypic properties and their epidemiological distribution. This is the first time that MALDI-TOF has been used as a rapid tool for typing K. pneumoniae clinical isolates. PMID:23620754

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Proteomic approach based on MALDI-TOF MS to detect powdered milk in fresh cow's milk.

    PubMed

    Calvano, Cosima Damiana; Monopoli, Antonio; Loizzo, Pasqua; Faccia, Michele; Zambonin, Carlo

    2013-02-27

    Milk and cheese are expensive foodstuffs, and their consumption is spread among the population because of their high nutritional value; for this reason they are often subjected to adulterations. Among the common illegal practices, the addition of powdered derivatives seems very difficult to detect because the adulterant materials have almost the same chemical composition of liquid milk. However, the high temperatures (180-200 °C) used for milk powder production could imply the occurrence of some protein modifications (e.g., glycation, lactosylation, oxidation, deamidation, dehydration). The modified proteins or peptides could then be used as markers for the presence of powdered milk. In this work, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze tryptic digests relevant to samples of raw liquid (without heat treatment), commercial liquid, and powdered cow's milk. Samples were subjected to two-dimensional gel electrophoresis (2-DE); differences among liquid and powder milk were detected at this stage and eventually confirmed by MALDI analysis of the in gel digested proteins. Some diagnostic peptides of powdered milk, attributed to modified whey proteins and/or caseins, were identified. Then, a faster procedure was optimized, consisting of the separation of caseins from milk whey and the subsequent in-solution digestion of the two fractions, with the advantage of obtaining almost the same information in a limited amount of time. Finally, analyses were carried out with the fast procedure on liquid milk samples adulterated with powdered milk at different percentages, and diagnostic peptides were detected down to 1% of adulteration level. PMID:22931122

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

    PubMed

    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

  16. 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

  17. The Use of MALDI-TOF-MS and In Silico Studies for Determination of Antimicrobial Peptides' Affinity to Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Mandal, Santi M.; Migliolo, Ludovico; Franco, Octavio L.

    2012-11-01

    Several methods have been proposed for determining the binding affinity of antimicrobial peptides (AMPs) to bacterial cells. Here the utilization of MALDI-TOF-MS was proposed as a reliable and efficient method for high throughput AMP screening. The major advantage of the technique consists of finding AMPs that are selective and specific to a wide range of Gram-negative and -positive bacteria, providing a simple reliable screening tool to determine the potential candidates for broad spectrum antimicrobial drugs. As a prototype, amp-1 and -2 were used, showing highest activity toward Gram-negative and -positive membranes respectively. In addition, in silico molecular docking studies with both peptides were carried out for the membranes. In silico results indicated that both peptides presented affinity for DPPG and DPPE phospholipids, constructed in order to emulate an in vivo membrane bilayer. As a result, amp-1 showed a higher complementary surface for Gram-negative while amp-2 showed higher affinity to Gram-positive membranes, corroborating MS analyses. In summary, results here obtained suggested that in vitro methodology using MALDI-TOF-MS in addition to theoretical studies may be able to improve AMP screening quality.

  18. Peptidomic analysis of Chinese shrimp ( Fenneropenaeus chinensis) hemolymph by magnetic bead-based MALDI-TOF MS

    NASA Astrophysics Data System (ADS)

    Wang, Baojie; Liu, Mei; Jiang, Keyong; Zhang, Guofan; Wang, Lei

    2013-03-01

    Peptides in shrimp hemolymph play an important role in the innate immune response. Analysis of hemolymph will help to detect and identify potential novel biomarkers of microbial infection. We used magnetic bead-based purification (ClinProt system) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to characterize shrimp hemolymph peptides. Shrimp serum and plasma were used as the source of samples for comparative analysis, and it was found that serum was more suitable for shrimp hemolymph peptidomic analysis. To screen potential specific biomarkers in serum of immune-challenged shrimps, we applied magnetic bead-based MALDI-TOF MS to serum samples from 10 immune-challenged and 10 healthy shrimps. The spectra were analyzed using FlexAnalysis 3.0 and ClinProTools 2.1 software. Thirteen peptide peaks significantly different between the two groups were selected as candidate biomarkers of lipopolysaccharide (LPS)-infection. The diagnostic model established by genetic algorithm using five of these peaks was able to discriminate LPS-challenged shrimps from healthy control shrimps with a sensitivity of 90% and a specificity of 100%. Our approach in MALDITOF MS-based peptidomics is a powerful tool for screening bioactive peptides or biomarkers derived from hemolymph, and will help to enable a better understanding of the innate immune response of shrimps.

  19. 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

  20. Metabolite Fingerprinting of Eugenia jambolana Fruit Pulp Extracts using NMR, HPLC-PDA-MS, GC-MS, MALDI-TOF-MS and ESI-MS/MS Spectrometry.

    PubMed

    Sharma, Ram Jee; Gupta, Ramesh C; Bansal, Arvind Kumar; Singh, Inder Pal

    2015-06-01

    Eugenia jambolana, commonly known as 'jamun' or Indian blackberry, is an important source of bioactive compounds. All parts of the plant like stem bark, leaves, flower, fruit pulp and seeds are traditionally used for many diseases. Metabolite profiling in medicinally important plants is critical to resolve the problems associated with standardization and quality control. Metabolite profiling of the fruit pulp of Jamun was performed by NMR, HPLC, MS, GC-MS and MALDI-TOF mass spectrometry. These hyphenated techniques helped in the identification of 68 chemically-diverse metabolites of the fruit pulp. These include anthocyanins, anthocyanidins, sugars, phenolics and volatile compounds. Five extracts of fruit pulp were prepared i.e. hexane, chloroform, ethylacetate, butanol and aqueous methanolic. Twenty-five metabolites identified and quantified in the n-butanol and aqueous-methanolic extracts of ripe jamun fruit by qNMR. LC-PDA-MS and MALDI-TOF spectrometry helped in deciphering thirty-nine metabolites out of which thirteen were quantified. PMID:26197529

  1. Detection of acid and hop shock induced responses in beer spoiling Lactobacillus brevis by MALDI-TOF MS.

    PubMed

    Schurr, Benjamin C; Behr, Jürgen; Vogel, Rudi F

    2015-04-01

    Due to the harsh environment, microorganisms encounter in beer, spoilage bacteria must be able to customise their metabolism and physiology in an order to master various kinds of perturbations. Proteomic approaches have been used to examine differences between various beer spoilage bacteria and between different stress conditions, such as acid and hop (Humulus lupulus) stress. However, these investigations cannot detect changes in low molecular weight (lmw) proteins (<150 amino acids). Therefore, for the first time, we herein present data from a proteomic study of lmw proteins for two Lactobacillus (L.) brevis strains exposed to acid stress or, respectively, two different qualities of hop induced stress. We used MALDI-TOF MS as analytical tool for the detection of lmw stress response proteins due to its high sensitivity and low throughput times. Comparing a hop-sensitive and a hop-tolerant strain, detection of the fatty acid biosynthesis-associated acyl carrier protein varied between different stress conditions and incubation times. The findings coincide with previous studies of our group regarding the fatty acid cell membrane composition of beer spoiling L. brevis. It is demonstrated that MALDI-TOF MS is a fast tool to detect and characterise stress situations in beer spoiling bacteria along the lmw sub-proteome. PMID:25475321

  2. Detection of sheep and goat milk adulterations by direct MALDI-TOF MS analysis of milk tryptic digests.

    PubMed

    Calvano, Cosima Damiana; De Ceglie, Cristina; Monopoli, Antonio; Zambonin, Carlo Giorgio

    2012-09-01

    In dairy field, one of the most common frauds is the adulteration of higher value types of milk (sheep's and goat's) with milk of lower value (cow's milk). This illegal practice has an economic advantage for milk producers and poses a threat for consumers' health because of the presence of hidden allergens as, for example, cow milk proteins, in particular, α(s1)-casein and β-lactoglobulin. The urgent need of sensitive techniques to detect this kind of fraud brought to the development of chromatographic, immunoenzymatic, electrophoretic and mass spectrometric assays. In the current work, we present a fast, reproducible and sensitive method based on the direct matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS analysis of milk tryptic digests for the detection of milk adulteration by evaluating specie-specific markers in the peptide profiles. Several pure raw and commercial milk samples and binary mixtures containing cows' and goats', cows' and sheep's and goats' and sheep's milk (concentrations of each milk varied from 0% to 100%) were prepared, and tryptic digests were analyzed by MALDI-TOF MS. The use of the new MALDI matrix α-cyano-4-chlorocinnamic acid allowed to detect cow and goat milk peptide markers up to 5% level of adulteration. Finally, from preliminary data, it seems that the strategy could be successfully applied also to detect similar adulterations in cheese samples. PMID:22972782

  3. High throughput detection of tetracycline residues in milk using graphene or graphene oxide as MALDI-TOF MS matrix.

    PubMed

    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. PMID:22644736

  4. 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.

  5. A Study of the Variation in the Salivary Peptide Profiles of Young Healthy Adults Acquired Using MALDI-TOF MS

    PubMed Central

    Brand, Henk; Imangaliyev, Sultan; Tsivtsivadze, Evgeni; van der Weijden, Fridus; de Jong, Ad; Paauw, Armand; Crielaard, Wim; Keijser, Bart; Veerman, Enno

    2016-01-01

    A cross-sectional observational study was conducted to evaluate the inter-individual variation in the MALDI-TOF MS peptide profiles of unstimulated whole saliva in a population of 268 systemically healthy adults aged 18–30 yr (150 males and 118 females) with no apparent caries lesions or periodontal disease. Using Spectral Clustering, four subgroups of individuals were identified within the study population. These subgroups were delimited by the pattern of variation in 9 peaks detected in the 2–15 kDa m/z range. An Unsupervised Feature Selection algorithm showed that P-C peptide, a 44 residue-long salivary acidic proline-rich protein, and three of its fragments (Fr. 1–25, Fr. 15–35 and Fr. 15–44) play a central role in delimiting the subgroups. Significant differences were found in the salivary biochemistry of the subgroups with regard to lysozyme and chitinase, two enzymes that are part of the salivary innate defense system (p < 0.001). These results suggest that MALDI-TOF MS salivary peptide profiles may relate information on the underlying state of the oral ecosystem and may provide a useful reference for salivary disease biomarker discovery studies. PMID:27258023

  6. 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. PMID:24321357

  7. 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…

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

    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. PMID:24862948

  9. The signal-to-noise ratio as a measure of HA oligomer concentration: a MALDI-TOF MS study.

    PubMed

    Busse, Katja; Averbeck, Marco; Anderegg, Ulf; Arnold, Klaus; Simon, Jan C; Schiller, Jürgen

    2006-06-12

    MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) was used to determine ng amounts of defined hyaluronan (HA) oligomers obtained by enzymatic digestion of high molecular weight HA with testicular hyaluronate lyase. The signal-to-noise (S/N) ratio of the positive and negative ion spectra represents a reliable concentration measure: Amounts of HA down to about 40 fmol could be determined and there is a linear correlation between the S/N ratio and the HA amount between about 0.8 pmol and 40 fmol. However, the detection limits depend considerably on the size of the HA oligomer with larger oligomers being less sensitively detectable. The advantages and drawbacks of the S/N ratio as concentration measure are discussed. PMID:16584713

  10. High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Li, Yan; Liu, Junyan; Deng, Chunhui; Zhang, Xiangmin

    2011-12-01

    In this work, a high throughput methodology for screening enzyme inhibitors has been demonstrated by combining enzyme immobilized magnetic carbonaceous microspheres and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with grapheme oxide as matrix. First, model enzyme acetylcholinesterase (AChE) was immobilized onto the 3-glycidoxypropyltrimethoxysilane (GLYMO)-modified magnetic carbonaceous (MC) microspheres, displaying a high enzyme activity and stability, and also facilitating the separation of enzyme from substrate and product. The efficiency of immobilized AChE was monitored by biochemical assay, which was carried out by mixing enzyme-immobilized MC microspheres with model substrate acetylcholine (ACh), and subsequent quantitative determination of substrate ACh and product choline using graphene oxide-based MALDI-TOF-MS with no background inference. The limit of detection (LOD) for ACh was 0.25 fmol/μL, and excellent linearity (R2 = 0.9998) was maintained over the range of 0.5 and 250 fmol/μL. Choline was quantified over the range of 0.05 and 15 pmol/μL, also with excellent linearity (R2 = 0.9994) and low LOD (0.15 fmol/μL). Good accuracy and precision were obtained for all concentrations within the range of the standard curves. All together, eight compounds (four known AChE inhibitors and four control chemical compounds with no AChE inhibit effect) were tested with our promoted methodology, and the obtained results demonstrated that our high throughput screening methodology could be a great help to the routine enzyme inhibitor screening.

  11. MALDI-TOF-MS Platform for Integrated Proteomic and Peptidomic Profiling of Milk Samples Allows Rapid Detection of Food Adulterations.

    PubMed

    Sassi, Mauro; Arena, Simona; Scaloni, Andrea

    2015-07-15

    Adulteration of ovine, caprine, and buffalo milks with more common bovine material occurs for economic reasons and seasonal availability. Frauds are also associated with the use of powdered milk instead of declared, fresh material. In this context, various analytical methods have been adapted to dairy science applications with the aim to evaluate adulteration of milk samples, although time-consuming, suitable only for speciation or thermal treatment analysis, or useful for a specific fraud type. An integrated MALDI-TOF-MS platform for the combined peptidomic and proteomic profiling of milk samples is here presented, which allows rapid detection of illegal adulterations due to the addition of either nondeclared bovine material to water buffalo, goat, and ovine milks or of powdered bovine milk to the fresh counterpart. Peptide and protein markers of each animal milk were identified after direct analysis of a large number of diluted skimmed and/or enriched diluted skimmed filtrate samples. In parallel, markers of thermal treatment were characterized in different types of commercial milks. Principal components scores of ad hoc prepared species- or thermal treatment-associated adulterated milk samples were subjected to partial least-squares regression, permitting a fast accurate estimate of the fraud extents in test samples at either protein and peptide level. With respect to previous reports on MALDI-TOF-MS protein profiling methodologies for milk speciation, this study extends that approach to the analysis of the thermal treatment and introduces an independent, complementary peptide profiling measurement, which integrates protein data with additional information on peptides, validating final results and ultimately broadening the method applicability. PMID:26098723

  12. The Construction and Evaluation of Reference Spectra for the Identification of Human Pathogenic Microorganisms by MALDI-TOF MS

    PubMed Central

    Xiao, Di; Ye, Changyun; Zhang, Huifang; Kan, Biao; Lu, Jingxing; Xu, Jianguo; Jiang, Xiugao; Zhao, Fei; You, Yuanhai; Yan, Xiaomei; Wang, Duochun; Hu, Yuan; Zhang, Maojun; Zhang, Jianzhong

    2014-01-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique for the rapid and high-throughput identification of microorganisms. There remains a dearth of studies in which a large number of pathogenic microorganisms from a particular country or region are utilized for systematic analyses. In this study, peptide mass reference spectra (PMRS) were constructed and evaluated from numerous human pathogens (a total of 1019 strains from 94 species), including enteric (46 species), respiratory (21 species), zoonotic (17 species), and nosocomial pathogens (10 species), using a MALDI-TOF MS Biotyper system (MBS). The PMRS for 380 strains of 52 species were new contributions to the original reference database (ORD). Compared with the ORD, the new reference database (NRD) allowed for 28.2% (from 71.5% to 99.7%) and 42.3% (from 51.3% to 93.6%) improvements in identification at the genus and species levels, respectively. Misidentification rates were 91.7% and 57.1% lower with the NRD than with the ORD for genus and species identification, respectively. Eight genera and 25 species were misidentified. For genera and species that are challenging to accurately identify, identification results must be manually determined and adjusted in accordance with the database parameters. Through augmentation, the MBS demonstrated a high identification accuracy and specificity for human pathogenic microorganisms. This study sought to provide theoretical guidance for using PMRS databases in various fields, such as clinical diagnosis and treatment, disease control, quality assurance, and food safety inspection. PMID:25181391

  13. 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

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

    PubMed Central

    2009-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. Identification of Bacillus strains by MALDI TOF MS using geometric approach

    PubMed Central

    Starostin, Konstantin V.; Demidov, Evgeny A.; Bryanskaya, Alla V.; Efimov, Vadim M.; Rozanov, Alexey S.; Peltek, Sergey E.

    2015-01-01

    Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html. PMID:26592761

  18. Identification of Bacillus strains by MALDI TOF MS using geometric approach.

    PubMed

    Starostin, Konstantin V; Demidov, Evgeny A; Bryanskaya, Alla V; Efimov, Vadim M; Rozanov, Alexey S; Peltek, Sergey E

    2015-01-01

    Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html. PMID:26592761

  19. Identification of Bacillus strains by MALDI TOF MS using geometric approach

    NASA Astrophysics Data System (ADS)

    Starostin, Konstantin V.; Demidov, Evgeny A.; Bryanskaya, Alla V.; Efimov, Vadim M.; Rozanov, Alexey S.; Peltek, Sergey E.

    2015-11-01

    Microorganism identification by MALDI TOF mass-spectrometry is based on the comparison of the mass spectrum of the studied organism with those of reference strains. It is a rapid and reliable method. However, commercial databases and programs are mostly designed for identification of clinically important strains and can be used only for particular mass spectrometer models. The need for open platforms and reference databases is obvious. In this study we describe a geometric approach for microorganism identification by mass spectra and demonstrate its capabilities by analyzing 24 strains belonging to the Bacillus pumilus group. This method is based on representing mass spectra as points on a multidimensional space, which allows us to use geometric distances to compare the spectra. Delimitation of microorganisms performed by geometric approach correlates well with the results of molecular phylogenetic analysis and clustering using Biotyper 3.1. All three methods used allowed us to reliably divide the strains into two groups corresponding to closely related species, Bacillus pumilus and Bacillus altitudinis. The method developed by us will be implemented in a Web interface designed for using open reference databases for microorganism identification. The data is available at http://www.bionet.nsc.ru/mbl/database/database.html.

  20. 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

  1. 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. PMID:27442502

  2. 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

  3. Screening of Anthocyanins and Anthocyanin-Derived Pigments in Red Wine Grape Pomace Using LC-DAD/MS and MALDI-TOF Techniques.

    PubMed

    Oliveira, Joana; Alhinho da Silva, Mara; Teixeira, Natércia; De Freitas, Victor; Salas, Erika

    2015-09-01

    Two phenolic extracts were made from a red wine grape pomace (GP) and fractionated first by sequential liquid-liquid extraction with organic solvents. The aqueous fraction was fractionated by low-pressure chromatography on Toyopearl HW-40 gel and on C18. Different fractions were obtained by sequential elution with aqueous/organic solvents, and then analyzed by liquid chromatography and mass spectrometry (LC-DAD/MS and MALDI-TOF). Over 50 anthocyanin-based pigments were detected by LC-DAD/MS in GP, mainly pyranoanthocyanins including A- and B-type vitisins and methylpyranoanthocyanins. The presence of oligomeric malvidin-3-O-coumaroylglucoside-based anthocyanins was also detected in GP using both LC-DAD/MS and MALDI-TOF. PMID:25912410

  4. Performances and Reliability of Bruker Microflex LT and VITEK MS MALDI-TOF Mass Spectrometry Systems for the Identification of Clinical Microorganisms

    PubMed Central

    Yaman, Gorkem; Ciftci, Ugur; Laleli, Yahya Rauf

    2015-01-01

    In clinical microbiology laboratories, routine microbial identification is mostly performed using culture based methodologies requiring 24 to 72 hours from culturing to identification. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology has been established as a cost effective, reliable, and faster alternative identification platform. In this study, we evaluated the reliability of the two available MALDI-TOF MS systems for their routine clinical level identification accuracy and efficiency in a clinical microbiology laboratory setting. A total of 1,341 routine phenotypically identified clinical bacterial and fungal isolates were selected and simultaneously analyzed using VITEK MS (bioMérieux, France) and Microflex LT (Bruker Diagnostics, Germany) MALDI-TOF MS systems. For any isolate that could not be identified with either of the systems and for any discordant result, 16S rDNA gene or ITS1/ITS2 sequencing was used. VITEK MS and Microflex LT correctly identified 1,303 (97.17%) and 1,298 (96.79%) isolates to the species level, respectively. In 114 (8.50%) isolates initial phenotypic identification was inaccurate. Both systems showed a similar identification efficiency and workflow robustness, and they were twice as more accurate compared to routine phenotypic identification in our sample pool. MALDITOF systems with their accuracy and robustness offer a good identification platform for routine clinical microbiology laboratories. PMID:26793718

  5. Measurement of blood protease kinetic parameters with self-assembled monolayer ligand binding assays and label-free MALDI-TOF MS.

    PubMed

    Patrie, Steven M; Roth, Michael J; Plymire, Daniel A; Maresh, Erica; Zhang, Junmei

    2013-11-01

    We report novel ligand binding assay (LBA) surface modalities that permit plasma protease catalytic efficiency (kcat/km) determination by MALDI-TOF MS without the use of liquid chromatography or internal standards such as chemical or metalized labels. Two model LBAs were constructed on planar self-assembled monolayers (SAMs) and used to evaluate the clinically relevant metalloprotease ADAMTS-13 kinetics in plasma. The SAM chemistries were designed to improve biosampling efficiency by minimization of nonspecific adsorption of abundant proteins present at ~100,000× the concentration of the endogenous enzyme. In the first protocol, in-solution digestion of the ADAMTS-13 substrate (vWFh) was performed with immunoaffinity enrichment of the reaction substrate and product to SAM arrays. The second configuration examined protease kcat/km via a surface digestion modality where different substrates were covalently immobilized to the SAM at controlled surface density for optimized protease screens. The results show the MALDI-TOF MS LBA platforms provide limits of quantitation to ~1% protease activity (~60 pM enzyme concentration) in <1 h analysis time, a ~16× improvement over other MS-based LBA formats. Implementation of a vacuum-sublimed MALDI matrix provided good MALDI-TOF MS intra- and interday repeatability, ~1.2 and ~6.6% RSD, respectively. Platform reliability permitted kcat/km determination without internal standards with observed values ~10× improved versus conventional fluorophoric assays. Application of the assays to 12 clinical plasma samples demonstrated proof-of-concept for clinical applications. Overall, this work demonstrates that rationally designed surface chemistries for MALDI-TOF MS may serve as an alternative, label-free methodology with potential for a wide range of biotechnology applications related to targeted enzyme molecular diagnostics. PMID:24107006

  6. Identification of metal-binding to proteins in seed samples using RF-HPLC-UV, GFAAS and MALDI-TOF-MS.

    PubMed

    Rigueira, Leila M B; Lana, Diogo A P D; Dos Santos, Daniel M; Pimenta, Adriano M; Augusti, Rodinei; Costa, Leticia M

    2016-11-15

    An extraction procedure using Tris-HCl buffer solution was employed in order to extract water-soluble proteins from seed samples of oat, wheat and soybean. Initially, the total protein concentration was determined by the Bradford method in each solution, after the extraction procedure. The soybean sample showed a higher concentration of total protein compared to the others. The protein extracts obtained were separated by reverse-phase chromatography (RP-HPLC-UV). The protein fractions were collected and analyzed by graphite furnace atomic absorption spectrometry (GFAAS) and matrix-assisted laser desorption/ionization (MALDI-TOF-MS) for determination of Cu, Fe, Mn and Zn and identification of proteins, respectively. The combination of techniques such as RP-HPLC-UV, GFAAS and MALDI-TOF-MS allowed the identification of several proteins bound to metals present in the seed samples. PMID:27283712

  7. Discrepancy in MALDI-TOF MS identification of uncommon Gram-negative bacteria from lower respiratory secretions in patients with cystic fibrosis

    PubMed Central

    AbdulWahab, Atqah; Taj-Aldeen, Saad J; Ibrahim, Emad Bashir; Talaq, Eman; Abu-Madi, Marawan; Fotedar, Rashmi

    2015-01-01

    Introduction Early identification of microbial organisms from respiratory secretions of patients with cystic fibrosis (CF) is important to guide therapeutic decisions. The objective was to compare the accuracy of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) relative to the conventional phenotypic method in identifying common bacterial isolates, including nonfermenting Gram-negative bacteria, in a cohort of patients with CF. Methods A total of 123 isolates from 50 patients with CF representing 14 bacterial species from respiratory specimens were identified using MALDI-TOF MS in parallel with conventional phenotypic methods. Discrepancies were confirmed by 16S ribosomal RNA (rRNA) gene sequencing in five Gram-negative isolates. Results The MALDI-TOF MS managed to identify 122/123 (99.2%) bacterial isolates to the genus level and 118/123 (95.9%) were identified to the species level. The MALDI-TOF MS results were 100% consistent to the species level with conventional phenotypic identification for isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pyogenes, Achromobacter xylosoxidans, Stenotrophomonas maltophilia, and other uncommon organisms such as Chryseobacterium gleum and Enterobacter cloacae. The 5/123 (4.6%) isolates misidentified were all Gram-negative bacteria. The isolation of E. cloacae and Haemophilus paraphrohaemolyticus may extend the potentially pathogenic list of organisms isolated from patients with CF. Conclusion Although the technique provides an early identification and antimicrobial therapy approach in patients with CF, limitation in the diagnosis of uncommon Gram-negative bacteria may exist. PMID:25995646

  8. 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

    2015-01-01

    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 (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. PMID:26391984

  9. Identification and growth dynamics of meat spoilage microorganisms in modified atmosphere packaged poultry meat by MALDI-TOF MS.

    PubMed

    Höll, Linda; Behr, Jürgen; Vogel, Rudi F

    2016-12-01

    Modified atmosphere packaging (MAP) is widely used in food industry to extend the microbiological shelf-life of meat. Typically, poultry meat has been packaged in a CO2/N2 atmosphere (with residual low O2). Recently, some producers use high O2 MAP for poultry meat to empirically reach comparable shelf lifes. In this work, we compared spoilage microbiota of skinless chicken breast in high (80% O2, 20% CO2) and low O2 MAP (65% N2 and 35% CO2). Two batches of meat were incubated in each atmosphere for 14 days at 4 °C and 10 °C. Atmospheric composition of each pack and colony forming units (25 °C, 48 h, BHI agar) of poultry samples were determined at seven timepoints. Identification of spoilage organisms was carried out by MALDI-TOF MS. Brochothrix thermosphacta, Carnobacterium sp. and Pseudomonas sp. were the main organisms found after eight days at 4 °C and 10 °C in high O2 MAP. In low O2 MAP, the main spoilage microbiota was represented by species Hafnia alvei at 10 °C, and genera Carnobacterium sp., Serratia sp., and Yersinia sp. at 4 °C. High O2 MAP is suggested as preferential gas because were less detrimental and pathogens like Yersinia were not observed. PMID:27554149

  10. Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS.

    PubMed

    Jansen, Bas C; Bondt, Albert; Reiding, Karli R; Lonardi, Emanuela; de Jong, Coen J; Falck, David; Kammeijer, Guinevere S M; Dolhain, Radboud J E M; Rombouts, Yoann; Wuhrer, Manfred

    2016-01-01

    Pregnancy requires partial suppression of the immune system to ensure maternal-foetal tolerance. Protein glycosylation, and especially terminal sialic acid linkages, are of prime importance in regulating the pro- and anti-inflammatory immune responses. However, little is known about pregnancy-associated changes of the serum N-glycome and sialic acid linkages. Using a combination of recently developed methods, i.e. derivatisation that allows the distinction between α2,3- and α2,6-linked sialic acids by high-throughput MALDI-TOF-MS and software-assisted data processing, we analysed the serum N-glycome of a cohort of 29 healthy women at 6 time points during and after pregnancy. A total of 77 N-glycans were followed over time, confirming in part previous findings while also revealing novel associations (e.g. an increase of FA2BG1S1(6), FA2G1S1(6) and A2BG2S2(6) with delivery). From the individual glycans we calculated 42 derived traits. With these, an increase during pregnancy and decrease after delivery was observed for both α2,3- and α2,6-linked sialylation. Additionally, a difference in the recovery speed after delivery was observed for α2,3- and α2,6-linked sialylation of triantennary glycans. In conclusion, our new high-throughput workflow allowed the identification of novel plasma glycosylation changes with pregnancy. PMID:27075729

  11. 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

  12. Two classifiers based on serum peptide pattern for prediction of HBV-induced liver cirrhosis using MALDI-TOF MS.

    PubMed

    Cao, Yuan; He, Kun; Cheng, Ming; Si, Hai-Yan; Zhang, He-Lin; Song, Wei; Li, Ai-Ling; Hu, Cheng-Jin; Wang, Na

    2013-01-01

    Chronic infection with hepatitis B virus (HBV) is associated with the majority of cases of liver cirrhosis (LC) in China. Although liver biopsy is the reference method for evaluation of cirrhosis, it is an invasive procedure with inherent risk. The aim of this study is to discover novel noninvasive specific serum biomarkers for the diagnosis of HBV-induced LC. We performed bead fractionation/MALDI-TOF MS analysis on sera from patients with LC. Thirteen feature peaks which had optimal discriminatory performance were obtained by using support-vector-machine-(SVM-) based strategy. Based on the previous results, five supervised machine learning methods were employed to construct classifiers that discriminated proteomic spectra of patients with HBV-induced LC from those of controls. Here, we describe two novel methods for prediction of HBV-induced LC, termed LC-NB and LC-MLP, respectively. We obtained a sensitivity of 90.9%, a specificity of 94.9%, and overall accuracy of 93.8% on an independent test set. Comparisons with the existing methods showed that LC-NB and LC-MLP held better accuracy. Our study suggests that potential serum biomarkers can be determined for discriminating LC and non-LC cohorts by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These two classifiers could be used for clinical practice in HBV-induced LC assessment. PMID:23509784

  13. Identification and characterization of a new IgE-binding protein in mackerel ( Scomber japonicus) by MALDI-TOF-MS

    NASA Astrophysics Data System (ADS)

    Wang, Bangping; Li, Zhenxing; Zheng, Lina; Liu, Yixuan; Lin, Hong

    2011-03-01

    As fish is one source of the `big eight' food allergens, the prevalence of fish allergy has increased over the past few years. In order to better understand fish allergy, it is necessary to identify fish allergens. Based on the sera from fish-allergenic patients, a 28 kDa protein from local mackerel ( Scomber japonicus), which has not been reported as a fish allergen, was found to be reactive with most of the patients' sera. The 28 kDa protein was analyzed by MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). Mascot search in NCBI database (Date: 08/07/2010) showed that the top protein matched, i.e. triosephosphate isomerase (TPI) from Xiphophorus maculatus and Poecilia reticulata, had a mowse (molecular weight search) score of 98. In addition, TPI from Epinephelus coioides also matched this mackerel protein with a mowse score of 96. Because TPI is considered as an allergen in other non-fish organisms, such as lychee, wheat, latex, archaeopotamobius ( Archaeopotamobius sibiriensis) and crangon ( Crangon crangon), we consider that it may also be an allergen in mackerel.

  14. Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS

    PubMed Central

    Jansen, Bas C.; Bondt, Albert; Reiding, Karli R.; Lonardi, Emanuela; de Jong, Coen J.; Falck, David; Kammeijer, Guinevere S. M.; Dolhain, Radboud J. E. M.; Rombouts, Yoann; Wuhrer, Manfred

    2016-01-01

    Pregnancy requires partial suppression of the immune system to ensure maternal-foetal tolerance. Protein glycosylation, and especially terminal sialic acid linkages, are of prime importance in regulating the pro- and anti-inflammatory immune responses. However, little is known about pregnancy-associated changes of the serum N-glycome and sialic acid linkages. Using a combination of recently developed methods, i.e. derivatisation that allows the distinction between α2,3- and α2,6-linked sialic acids by high-throughput MALDI-TOF-MS and software-assisted data processing, we analysed the serum N-glycome of a cohort of 29 healthy women at 6 time points during and after pregnancy. A total of 77 N-glycans were followed over time, confirming in part previous findings while also revealing novel associations (e.g. an increase of FA2BG1S1(6), FA2G1S1(6) and A2BG2S2(6) with delivery). From the individual glycans we calculated 42 derived traits. With these, an increase during pregnancy and decrease after delivery was observed for both α2,3- and α2,6-linked sialylation. Additionally, a difference in the recovery speed after delivery was observed for α2,3- and α2,6-linked sialylation of triantennary glycans. In conclusion, our new high-throughput workflow allowed the identification of novel plasma glycosylation changes with pregnancy. PMID:27075729

  15. Detection of Leishmania donovani infection using magnetic beads-based serum peptide profiling by MALDI-TOF MS in mice model.

    PubMed

    Li, Lixia; Li, Jiping; Jin, Hongtao; Shang, Limin; Li, Bo; Wei, Feng; Liu, Quan

    2012-03-01

    Leishmaniasis is an important parasitic disease, and definite diagnosis using a specific and sensitive method is the first step to cure the disease. Here, we present a novel diagnostic strategy based on serum peptide profiling by magnetic beads and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The serum peptides from the Leishmani donovani-infected and healthy mice were enriched by the optimized magnetic beads. The mass spectrograms were acquired by MALDI-TOF MS and analyzed by the ClinProTools bioinformatics software from Bruker Daltonics. The diagnostic model of serum peptide profiling produced by the ClinProTools software could correctly detect L. donovani infection in mice from the third day post-infection, with the accuracy of 94.1%, sensitivity of 92.4%, and specificity of 97.1%, respectively. The results of the present study suggested that the serum peptide profiling by MALDI-TOF MS is a novel potential tool for the clinical diagnosis of leishmaniasis. PMID:21850454

  16. 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. PMID:26002944

  17. Early diagnosis of Irkut virus infection using magnetic bead-based serum peptide profiling by MALDI-TOF MS in a mouse model.

    PubMed

    Li, Nan; Liu, Ye; Hao, Zhuo; Zhang, Shoufeng; Hu, Rongliang; Li, Jiping

    2014-01-01

    Early diagnosis is important for the prompt post-exposure prophylaxis of lyssavirus infections. To diagnose Irkut virus (IRKV) infection during incubation in mice, a novel method using magnetic bead-based serum peptide profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been established. For this test, serum peptides were concentrated by adsorption to and elution from the magnetic bead-based weak cation ion exchanger. Mass spectrograms obtained by MALDI-TOF MS were analyzed using ClinProTools bioinformatics software. Construction of the diagnostic model was performed using serum samples from mice infected with IRKV and rabies virus (RABV) BD06, Flury-LEP, and SRV9 (as controls). The method accurately diagnosed sera 2, 4 and 8 days after IRKV and RABV infections. The sensitivity, specificity, and total accuracy of diagnosis were 86.7%, 95.2%, and 92.9%, respectively. However, IRKV could not be differentiated from RABV 1 day after infection. The results of the present study indicate that serum peptide profiling by MALDI-TOF MS is a promising technique for the early clinical diagnosis of lyssavirus infections and needs to be further tested in humans and farm animals. PMID:24670473

  18. 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. PMID:27004481

  19. Optimization of MALDI-TOF MS Detection for Enhanced Sensitivity of Affinity-Captured Proteins Spanning a 100 kDa Mass Range

    PubMed Central

    Gatlin-Bunai, Christine L.; Cazares, Lisa H.; Cooke, William E.; Semmes, Oliver J.; Malyarenko, Dariya I.

    2007-01-01

    Analysis of complex biological samples by MALDI-TOF mass spectrometry has been generally limited to the detection of low-mass protein (or protein fragment) peaks. We have extended the mass range of MALDI-TOF high-sensitivity detection by an order of magnitude through the combined optimization of instrument parameters, data processing, and sample preparation procedures for affinity capture. WCX, C3, and IMAC magnetic beads were determined to be complementary and most favorable for broad mass range protein profiling. Key instrument parameters for extending mass range included adjustment of the ADC offset and preamplifier filter values of the TOF detector. Data processing was improved by a combination of constant and quadratic down-sampling, preceded by exponential baseline subtraction, to increase sensitivity of signal peaks. This enhancement in broad mass range detection of protein signals will be of direct benefit in MS expression profiling studies requiring full linear range mass detection. PMID:17918874

  20. Analyses of black fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): species-level identification of clinical isolates of Exophiala dermatitidis.

    PubMed

    Kondori, Nahid; Erhard, Marcel; Welinder-Olsson, Christina; Groenewald, Marizeth; Verkley, Gerard; Moore, Edward R B

    2015-01-01

    Conventional mycological identifications based on the recognition of morphological characteristics can be problematic. A relatively new methodology applicable for the identification of microorganisms is based on the exploitation of taxon- specific mass patterns recorded from abundant cell proteins directly from whole-cell preparations, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This study reports the application of MALDI-TOF MS for the differentiation and identifications of black yeasts, isolated from the respiratory tracts of patients with cystic fibrosis (CF). Initial phenotypic and DNA sequence-based analyses identified these isolates to be Exophiala dermatitidis. The type strains of E. dermatitidis (CBS 207.35(T)) and other species of Exophiala were included in the MALDI-TOF MS analyses to establish the references for comparing the mass spectra of the clinical isolates of Exophiala. MALDI-TOF MS analyses exhibited extremely close relationships among the clinical isolates and with the spectra generated from the type strain of E. dermatitidis. The relationships observed between the E. dermatitidis strains from the MALDI-TOF MS profiling analyses were supported by DNA sequence-based analyses of the rRNA ITS1 and ITS2 regions. These data demonstrated the applicability of MALDI-TOF MS as a reliable, rapid and cost-effective method for the identification of isolates of E. dermatitidis and other clinically relevant fungi and yeasts that typically are difficult to identify by conventional methods. PMID:25790495

  1. Development of aptamer-conjugated magnetic graphene/gold nanoparticle hybrid nanocomposites for specific enrichment and rapid analysis of thrombin by MALDI-TOF MS.

    PubMed

    Xiong, Ya; Deng, Chunhui; Zhang, Xiangmin

    2014-11-01

    Simple, rapid and sensitive analysis of thrombin (a tumor biomarker) in complex samples is quite clinical relevant and essential for the development of disease diagnosis and pharmacotherapy. Herein, we developed a novel method based on aptamer-conjugated magnetic graphene/gold nanoparticles nanocomposites (MagG@Au) for specific enrichment and rapid analysis of thrombin in biological samples using MALDI-TOF-MS. At first, gold nanoparticles were compactly deposited on PDDA functionalized magnetic graphene through electrostatic interaction. Afterwards, aptamer was easily conjugated to gold nanoparticles via Au-S bond formation. The as-made aptamer-conjugated nanocomposites took advantage of the magnetism of magnetic graphene, the high affinity and specificity of aptamer, facilitating a high-efficient separation and enrichment of thrombin. More importantly, due to the large surface area of the hybrid substrate, the average coverage density of aptamer achieved 0.34 nmol/mg, which enhanced the thrombin binding capacity and the recovery of thrombin in real samples. In turn, the enriched thrombin attributed to the sensitive output of MALDI-TOF mass spectrometry signal, 0.085 ng μL(-1) (2.36 nM) thrombin could be detected. This proposed method has a relatively wide linear relation ranging from 0.1 ng μL(-1) to 10 ng μL(-1), and satisfactory specificity. The proposed high-throughput method based on MALDI-TOF MS is expected to the application in the disease biomarker detection and clinical diagnosis. PMID:25127596

  2. 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. PMID:21503840

  3. Comparison of MALDI-TOF MS and AFLP for strain typing of ESBL-producing Escherichia coli.

    PubMed

    Veenemans, J; Welker, M; van Belkum, A; Saccomani, M C; Girard, V; Pettersson, A; Verhulst, C; Kluytmans-Vandenbergh, M; Kluytmans, J

    2016-05-01

    Typing of bacterial isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) potentially provides an efficient on-site method to monitor the spread of antibiotic-resistant bacteria and rapidly detect outbreaks. We compared MALDI-MS typing results to those of amplified fragment length polymorphism (AFLP) in a collection of 52 ESBL-producing Escherichia coli, isolated in a Dutch nursing home with an on-going outbreak of ST131 E. coli. Specific MALDI types were defined based on spectral data from four replicate colony samples of isolates grown on Columbia agar using multivariate statistical procedures. Type-specific superspectra were computed for four E .coli MALDI-types and tested for the potential of rapid and automated typing. The effect of different incubation conditions on typing performance was tested by analysing five isolates incubated for 24 h and 48 h on five different media. Types defined based on MALDI spectra were largely in agreement with the AFLP results, although some MALDI types comprised of more than one AFLP type. In particular, isolates belonging to ST131 showed distinct mass patterns. The proportion of isolates correctly assigned was substantially lower for isolates incubated on Sabouraud-dextrose and Drigalski agars for 24 h, and for those incubated for 48 h (all media). Our results show that the identification of type-specific peaks potentially allows direct typing of isolates belonging to specific clonal lineages. Both incubation time and media affected type assignment, suggesting that there is a need for a careful standardization of incubation time and culturing conditions when developing MALDI-typing schemes for E. coli. PMID:26922068

  4. MALDI TOF MS: An Exobiology Surface-Science Approach for Europa

    NASA Technical Reports Server (NTRS)

    Gerakines, Perry A.; Wdowiak, Thomas J.

    2002-01-01

    If Europa is to be of primary exobiological interest, namely as a habitat for extant life, it is obvious that: (i) a hydrosphere must prevail beneath the cryosphere for a long time, (ii) internal energy sources must be present in a sufficient state of activity, and (iii) a reasonable technical means must be available for assessing if indeed life does exist in the hypothesized hydrosphere. This discussion focuses on technological issues, because the compounding evidence about Europa indicates that the first two are highly likely to be true. We present a consideration of time-of-flight mass spectroscopy (TOF MS) conducted in-situ on the cryosphere surface of Europa during a landed robotic mission. We assert that this is a reasonable technical means not only for exploring the composition of the cryosphere itself, but also for locating any biomolecular indicators of extant life brought to the surface through cryosphere activity. We also describe a MALDI (MAtrix Laser Desorption and Ionization) TOF MS system that we are constructing as a proof-of-concept prototype for conducting TOF MS measurements on Europa.

  5. Rapid urine preparation prior to identification of uropathogens by MALDI-TOF MS.

    PubMed

    Veron, L; Mailler, S; Girard, V; Muller, B H; L'Hostis, G; Ducruix, C; Lesenne, A; Richez, A; Rostaing, H; Lanet, V; Ghirardi, S; van Belkum, A; Mallard, F

    2015-09-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-ToF MS) has been introduced in clinical routine microbiology laboratories. For the rapid diagnosis of urinary tract infections, culture-independent methods prior MALDI-mediated identification have been described. Here, we describe a comparison of three of these methods based on their performance of bacterial identification and their potential as a routine tool for microbiology labs : (i) differential centrifugation, (ii) urine filtration and (iii) a 5-h bacterial cultivation on solid culture media. For 19 urine samples, all methods were directly compared and correct bacterial species identification by MALDI was used as performance indicator. A higher percentage of correct MALDI identification was obtained after filtration (78.9 %) and the growth-based method (84.2 %) as compared to differential centrifugation (68.4 %). Additional testing of 76 mono-microbial specimens (bacteriuria > 10(5) CFU/mL) confirmed the good performance of short growth with a 90.8 % correct MALDI score, with a potentially better fit to the routine workflow of microbiology labs. PMID:26054715

  6. Top-down proteomic identification of furin-cleaved alpha-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method has been developed to identify the alpha-subunit of shiga toxin 2 (alpha-Stx2) from Escherichia coli O157:H7 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics using web-based software develo...

  7. Induction and identification of disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 (beta-Stx2) from Escherichia coli O157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic an...

  8. Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC.

    PubMed

    Yang, Huan; Li, Xu; Li, Xue; Yu, Huimin; Shen, Zhongyao

    2015-03-01

    A three-stage linear gradient strategy using reverse-phase high-performance liquid chromatography (HPLC) was optimized for rapid, high-quality, and simultaneous purification of the lipopeptide isoforms of iturin, fengycin, and surfactin, which may differ in composition by only a single amino acid and/or the fatty acid residue. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was applied to detect the lipopeptides harvested from each reversed-phase HPLC peak. Amino acid analysis based on phenyl isothiocyanate derivatization was further used for confirmation of the amino acid species and molar ratio in a certain HPLC fraction. By this MALDI-TOF-MS/MS coupled with amino acid analysis, it was revealed that iturin at m/z 1,043 consists of a circular Asn-Tyr-Asn-Gln-Pro-Asn-Ser peptide and C14 β-OH fatty acid. Surfactin homologs from Bacillus subtilis THY-7 at m/z 1,030, 1,044, 1,058, and 1,072 possess a circular Glu-Leu-Leu-Val-Asp-Leu-Leu peptide and the β-OH fatty acid with a different length (C13-C16). Fengycin species at m/z 1,463 and 1,477 are homologs possessing the circular peptide Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile linked to a C16 or C17 γ-OH fatty acid, whereas fengycin at m/z 1,505 contains a Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile sequence with a Val instead of Ala at position 6. The method developed in this work provided an efficient approach for characterization of diverse lipopeptide isoforms from the iturin, fengycin, and surfactin families. PMID:25662934

  9. Performance of mass spectrometric identification of bacteria and yeasts routinely isolated in a clinical microbiology laboratory using MALDI-TOF MS

    PubMed Central

    Wang, Weiping; Xi, Haiyan; Huang, Mei; Wang, Jie; Fan, Ming; Chen, Yong; Shao, Haifeng

    2014-01-01

    Background Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to identifying bacterial and yeast strains. The aim of this study was to evaluate the clinical performance of the VITEK® MS system in the identification of bacteria and yeast strains routinely isolated from clinical samples. Methods We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria and yeasts regardless of phylum or source of isolation. Discordant results were resolved with 16S rDNA or internal transcribed spacer (ITS) gene sequencing. Colonies (a single deposit on a MALDI disposable target without any prior extraction step) were analyzed using the VITEK® MS system. Peptide spectra acquired by the system were compared with the VITEK® MS IVD database Version 2.0, and the identification scores were recorded. Results Of the 1,181 isolates (1,061 bacterial isolates and 120 yeast isolates) analyzed, 99.5% were correctly identified by MALDI-TOF mass spectrometry; 95.7% identified to the species level, 3.6% identified to the genus level, and 0.3% identified within a range of species belonging to different genera. Conversely, 0.1% of isolates were misidentified and 0.4% were unidentified, partly because the species were not included in the database. Re-testing using a second deposit provided a successful identification for 0.5% of isolates unidentified with the first deposit. Our results show that the VITEK® MS system has exceptional performance in identifying bacteria and yeast by comparing acquired peptide spectra to those contained in its database. Conclusions MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive method for bacterial and yeast identification. Our results demonstrate that the VITEK® MS system is a fast and reliable technique, and has the potential to replace conventional phenotypic

  10. High Molecular Weight Typing with MALDI-TOF MS - A Novel Method for Rapid Typing of Clostridium difficile

    PubMed Central

    Rizzardi, Kristina; Åkerlund, Thomas

    2015-01-01

    Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations. PMID:25923527

  11. 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 Central

    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

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

    PubMed Central

    Heras Cañas, V.; Pérez Ramirez, M.D.; Bermudez Jiménez, F.; Rojo Martin, M.D.; Miranda Casas, C.; Marin Arriaza, M.; Navarro Marí, J.M.

    2015-01-01

    Lactococcus garvieae is a Gram-positive, catalase negative coccus arranged in pairs or short chains, well-known as a fish pathogen. We report a case of Infective Endocarditis (IE) by L. garvieae in a native valve from a 68-year-old male with unknown history of contact with raw fish and an extensive history of heart disease. This case highlights the reliability of MALDI-TOF MS compared to conventional methods in the identification of rare microorganisms like this. PMID:25949815

  13. 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

  14. Usefulness of CHROMagar Candida Medium, Biochemical Methods--API ID32C and VITEK 2 Compact and Two MALDI-TOF MS Systems for Candida spp. Identification.

    PubMed

    Stefaniuk, Elzbieta; Baraniak, Anna; Fortuna, Monika; Hryniewicz, Waleria

    2016-01-01

    This study was conducted to compare of the yeasts identification results obtained with two new systems using the MALDI-TOF MS technique with the ones obtained using the routine identification methods of Candida spp. in clinical microbiology laboratories. All 124 Candida spp. isolates were recovered from the routine examination of clinical specimens in microbiological laboratories and collected in the Centre of Quality Control in Microbiology in Warsaw (Poland). Our findings confirm the high agreement (98%) of fungal identification using the standard, biochemistry laboratory methods and mass spectrometry technique. PMID:27282002

  15. Strain-level bacterial identification by CeO2-catalyzed MALDI-TOF MS fatty acid analysis and comparison to commercial protein-based methods

    PubMed Central

    Cox, C. R.; Jensen, K. R.; Saichek, N. R.; Voorhees, K. J.

    2015-01-01

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid approach for clinical bacterial identification. However, current protein-based commercial bacterial ID methods fall short when differentiating closely related species/strains. To address this shortcoming, we employed CeO2-catalyzed fragmentation of lipids to produce fatty acids using the energy inherent to the MALDI laser as a novel alternative to protein profiling. Fatty acid profiles collected from Enterobacteriaceae, Acinetobacter, and Listeria using CeO2-catalyzed metal oxide laser ionization (MOLI MS), processed by principal component analysis, and validated by leave–one-out cross-validation (CV), showed 100% correct classification at the species level and 98% at the strain level. In comparison, protein profile data from the same bacteria yielded 32%, 54% and 67% mean species-level accuracy using two MALDI-TOF MS platforms, respectively. In addition, several pathogens were misidentified by protein profiling as non-pathogens and vice versa. These results suggest novel CeO2-catalyzed lipid fragmentation readily produced (i) taxonomically tractable fatty acid profiles by MOLI MS, (ii) highly accurate bacterial classification and (iii) consistent strain-level ID for bacteria that were routinely misidentified by protein-based methods. PMID:26190224

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

    PubMed

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

    2012-03-01

    This study focuses on acquiring information on the degradation process of proteinaceous binders due to ultra violet (UV) radiation and possible interactions owing to the presence of historical mineral pigments. With this aim, three different paint model samples were prepared according to medieval recipes, using rabbit glue as proteinaceus binders. One of these model samples contained only the binder, and the other two were prepared by mixing each of the pigments (cinnabar or azurite) with the binder (glue tempera model samples). The model samples were studied by applying Principal Component Analysis (PCA) to their mass spectra obtained with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). The complementary use of Fourier Transform Infrared Spectroscopy to study conformational changes of secondary structure of the proteinaceous binder is also proposed. Ageing effects on the model samples after up to 3000 h of UV irradiation were periodically analyzed by the proposed approach. PCA on MS data proved capable of identifying significant changes in the model samples, and the results suggested different aging behavior based on the pigment present. This research represents the first attempt to use this approach (PCA on MALDI-TOF-MS data) in the field of Cultural Heritage and demonstrates the potential benefits in the study of proteinaceous artistic materials for purposes of conservation and restoration. PMID:22431458

  17. A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

    PubMed Central

    Lévesque, Simon; Dufresne, Philippe J.; Soualhine, Hafid; Domingo, Marc-Christian; Bekal, Sadjia; Lefebvre, Brigitte; Tremblay, Cécile

    2015-01-01

    Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97

  18. The influence of matrix and laser energy on the molecular mass distribution of synthetic polymers obtained by MALDI-TOF-MS

    NASA Astrophysics Data System (ADS)

    Wetzel, Stephanie J.; Guttman, Charles M.; Girard, James E.

    2004-11-01

    The molecular mass distribution (MMD) obtained in synthetic polymer characterization by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) may be biased by preferential desorption/ionization of low mass polymer molecules, preferential ion attachment to larger polymers, or degradation and fragmentation due to the desorption process. In this study we focus on the effect of matrix and laser energy on the MMD of four synthetic polymers of low polydispersity with varying thermal stabilities. The four polymers considered were polystyrene (PS), poly(ethylene glycol) (PEG), poly(methyl methacrylate) (PMMA) and poly(tetrahydrofuran) (PTHF). The matrix in which the polymer is analyzed may also influence the laser energy effect of MALDI and was also considered in this paper. Three common matrixes were considered, dithranol, all trans-retinoic acid (RA) and 2,5-dihydroxybenzoic acid (DHB). Statistical analyses of the molecular mass distributions, obtained by varying laser energy and matrixes, reveal trends that can be used to describe the influences of matrix and laser energy on MALDI-TOF-MS data measurement of synthetic polymers. The statistical analysis revealed that the matrix has a greater effect on the polymer MMD than was expected. Polymers analyzed in DHB yielded lower mass moments than polymers analyzed in RA and dithranol. The effects of laser power on the MMD of the polymers were found to be matrix dependent.

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

    PubMed Central

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

    2015-01-01

    MALDI-TOF MS profiling has been shown to be a rapid and reliable method to characterize pure cultures of bacteria. Currently, there is keen interest in using this technique to identify bacteria in mixtures. Promising results have been reported with two- or three-isolate model systems using biomarker-based approaches. In this work, we applied MALDI-TOF MS-based methods to a more complex model mixture containing six bacteria. We employed: 1) a biomarker-based approach that has previously been shown to be useful in identification of individual bacteria in pure cultures and simple mixtures and 2) a similarity coefficient-based approach that is routinely and nearly exclusively applied to identification of individual bacteria in pure cultures. Both strategies were developed and evaluated using blind-coded mixtures. With regard to the biomarker-based approach, results showed that most peaks in mixture spectra could be assigned to those found in spectra of each component bacterium; however, peaks shared by two isolates as well as peaks that could not be assigned to any individual component isolate were observed. For two-isolate blind-coded samples, bacteria were correctly identified using both similarity coefficient- and biomarker-based strategies, while for blind-coded samples containing more than two isolates, bacteria were more effectively identified using a biomarker-based strategy. PMID:26537565

  20. VibrioBase: A MALDI-TOF MS database for fast identification of Vibrio spp. that are potentially pathogenic in humans.

    PubMed

    Erler, René; Wichels, Antje; Heinemeyer, Ernst-August; Hauk, Gerhard; Hippelein, Martin; Reyes, Nadja Torres; Gerdts, Gunnar

    2015-02-01

    Mesophilic marine bacteria of the family Vibrionaceae, specifically V. cholerae, V. parahaemolyticus and V. vulnificus, are considered to cause severe illness in humans. Due to climate-change-driven temperature increases, higher Vibrio abundances and infections are predicted for Northern Europe, which in turn necessitates environmental surveillance programs to evaluate this risk. We propose that whole-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling is a promising tool for the fast and reliable species classification of environmental isolates. Because the reference database does not contain sufficient Vibrio spectra we generated the VibrioBase database in this study. Mass spectrometric data were generated from 997 largely environmental strains and filed in this new database. MALDI-TOF MS clusters were assigned based on the species classification obtained by analysis of partial rpoB (RNA polymerase beta-subunit) sequences. The affiliation of strains to species-specific clusters was consistent in 97% of all cases using both approaches, and the extended VibrioBase generated more specific species identifications with higher matching scores compared to the commercially available database. Therefore, we have made the VibrioBase database freely accessible, which paves the way for detailed risk assessment studies of potentially pathogenic Vibrio spp. from marine environments. PMID:25466918

  1. Comparison of VITEK2, MALDI-TOF MS, and 16S rDNA sequencing for identification of Myroides odoratus and Myroides odoratimimus.

    PubMed

    Schröttner, Percy; Rudolph, Wolfram W; Eing, Bodo R; Bertram, Sebastian; Gunzer, Florian

    2014-06-01

    The genus Myroides comprises the 2 medically relevant species Myroides odoratus and Myroides odoratimimus that are rare opportunistic pathogens and cause infections in immunocompromised patients. A fast identification of Myroides is of importance because these bacterial strains show multiple resistance against antibiotics and therefore limit treatment options. They are associated, for instance, with urinary tract infections, sepsis, meningitis, pneumonia, and infectious cellulitis. Since more and more Myroides spp. are being described, additional potentially pathogenic bacteria may be identified in the future demanding the need for fast and reliable identification methods at species level. However, to date, only molecular approaches meet these demands. In this study, we, therefore, attempt to define an appropriate method other than DNA fingerprinting that will permit a comparable efficacy and, possibly, a more economical strain identification. For this purpose, we compared 2 widely used automated diagnostic systems (VITEK 2 [bioMérieux, Nürtingen, Germany] and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) [Bruker Daltonics, Bremen, Germany]) and correlated the results to 16S rDNA sequencing data. In total, we analyzed 22 strains collected in the course of routine diagnostics. In this study, we demonstrate that VITEK 2 reliably identifies the genus Myroides but cannot differentiate between M. odoratimimus and M. odoratus. In contrast to this, both MALDI-TOF MS and 16S rDNA sequencing efficiently distinguish between the 2 species. PMID:24666701

  2. Identification of Differential Protein Expression in Hepatocellular Carcinoma Induced Wistar Albino Rats by 2D Electrophoresis and MALDI-TOF-MS Analysis.

    PubMed

    Vedarethinam, Vadanasundari; Dhanaraj, Karthik; Soundherrajan, Ilavenil; Sivanesan, Ravikumar

    2016-04-01

    Hepato cellular carcinoma (HCC) is a type of malignant tumor. To investigate the proteins in cancer molecular mechanism and its role in HCC, we have used proteomic tools such as 2DE and MALDI-TOF-MS. Our investigation ravels that, plasma α-fetoprotein and carcinoembryonic antigen levels were elevated in DEN induced rats and gradually decreased after the treatment with 1,3BPMU. 2DE and MALDI-TOF-MS tool offers to identify the up and down regulation of proteins in HCC. Proteomic study reveals that, five differentially expressed proteins were identified in DEN induced rats and 1,3BPMU treated rats i.e. three up regulated protein such as T kininogen, NDPKB, PRMT1 (DEN induced rats), RGS19 and PAF (1,3BPMU treated rats) in 3BPMU treated rats, activation of transcription of a single gene from multiple promoters provides flexibility in the controlled gene expression. The regulations of hepatocyte stimulating factor were slow down the proliferation of hepatic cell and uncontrolled hepatic cell growth and also molecular signals strongly argue for a patho-physiological role in liver metastasis to control the cell aggression. This indicates that, anti cancer property of 1,3BPMU can be used as potent anti cancer agent. The present study also shows the proteomic approach helps to elucidate the tumor maker as well as regulatory marker proteins in HCC. PMID:27069327

  3. A MALDI-TOF MS method for the simultaneous and quantitative analysis of neutral and sialylated glycans of CHO-expressed glycoproteins.

    PubMed

    Tep, Samnang; Hincapie, Marina; Hancock, William S

    2012-01-10

    The development of a MALDI-TOF MS method for the quantitative analysis of the glycosylation of CHO-expressed biotherapeutic glycoproteins shall be presented. The method utilizes a well-established chemistry, reductive amination of glycans, to derivatize glycans with either a light analog ((12)C(7) anthranilic acid) or a heavy analog ((13)C(7) anthranilic acid) to allow for the direct comparison of the alternately-labeled glycans by MALDI-TOF MS. The method allows for the simultaneous analysis of neutral and sialylated glycans and displays a linear dynamic range over two orders of magnitude with sub-picomolar sensitivity. Additionally, because the glycans are derivatized with anthranilic acid, which is a very sensitive fluorophore, the glycans can be analyzed by chromatography with fluorescence detection. The need for this type of method is highlighted by the biotechnology/biopharmaceutical industry's continuous drive towards fully understanding process control. By providing this type of quantitative data, glycosylation changes of the expressed protein can be easily observed thereby helping to further advance the understanding of a major aspect of the biopharmaceutical process. PMID:22138464

  4. MALDI-TOF MS Imaging evidences spatial differences in the degradation of solid polycaprolactone diol in water under aerobic and denitrifying conditions.

    PubMed

    Rivas, Daniel; Ginebreda, Antoni; Pérez, Sandra; Quero, Carmen; Barceló, Damià

    2016-10-01

    Degradation of solid polymers in the aquatic environment encompasses a variety of biotic and abiotic processes giving rise to heterogeneous patterns across the surface of the material, which cannot be investigated using conventional Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that only renders an "average" picture of the sample. In that context, MALDI-TOF MS Imaging (MALDI MSI) provides a rapid and efficient tool to study 2D spatial changes occurred in the chemical composition of the polymer surface. Commercial polycaprolactone diol (average molecular weight of 1250Da) was selected as test material because it had been previously known to be amenable to biological degradation. The test oligomer probe was incubated under aerobic and denitrifying conditions using synthetic water and denitrifying mixed liquor obtained from a wastewater treatment plant respectively. After ca. seven days of exposure the mass spectra obtained by MALDI MSI showed the occurrence of chemical modifications in the sample surface. Observed heterogeneity across the probe's surface indicated significant degradation and suggested the contribution of biotic processes. The results were investigated using different image processing tools. Major changes on the oligomer surface were observed when exposed to denitrifying conditions. PMID:27213667

  5. 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. PMID:27296834

  6. Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

    PubMed Central

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

    2014-01-01

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

  7. Phosphorylation of extracellular matrix tenascin-X detected by differential mass tagging followed by nanoLC-MALDI-TOF/TOF-MS/MS using ProteinPilot software.

    PubMed

    Matsumoto, Ken-Ichi

    2012-01-01

    Reversible protein phosphorylation represents a major mechanism of signal transduction in a variety of cellular functions. An understanding of proteome-wide phosphorylation dynamics is important to obtain an overview of the whole signal transduction network. However, a systematic analysis for differentially expressed phosphoproteins under serum-stimulated response is lacking. Here, an easy and fast approach for the identification of differentially expressed phosphoproteins was used. After enrichment of phosphoproteins from serum-stimulated cell lysates by immobilized metal affinity chromatography, a quantitative proteomic approach with isobaric tag for absolute and relative quantitation labeling in combination with nanoLC-MALDI-TOF/TOF-MS/MS followed by ProteinPilot analysis was used. Consequently, 506 differentially expressed phosphoproteins were identified. Among them, 22 proteins that had a reproducible phosphorylation site at Ser or Thr were identified. Out of these 22 phosphoproteins, 7 are mainly involved in splicing. Among the 22 proteins, it was found that extracellular matrix tenascin-X is phosphorylated, although there is little quantitative change by the serum stimulation. MS/MS analysis revealed a novel phosphorylation site of tenascin-X, Thr1841, located in the loop region between the 10th and 11th fibronectin type III repeats. The phosphorylation of tenascin-X would be considered in clarifying its function in the future. PMID:21967672

  8. 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

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

    2015-10-01

    Enterobacter cloacae is among the most important pathogens responsible for nosocomial infections and outbreaks. In this study, 77 Enterobacter isolates were collected: 27 isolates from Algerian hospitals (in Constantine, Annaba, and Skikda) and 50 isolates from Marseille, France. All strains were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility testing was performed by the disk diffusion method. PCR was used to detect extended-spectrum-beta-lactamase (ESBL)-encoding, fluoroquinolone resistance-encoding, and aminoglycoside-modifying enzyme (AME) genes. Epidemiological typing was performed using MALDI-TOF MS with data mining approaches, along with multilocus sequence typing (MLST). Sixty-eight isolates (27 from Algeria, 41 from Marseille) were identified by MALDI-TOF MS as E. cloacae. Resistance to antibiotics in the Algerian isolates was significantly higher than that in the strains from Marseille, especially for beta-lactams and aminoglycosides. Eighteen of the 27 Algerian isolates and 11 of the 41 Marseille isolates possessed at least one ESBL-encoding gene: blaCTX-M and/or blaTEM. AME genes were detected in 20 of the 27 Algerian isolates and 8 of the 41 Marseille isolates [ant(2″)-Ia, aac(6')-Ib-cr, aadA1, aadA2, and armA]. Conjugation experiments showed that armA was carried on a transferable plasmid. MALDI-TOF typing showed three separate clusters according to the geographical distribution and species level. An MLST-based phylogenetic tree showed a clade of 14 E. cloacae isolates from a urology unit clustering together in the MALDI-TOF dendrogram, suggesting the occurrence of an outbreak in this unit. In conclusion, the ability of MALDI-TOF to biotype strains was confirmed, and surveillance measures should be implemented, especially for Algerian patients hospitalized in France. PMID:26239991

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

    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

  10. A Designed Experiments Approach to Optimizing MALDI-TOF MS Spectrum Processing Parameters Enhances Detection of Antibiotic Resistance in Campylobacter jejuni

    PubMed Central

    Penny, Christian; Grothendick, Beau; Zhang, Lin; Borror, Connie M.; Barbano, Duane; Cornelius, Angela J.; Gilpin, Brent J.; Fagerquist, Clifton K.; Zaragoza, William J.; Jay-Russell, Michele T.; Lastovica, Albert J.; Ragimbeau, Catherine; Cauchie, Henry-Michel; Sandrin, Todd R.

    2016-01-01

    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 study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the

  11. A Designed Experiments Approach to Optimizing MALDI-TOF MS Spectrum Processing Parameters Enhances Detection of Antibiotic Resistance in Campylobacter jejuni.

    PubMed

    Penny, Christian; Grothendick, Beau; Zhang, Lin; Borror, Connie M; Barbano, Duane; Cornelius, Angela J; Gilpin, Brent J; Fagerquist, Clifton K; Zaragoza, William J; Jay-Russell, Michele T; Lastovica, Albert J; Ragimbeau, Catherine; Cauchie, Henry-Michel; Sandrin, Todd R

    2016-01-01

    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 study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the

  12. Epigenetic Activation of Antibacterial Property of an Endophytic Streptomyces coelicolor Strain AZRA 37 and Identification of the Induced Protein Using MALDI TOF MS/MS.

    PubMed

    Kumar, Jitendra; Sharma, Vijay K; Singh, Dheeraj K; Mishra, Ashish; Gond, Surendra K; Verma, Satish K; Kumar, Anuj; Kharwar, Ravindra Nath

    2016-01-01

    The endophytic Streptomyces coelicolor strain AZRA 37 was isolated from the surface sterilized root of Azadirachta indica A. Juss., commonly known as neem plant in India. Since only a few reports are available regarding epigenetic modulations of microbial entities, S. coelicolor was treated with different concentrations of 5-azacytidine for this purpose and evaluated for its antibacterial potential against five human pathogenic bacteria (Aeromonas hydrophila IMS/GN11, Enterococcus faecalis IMS/GN7, Salmonella typhi MTCC 3216, Shigella flexneri ATCC 12022 and Staphylococcus aureus ATCC 25923). The crude extract obtained from cultures treated with 25 μM concentration of 5-azacytidine, was found effective against all five pathogenic bacteria tested while the untreated control was only active against 3 pathogenic bacteria. HPLC analysis of crude compounds from treated cultures showed a greater number of compounds than that of the control. Extraction of whole cell protein and its SDS PAGE analysis showed an additional major protein band in 25 μM 5-azacytidine treated culture and MALDI TOF MS/MS analysis revealed that this protein belongs to the porin family. PMID:26844762

  13. Epigenetic Activation of Antibacterial Property of an Endophytic Streptomyces coelicolor Strain AZRA 37 and Identification of the Induced Protein Using MALDI TOF MS/MS

    PubMed Central

    Kumar, Jitendra; Sharma, Vijay K.; Singh, Dheeraj K.; Mishra, Ashish; Gond, Surendra K.; Verma, Satish K.; Kumar, Anuj; Kharwar, Ravindra Nath

    2016-01-01

    The endophytic Streptomyces coelicolor strain AZRA 37 was isolated from the surface sterilized root of Azadirachta indica A. Juss., commonly known as neem plant in India. Since only a few reports are available regarding epigenetic modulations of microbial entities, S. coelicolor was treated with different concentrations of 5-azacytidine for this purpose and evaluated for its antibacterial potential against five human pathogenic bacteria (Aeromonas hydrophila IMS/GN11, Enterococcus faecalis IMS/GN7, Salmonella typhi MTCC 3216, Shigella flexneri ATCC 12022 and Staphylococcus aureus ATCC 25923). The crude extract obtained from cultures treated with 25 μM concentration of 5-azacytidine, was found effective against all five pathogenic bacteria tested while the untreated control was only active against 3 pathogenic bacteria. HPLC analysis of crude compounds from treated cultures showed a greater number of compounds than that of the control. Extraction of whole cell protein and its SDS PAGE analysis showed an additional major protein band in 25 μM 5-azacytidine treated culture and MALDI TOF MS/MS analysis revealed that this protein belongs to the porin family. PMID:26844762

  14. Isolation and identification of flavour peptides from Puffer fish (Takifugu obscurus) muscle using an electronic tongue and MALDI-TOF/TOF MS/MS.

    PubMed

    Zhang, Mei-Xiu; Wang, Xi-Chang; Liu, Yuan; Xu, Xing-Lian; Zhou, Guang-Hong

    2012-12-01

    To clarify the key flavour peptides that account for the cooked taste of puffer fish, this study was performed to examine flavour peptides extracted from the flesh of puffer fish (Takifugu obscurus). Peptides fractions (P1, P2, P3, P4 and P5) were purified from an aqueous extract of T. obscurus muscle by ultrafiltration and Sephadex G-15 gel filtration chromatography (GFC). P2 was further fractionated into P2a, P2b, and P2c by reverse phase high performance liquid chromatography (RP-HPLC). Fraction P2b elicited umami and sweet taste. The amino acid sequence of P2b subfraction was identified as Tyr-Gly-Gly-Thr-Pro-Pro-Phe-Val (836.4Da) by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF/TOF MS/MS). Hydrophilic amino acids residues Tyr, Gly, Gly, Thr, and Phe are likely to contribute to the umami and sweet taste of this octapeptide. The results of this study suggest this peptide is one of important components of the 'mellowness' and 'tenderness' taste of the T. obscurus. PMID:22953881

  15. Novel PDD-PDT system based on spectrophotometric real-time fluorescence monitoring and MALDI-TOF-MS analysis of tumors

    NASA Astrophysics Data System (ADS)

    Yoshida, Takato O.; Kohno, Eiji; Dodeller, Marc; Sakurai, Takashi; Yamamoto, Seiji; Terakawa, Susumu

    2009-06-01

    In the PDT practice for tumor patients, the dose and irradiation time for the treatment are chosen by experience and not by real need. To establish advanced PDD-PDT model system for patients, we developed a method for monitoring the cell-death based on a spectrophotometric real-time change in fluorescence in HeLa-tumors during Photofrin®-PDT and ALA-PDT. Here, we describe the results of application of the new PDD-PDT system to human tumors. The fluorescence spectra obtained from human tumors were analyzed by the differential spectral analysis. The mass-spectral changes of tumor tissues during PDD-PDT were also examined by MALDI-TOF-MS/MS. The first author's seborrheic keratosis was monitored with this system during the PDD-PDT with a topically applied ALA-ointment. The changes in fluorescence spectrum were successfully detected, and the tumor regressed completely within 5 months. The differential spectral analysis of PDD-PDT-fluorescence monitoring spectra of tumors and isolated mitochondria showed a marked decrease of three peaks in the red region indicative of the PDD (600 - 720 nm), and a transient rise followed by a decline of peaks in the green region indicative of the PDT (450 - 580 nm). The MALDI-TOF-MS analysis of PDD-PDT HeLa-tumors showed a consumption of Photofrin-deuteroporphyrin and ALA-PpIX, and decreases in protein mass in the range of 4,000 - 16,000 Da, m/z 4929, 8564, 10089, 15000, and an increase in m/z 7002 in a Photofrin® PDD-PDT monitoring tumor.

  16. A Sensitive and Effective Proteomic Approach to Identify She-Donkey’s and Goat’s Milk Adulterations by MALDI-TOF MS Fingerprinting

    PubMed Central

    Di Girolamo, Francesco; Masotti, Andrea; Salvatori, Guglielmo; Scapaticci, Margherita; Muraca, Maurizio; Putignani, Lorenza

    2014-01-01

    She-donkey’s milk (DM) and goat’s milk (GM) are commonly used in newborn and infant feeding because they are less allergenic than other milk types. It is, therefore, mandatory to avoid adulteration and contamination by other milk allergens, developing fast and efficient analytical methods to assess the authenticity of these precious nutrients. In this experimental work, a sensitive and robust matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling was designed to assess the genuineness of DM and GM milks. This workflow allows the identification of DM and GM adulteration at levels of 0.5%, thus, representing a sensitive tool for milk adulteration analysis, if compared with other laborious and time-consuming analytical procedures. PMID:25110863

  17. A sensitive and effective proteomic approach to identify she-donkey's and goat's milk adulterations by MALDI-TOF MS fingerprinting.

    PubMed

    Di Girolamo, Francesco; Masotti, Andrea; Salvatori, Guglielmo; Scapaticci, Margherita; Muraca, Maurizio; Putignani, Lorenza

    2014-01-01

    She-donkey's milk (DM) and goat's milk (GM) are commonly used in newborn and infant feeding because they are less allergenic than other milk types. It is, therefore, mandatory to avoid adulteration and contamination by other milk allergens, developing fast and efficient analytical methods to assess the authenticity of these precious nutrients. In this experimental work, a sensitive and robust matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling was designed to assess the genuineness of DM and GM milks. This workflow allows the identification of DM and GM adulteration at levels of 0.5%, thus, representing a sensitive tool for milk adulteration analysis, if compared with other laborious and time-consuming analytical procedures. PMID:25110863

  18. Rapid Discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by Fluorescence In Situ Hybridization (FISH) and Two Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Platforms

    PubMed Central

    Frickmann, Hagen; Christner, Martin; Donat, Martina; Berger, Anja; Essig, Andreas; Podbielski, Andreas; Hagen, Ralf Matthias; Poppert, Sven

    2013-01-01

    Background Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level. Methodology A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction. Principal Findings Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system's database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients. PMID:23646201

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. Preparation of C60-functionalized magnetic silica microspheres for the enrichment of low-concentration peptides and proteins for MALDI-TOF MS analysis.

    PubMed

    Chen, Hemei; Qi, Dawei; Deng, Chunhui; Yang, Penyuan; Zhang, Xiangmin

    2009-01-01

    In this work, for the first time, a novel C60-functionalized magnetic silica microsphere (designated C60-f-MS) was synthesized by radical polymerization of C60 molecules on the surface of magnetic silica microspheres. The resulting C60-f-MS microsphere has magnetite core and thin C60 modified silica shell, which endow them with useful magnetic responsivity and surface affinity toward low-concentration peptides and proteins. As a result of their excellent magnetic property, the synthesized C60-f-MS microspheres can be easily separated from sample solution without ultracentrifuge. The C60-f-MS microspheres were successfully applied to the enrichment of low-concentration peptides in tryptic protein digest and human urine via a MALDI-TOF MS analysis. Moreover, they were demonstrated to have enrichment efficiency for low-concentration proteins. Due to the novel materials maintaining excellent magnetic properties and admirable adsorption, the process of enrichment and desalting is very fast (only 5 min), convenient and efficient. As it has been demonstrated in the study, newly developed fullerene-derivatized magnetic silica materials are superior to those already available in the market. The facile and low-cost synthesis as well as the convenient and efficient enrichment process of the novel C60-f-MS microspheres makes it a promising candidate for isolation of low-concentration peptides and proteins even in complex biological samples such as serum, plasma, and urine or cell lysate. PMID:19086100

  1. Bacillus subtilis subsp. subtilis CBMDC3f with antimicrobial activity against Gram-positive foodborne pathogenic bacteria: UV-MALDI-TOF MS analysis of its bioactive compounds.

    PubMed

    Torres, M J; Petroselli, G; Daz, M; Erra-Balsells, R; Audisio, M C

    2015-06-01

    In this work a new Bacillus sp. strain, isolated from honey, was characterized phylogenetically. Its antibacterial activity against three relevant foodborne pathogenic bacteria was studied; the main bioactive metabolites were analyzed using ultraviolet matrix assisted laser desorption-ionization mass spectrometry (UV-MALDI MS). Bacillus CBMDC3f was phylogenetically characterized as Bacillus subtilis subsp. subtilis after rRNA analysis of the 16S subunit and the gyrA gene (access codes Genbank JX120508 and JX120516, respectively). Its antibacterial potential was evaluated against Listeria monocytogenes (9 strains), B. cereus (3 strains) and Staphylococcus aureus ATCC29213. Its cell suspension and cell-free supernatant (CFS) exerted significant anti-Listeria and anti-S. aureus activities, while the lipopeptides fraction (LF) also showed anti-B. cereus effect. The UV-MALDI-MS analysis revealed surfactin, iturin and fengycin in the CFS, whereas surfactin predominated in the LF. The CFS from CBMDC3f contained surfactin, iturin and fengycin with four, two and four homologues per family, respectively, whereas four surfactin, one iturin and one fengycin homologues were identified in the LF. For some surfactin homologues, their UV-MALDI-TOF/TOF (MS/MS; Laser Induced Decomposition method, LID) spectra were also obtained. Mass spectrometry analysis contributed with relevant information about the type of lipopeptides that Bacillus strains can synthesize. From our results, surfactin would be the main metabolite responsible for the antibacterial effect. PMID:25820813

  2. Ion detection with a cryogenic detector compared to a microchannel plate detector in MALDI TOF-MS

    SciTech Connect

    Benner, W H; Frank, M; Labov, S; Westmacott, G; Zhong, F

    1999-06-29

    Detection of molecular ions in mass spectrometry is typically accomplished by an ion colliding with a surface and then amplifying the emitted secondary electrons. It is well established that the secondary electron yield decreases as the mass of the primary ion increases [1-3], thus limiting the detection efficiency of large molecular ions. One way around this limitation is to use secondary ion detectors because the emission efficiency of secondary ions does not seem to decrease for increasing primary ion mass [1]. However this technique has limitations in timing resolution because of the mass spread of the emitted secondary ions. To find other ways around high mass detection limitations it is important to understand existing mechanisms of detection and to explore alternative detector types. To this end, a superconducting tunnel junction (STJ) detector was used in measuring the secondary electron emission efficiency, se, for a MCP detector. STJ detectors are energy sensitive and do not rely on secondary emission to produce a signal. Using a linear MALDI-TOF mass spectrometer, a STJ detector is mounted directly behind the hole in an annular MCP detector. This mounting arrangement allows ions to be detected simultaneously by each detector. The STJ detector sits in a liquid helium cryostat and is operated at 1.3 K to minimize thermal noise (see [4,5] for more details). Primary ions passing through the center hole of the MCP detector collide with the 0.04 mm{sup 2} STJ surface and generate a detector-pulse that is approximately proportional to the ion's total energy. A mask with a small hole in it was placed in front of the MCP detector so that the MCP and STJ detectors have approximately the same effective active areas. The ion beam diameter near the MCP is over 2.5 cm (measured with a MCP-phosphorus screen detector) and the axial separation of the two detectors is about 4 mm. Both detectors were operated in pulse-counting mode and set to have the same effective

  3. Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat

    PubMed Central

    2010-01-01

    Background Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF × SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries. Results At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles. Conclusions PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of

  4. Clinical Impact of MALDI-TOF MS Identification and Rapid Susceptibility Testing on Adequate Antimicrobial Treatment in Sepsis with Positive Blood Cultures

    PubMed Central

    Verroken, Alexia; Defourny, Lydwine; le Polain de Waroux, Olivier; Belkhir, Leïla; Laterre, Pierre-François; Delmée, Michel; Glupczynski, Youri

    2016-01-01

    Shortening the turn-around time (TAT) of positive blood culture (BC) identification (ID) and susceptibility results is essential to optimize antimicrobial treatment in patients with sepsis. We aimed to evaluate the impact on antimicrobial prescription of a modified workflow of positive BCs providing ID and partial susceptibility results for Enterobacteriaceae (EB), Pseudomonas aeruginosa and Staphylococcus aureus on the day of BC positivity detection. This study was divided into a pre-intervention period (P0) with a standard BC workflow followed by 2 intervention periods (P1, P2) with an identical modified workflow. ID was performed with MALDI-TOF MS from blood, on early or on overnight subcultures. According to ID results, rapid phenotypic assays were realized to detect third generation cephalosporin resistant EB/P. aeruginosa or methicillin resistant S. aureus. Results were transmitted to the antimicrobial stewardship team for patient’s treatment revision. Times to ID, to susceptibility results and to optimal antimicrobial treatment (OAT) were compared across the three study periods. Overall, 134, 112 and 154 positive BC episodes in P0, P1 and P2 respectively were included in the analysis. Mean time to ID (28.3 hours in P0) was reduced by 65.3% in P1 (10.2 hours) and 61.8% in P2 (10.8 hours). Mean time to complete susceptibility results was reduced by 27.5% in P1 and 27% in P2, with results obtained after 32.4 and 32.6 hours compared to 44.7 hours in P0. Rapid tests allowed partial susceptibility results to be obtained after a mean time of 11.8 hours in P1 and 11.7 hours in P2. Mean time to OAT was decreased to 21.6 hours in P1 and to 17.9 hours in P2 compared to 36.1 hours in P0. Reducing TAT of positive BC with MALDI-TOF MS ID and rapid susceptibility testing accelerated prescription of targeted antimicrobial treatment thereby potentially improving the patients’ clinical outcome. PMID:27228001

  5. 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

  6. Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores

    PubMed Central

    Weller, Simon A.; Stokes, Margaret G. M.; Lukaszewski, Roman A.

    2015-01-01

    A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 106-108cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis. PMID:26633884

  7. Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores.

    PubMed

    Weller, Simon A; Stokes, Margaret G M; Lukaszewski, Roman A

    2015-01-01

    A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10(6)-10(8) cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis. PMID:26633884

  8. MALDI-TOF MS characterization of glycation products of whey proteins in a glucose/galactose model system and lactose-free milk.

    PubMed

    Carulli, Saverio; Calvano, Cosima D; Palmisano, Francesco; Pischetsrieder, Monika

    2011-03-01

    The major modifications induced by thermal treatment of whey proteins α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) in a model system mimicking lactose-free milk (L(-) sugar mix) were investigated by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The analysis of the intact α-La revealed species with up to 7 and 14 adducts from lactose and sugar mix, respectively, whereas for β-Lg 3 and up to 5 sugar moieties were observed in the case of lactose and sugar mix experiments, respectively. A partial enzymatic hydrolysis with endoproteinase AspN prior to mass spectrometric analysis allowed the detection of further modifications and their localization in the amino acid sequence. Using α-cyano-4-chlorocinnamic acid as MALDI matrix, it could be shown that heating α-La and β-Lg with glucose or galactose led to the modification of lysine residues that are not glycated by lactose. The higher glycation degree of whey proteins in a lactose-free milk system relative to normal milk with lactose reflects the higher reactivity of monosaccharides compared to the parent disaccharide. Finally, the analysis of the whey extract of a commercial lactose-free milk sample revealed that the two whey proteins were present as three main forms (native, single, and double hexose adducts). PMID:21319853

  9. Chlorinated and brominated phosphatidylcholines are generated under the influence of the Fenton reagent at low pH-a MALDI-TOF MS study.

    PubMed

    Wu, Jianqing; Teuber, Kristin; Eibisch, Mandy; Fuchs, Beate; Schiller, Jürgen

    2011-01-01

    Lipid (phospholipid) oxidation is an increasingly important research topic due to the significant physiological relevance. The Fenton reaction, i.e. the transition metal catalyzed decomposition of H(2)O(2) is frequently used to generate hydroxyl radicals (HO*). Lipids with unsaturated fatty acyl residues are primarily converted by HO* radicals into peroxides. In contrast, chloro- and bromohydrins as well as dihalogenides are formed by the addition of HOCl or HOBr to the olefinic groups of the fatty acyl residues of lipids or under the influence of the enzyme myeloperoxidase (MPO) from Cl(-) and H(2)O(2). We will show here by using MALDI-TOF MS for product analysis that halogenated products may also be generated in the presence of the Fenton reagent, if either FeCl(2) or FeBr(2) is used. In the presence of FeSO(4), however, peroxides are exclusively generated. It will also be shown that the generation of halogen-containing products is a competing reaction with the cleavage of the double bond under generation of the corresponding aldehyde or carboxylic acid that is favored at prolonged incubation times and at elevated pH. PMID:20932962

  10. Thin-layer chromatography combined with MALDI-TOF-MS and 31P-NMR to study possible selective bindings of phospholipids to silica gel.

    PubMed

    Teuber, Kristin; Riemer, Thomas; Schiller, Jürgen

    2010-12-01

    High-performance thin-layer chromatography (HPTLC) is a highly established separation method in the field of lipid and (particularly) phospholipid (PL) research. HPTLC is not only used to identify certain lipids in a mixture but also to isolate lipids (preparative TLC). To do this, the lipids are separated and subsequently re-eluted from the silica gel. Unfortunately, it is not yet known whether all PLs are eluted to the same extent or whether some lipids bind selectively to the silica gel. It is also not known whether differences in the fatty acyl compositions affect the affinities to the stationary phase. We have tried to clarify these questions by using a readily available extract from hen egg yolk as a selected example of a lipid mixture. After separation, the complete lanes or selected spots were eluted from the silica gel and investigated by a combination of MALDI-TOF MS and (31)P NMR spectroscopy. The data obtained were compared with the composition of the total extract (without HPTLC). Although there were significant, solvent-dependent losses in the amount of each lipid, the relative composition of the mixture remained constant; there were also only very slight changes in the fatty acyl compositions of the individual PL classes. Therefore, lipid isolation by TLC may be used without any risk of major sample alterations. PMID:20694807

  11. MNSs genotyping by MALDI-TOF MS shows high concordance with serology, allows gene copy number testing and reveals new St(a) alleles.

    PubMed

    Meyer, Stefan; Vollmert, Caren; Trost, Nadine; Sigurdardottir, Sonja; Portmann, Claudia; Gottschalk, Jochen; Ries, Judith; Markovic, Alexander; Infanti, Laura; Buser, Andreas; Amar El Dusouqui, Soraya; Rigal, Emmanuel; Castelli, Damiano; Weingand, Bettina; Maier, Andreas; Mauvais, Simon M; Sarraj, Amira; Braisch, Monica C; Thierbach, Jutta; Hustinx, Hein; Frey, Beat M; Gassner, Christoph

    2016-08-01

    Results of genotyping with true high-throughput capability for MNSs antigens are underrepresented, probably because of technical issues, due to the high level of nucleotide sequence homology of the paralogous genes GYPA, GYPB and GYPE. Eight MNSs-specific single nucleotide polymorphisms (SNP) were detected using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) in 5800 serologically M/N and S/s pre-typed Swiss blood donors and 50 individuals of known or presumptive black African ethnicity. Comparison of serotype with genotype delivered concordance rates of 99·70% and 99·90% and accuracy of genotyping alone of 99·88% and 99·95%, for M/N and S/s, respectively. The area under the curve of peak signals was measured in intron 1 of the two highly homologous genes GYPB and GYPE and allowed for gene copy number variation estimates in all individuals investigated. Elevated GYPB:GYPE ratios accumulated in several carriers of two newly observed GYP*401 variants, termed type G and H, both encoding for the low incidence antigen St(a). In black Africans, reduced GYPB gene contents were proven in pre-typed S-s-U- phenotypes and could be reproduced in unknown specimens. Quantitative gene copy number estimates represented a highly attractive supplement to conventional genotyping, solely based on MNSs SNPs. PMID:27072601

  12. Selective Enrichment and MALDI-TOF MS Analysis of Small Molecule Compounds with Vicinal Diols by Boric Acid-Functionalized Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Zheng, Xiaoling; Ni, Yanli

    2015-08-01

    In this study, a 4-vinylphenylboronic acid-functionalized graphene oxide (GO) material was prepared via atom-transfer radical polymerization (ATRP) method and applied for the first time as a novel matrix for the selective enrichment and analysis of small-molecule compounds with vicinal diols, which have been the focus of intense research in the field of life science, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in positive-ion mode. There are two main factors playing a decisive role in assisting laser D/I process comparing to some traditional matrices: (1) GO provides π-conjugated system by itself for laser absorption and energy transfer; (2) the modified 4-vinylphenylboronic acid can selectively capture small-molecule compounds with vicinal diols. The results demonstrate that the novel material has distinct advantages over previously reported matrices in enriching and assisting the highly efficient ionization of target molecules for mass spectrometry analysis. This work indicates a new application branch for graphene-based matrices and provides an alternative solution for small-molecules analysis.

  13. Atypical yeasts identified as Saccharomyces cerevisiae by MALDI-TOF MS and gene sequencing are the main responsible of fermentation of chicha, a traditional beverage from Peru.

    PubMed

    Vallejo, Juan Andrés; Miranda, Patricia; Flores-Félix, José David; Sánchez-Juanes, Fernando; Ageitos, José M; González-Buitrago, José Manuel; Velázquez, Encarna; Villa, Tomás G

    2013-12-01

    Chicha is a drink prepared in several Andean countries from Inca's times by maize fermentation. Currently this fermentation is carried out in familiar artesanal "chicherías" that make one of the most known types of chicha, the "chicha de jora". In this study we isolate and identify the yeasts mainly responsible of the fermentation process in this type of chicha in 10 traditional "chicherías" in Cusco region in Peru. We applied by first time MALDI-TOF MS analysis for the identification of yeast of non-clinic origin and the results showed that all of yeast strains isolated belong to the species Saccharomyces cerevisiae. These results agree with those obtained after the analysis of the D1/D2 and 5.8S-ITS regions. However the chicha strains have a phenotypic profile that differed in more than 40% as compared to that of current S. cerevisiae strains. To the best of our knowledge this is the first report concerning the yeasts involved in chicha fermentation. PMID:24120265

  14. MALDI-TOF MS and CD Spectral Analysis for Identification and Structure Prediction of a Purified, Novel, Organic Solvent Stable, Fibrinolytic Metalloprotease from Bacillus cereus B80

    PubMed Central

    Saxena, Rajshree

    2015-01-01

    The ability to predict protein function from structure is becoming increasingly important; hence, elucidation and determination of protein structure become the major steps in proteomics. The present study was undertaken for identification of metalloprotease produced by Bacillus cereus B80 and recognition of characteristics that can be industrially exploited. The enzyme was purified in three steps combining precipitation and chromatographic methods resulting in 33.5% recovery with 13.1-fold purification of enzyme which was detected as a single band with a molecular mass of 26 kDa approximately in SDS-PAGE and zymogram. The MALDI-TOF MS showed that the enzyme exhibited 70–93% similarity with zinc metalloproteases from various strains Bacillus sp. specifically from Bacillus cereus group. The sequence alignment revealed the presence of zinc-binding region VVVHEMCHMV in the most conserved C terminus region. Secondary structure of the enzyme was obtained by CD spectra and I-TASSER. The enzyme kinetics revealed a Michaelis constant (Km) of 0.140 μmol/ml and Vmax of 2.11 μmol/min. The application studies showed that the enzyme was able to hydrolyze various proteins with highest affinity towards casein followed by BSA and gelatin. The enzyme exhibited strong fibrinolytic, collagenolytic, and gelatinolytic properties and stability in various organic solvents. PMID:25802851

  15. Selective Enrichment and MALDI-TOF MS Analysis of Small Molecule Compounds with Vicinal Diols by Boric Acid-Functionalized Graphene Oxide.

    PubMed

    Zhang, Jing; Zheng, Xiaoling; Ni, Yanli

    2015-08-01

    In this study, a 4-vinylphenylboronic acid-functionalized graphene oxide (GO) material was prepared via atom-transfer radical polymerization (ATRP) method and applied for the first time as a novel matrix for the selective enrichment and analysis of small-molecule compounds with vicinal diols, which have been the focus of intense research in the field of life science, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in positive-ion mode. There are two main factors playing a decisive role in assisting laser D/I process comparing to some traditional matrices: (1) GO provides π-conjugated system by itself for laser absorption and energy transfer; (2) the modified 4-vinylphenylboronic acid can selectively capture small-molecule compounds with vicinal diols. The results demonstrate that the novel material has distinct advantages over previously reported matrices in enriching and assisting the highly efficient ionization of target molecules for mass spectrometry analysis. This work indicates a new application branch for graphene-based matrices and provides an alternative solution for small-molecules analysis. PMID:25990923

  16. 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. PMID:25938749

  17. Biomarker Discovery and Redundancy Reduction towards Classification using a Multi-factorial MALDI-TOF MS T2DM Mouse Model Dataset

    PubMed Central

    2011-01-01

    Background Diabetes like many diseases and biological processes is not mono-causal. On the one hand multi-factorial studies with complex experimental design are required for its comprehensive analysis. On the other hand, the data from these studies often include a substantial amount of redundancy such as proteins that are typically represented by a multitude of peptides. Coping simultaneously with both complexities (experimental and technological) makes data analysis a challenge for Bioinformatics. Results We present a comprehensive work-flow tailored for analyzing complex data including data from multi-factorial studies. The developed approach aims at revealing effects caused by a distinct combination of experimental factors, in our case genotype and diet. Applying the developed work-flow to the analysis of an established polygenic mouse model for diet-induced type 2 diabetes, we found peptides with significant fold changes exclusively for the combination of a particular strain and diet. Exploitation of redundancy enables the visualization of peptide correlation and provides a natural way of feature selection for classification and prediction. Classification based on the features selected using our approach performs similar to classifications based on more complex feature selection methods. Conclusions The combination of ANOVA and redundancy exploitation allows for identification of biomarker candidates in multi-dimensional MALDI-TOF MS profiling studies with complex experimental design. With respect to feature selection our method provides a fast and intuitive alternative to global optimization strategies with comparable performance. The method is implemented in R and the scripts are available by contacting the corresponding author. PMID:21554713

  18. Conservation of honey bee (Apis mellifera) sperm phospholipids during storage in the bee queen--a TLC/MALDI-TOF MS study.

    PubMed

    Wegener, Jakob; Zschörnig, Kristin; Onischke, Kristin; Fuchs, Beate; Schiller, Jürgen; Müller, Karin

    2013-02-01

    The honey bee (Apis mellifera) is characterized by a high degree of phenotypic plasticity of senescence-related processes, and has therefore become a model organism of gerontological research. Sperm of honey bee drones can remain fertile for several years within the storage organ of queens. The reason for this longevity is unknown, but the suppression of lipid peroxidation seems to play a decisive role. Here, we examined the questions of whether spermatheca- and in vitro-stored honey bee sperm are indeed resistant to lipid peroxidation, and whether the nature of sperm lipids could explain this resistance. The lipid composition of bee sperm was determined by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) combined with thin-layer chromatography (TLC). The positive ion mass spectra of drone sperm lipids are dominated by two glycerophosphocholine (GPC) species, although small amounts of sphingomyelins (SM) and glycerophosphoethanolamines (GPE) are also detectable after TLC. Alkyl/acyl and alkenyl/acyl compounds of GPC, and alkyl/acyl as well as diacyl compounds of GPE were detected containing oleyl, oleoyl, palmityl and palmitoyl as the most abundant residues. Assignments of all compounds have been additionally verified by enzymatic digestion and exposition to HCl. During incubation of sperm in the presence of air, characteristic lipid oxidation products such as lysophosphatidylcholine (LPC) appear. Inside the spermatheca, however, sperm lipids are obviously protected from oxidation and their composition does not change, even if they are stored over years. Our data support the view that the membrane composition of honey bee sperm could help to explain the extraordinary longevity of these cells. PMID:23279974

  19. High-Throughput Identification and Screening of Novel Methylobacterium Species Using Whole-Cell MALDI-TOF/MS Analysis

    PubMed Central

    Tani, Akio; Sahin, Nurettin; Matsuyama, Yumiko; Enomoto, Takashi; Nishimura, Naoki; Yokota, Akira; Kimbara, Kazuhide

    2012-01-01

    Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing. PMID:22808262

  20. Fractionation and proteomic analysis of the Walterinnesia aegyptia snake venom using OFFGEL and MALDI-TOF-MS techniques.

    PubMed

    Abd El Aziz, Tarek Mohamed; Bourgoin-Voillard, Sandrine; Combemale, Stéphanie; Beroud, Rémy; Fadl, Mahmoud; Seve, Michel; De Waard, Michel

    2015-10-01

    Animal venoms are complex mixtures of more than 100 different compounds, including peptides, proteins, and nonprotein compounds such as lipids, carbohydrates, and metal ions. In addition, the existing compounds show a wide range of molecular weights and concentrations within these venoms, making separation and purification procedures quite tedious. Here, we analyzed for the first time by MS the advantages of using the OFFGEL technique in the separation of the venom components of the Egyptian Elapidae Walterinnesia aegyptia snake compared to two classical methods of separation, SEC and RP-HPLC. We demonstrate that OFFGEL separates venom components over a larger scale of fractions, preserve respectable resolution with regard to the presence of a given compound in adjacent fractions and allows the identification of a greater number of ions by MS (102 over 134 total ions). We also conclude that applying several separating techniques (SEC and RP-HPLC in addition to OFFGEL) provides complementary results in terms of ion detection (21 more for SEC and 22 more with RP-HPLC). As a result, we provide a complete list of 134 ions present in the venom of W. aegyptia by using all these techniques combined. PMID:26178575

  1. 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.

    PubMed

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

    2015-10-01

    Cadmium is a toxic heavy metal present in the environment and in industrial materials. Cadmium has demonstrated carcinogenic activity that induces cell transformation, but how this occurs is unclear. We used 2D-DIGE and MALDI TOF/TOF MS combined with bioinformatics and immunoblotting to investigate the molecular mechanism of cadmium transformation. We found that small GTPases were critical for transformation. Additionally, proteins involved in mitochondrial transcription, DNA repair, and translation also had altered expression patterns in cadmium treated cells. Collectively, our results suggest that activation of small GTPases contributes to cadmium-induced transformation of colon cells. PMID:26220685

  2. Phosphatidylcholines and -ethanolamines can be easily mistaken in phospholipid mixtures: a negative ion MALDI-TOF MS study with 9-aminoacridine as matrix and egg yolk as selected example.

    PubMed

    Fuchs, Beate; Bischoff, Annabell; Süss, Rosmarie; Teuber, Kristin; Schürenberg, Martin; Suckau, Detlev; Schiller, Jürgen

    2009-12-01

    Phospholipids (PL) are increasingly analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). As in the case of polar molecules, however, the careful selection of the matrix is crucial for optimum results. 9-Aminoacridine (9-AA) was recently suggested as the matrix of choice to analyze PL mixtures because of (a) the improved sensitivity and (b) the reduction of suppression effects compared to other matrices. However, the distinction of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the negative ion mode is obscured as PC is also detectable as -CH3+ ion if 9-AA is used as matrix. This may result in the erroneous assignment of PC as a PE species. Using an organic extract from hen egg yolk as example it will be shown that the contribution of PC must be taken into consideration if the negative ion mass spectra are used to evaluate the fatty acyl compositions of PE mixtures. 9-AA can as well be used in hyphenated thin-layer chromatography (TLC)-MALDI-TOF MS where PC and PE are chromatographically well separated for unequivocal assignments. PMID:19690837

  3. Optimization of experimental and modelling parameters for the differentiation of beverage spoiling yeasts by Matrix-Assisted-Laser-Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in response to varying growth conditions.

    PubMed

    Usbeck, Julia C; Kern, Carola C; Vogel, Rudi F; Behr, Jürgen

    2013-12-01

    The growth of spoiling yeasts in beverages results in reduced quality, economic and image losses. Therefore, biochemical and DNA-based identification methods have been developed but are mostly time-consuming and laborious. Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) could deliver discriminative peptide mass fingerprints within minutes and could thus be a rapid and reliable tool for identification and differentiation. However, routine analysis of yeasts by MALDI-TOF MS is yet impaired by low reproducibility and effects of different physiological states of organisms on the reliability of the identification method are still controversial. The aim of this study was to optimize sample preparation and measurement parameterization using three spoilage yeasts (Saccharomyces cerevisiae var. diastaticus, Wickerhamomyces anomalus and Debaryomyces hansenii). The influence of environmental or physiological parameters including oxygen availability, different nutrients, cell density and growth phase were analysed and revealed small differences in mass fingerprints. Yeasts grown in the presence or absence of oxygen were precisely differentiated along these differences in mass fingerprints and a crude classification of growth phase was possible. Cell concentration did not affect the spectra distinctly, neither qualitatively nor quantitatively, and an influence of available nutrients could not be measured in each case. However, core mass peaks remained constant under all tested conditions enabling reliable identification. PMID:24010620

  4. Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study.

    PubMed

    Schiller, J; Arnhold, J; Benard, S; Reichl, S; Arnold, K

    1999-05-31

    Matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) has been used to investigate degradation products of two selected polysaccharides of cartilage (chondroitin sulfate and hyaluronic acid). Testicular hyaluronate lyase and chondroitin ABC lyase were used for enzymic digestion of both polysaccharides as well as of cartilage specimens. Polysaccharide solutions and cartilage supernatants were assayed by positive and negative MALDI-TOF MS. Especially chondroitin ABC lyase produced high amounts of digestion products (unsaturated di- and tetrasaccharides) from polysaccharides as well as from cartilage, clearly monitored by MALDI-TOF MS. It is concluded that MALDI-TOF MS provides a precise and fast tool for the determination of oligosaccharides since no previous derivatization is required. PMID:10576924

  5. Direct MALDI-TOF mass spectrometric peptide profiling of neuroendocrine tissue of Drosophila.

    PubMed

    Wegener, Christian; Neupert, Susanne; Predel, Reinhard

    2010-01-01

    Direct MALDI-TOF mass spectrometric peptide profiling is increasingly used to analyze the peptide complement in the nervous system of a variety of invertebrate animals, from leech to Aplysia and many arthropod species, especially insects and crustaceans. Proper sample preparation is often the most crucial step to obtain the necessary data. Here, we describe protocols for the use of MALDI-TOF mass spectrometry to directly analyze the peptidome of neuroendocrine tissues of insects, particularly Drosophila melanogaster, by MALDI-TOF MS. PMID:20013204

  6. Characterization of Enterobacteria using MALDI-TOF mass spectrometry.

    PubMed

    Pribil, Patrick; Fenselau, Catherine

    2005-09-15

    A method is proposed for the rapid classification of Gram-negative Enterobacteria using on-slide solubilization and trypsin digestion of proteins, followed by MALDI-TOF MS analysis. Peptides were identified from tryptic digests using microsequencing by tandem mass spectrometry and database searches. Proteins from the outer membrane family (OMP) were consistently identified in the Enterobacteria Escherichia coli, Enterobacter cloacae, Erwinia herbicola, and Salmonella typhimurium. Database searches indicate that these OMP peptides observed are unique to the Enterobacteria order. PMID:16159146

  7. Source-Identifying Biomarker Ions between Environmental and Clinical Burkholderia pseudomallei Using Whole-Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

    PubMed Central

    Srisanga, Kitima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

    2014-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, which is an endemic disease in Northeast Thailand and Northern Australia. Environmental reservoirs, including wet soils and muddy water, serve as the major sources for contributing bacterial infection to both humans and animals. The whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has recently been applied as a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In this present study, we employed a whole-cell MALDI-TOF MS approach for assessing its potency in clustering a total of 11 different B. pseudomallei isolates (consisting of 5 environmental and 6 clinical isolates) with respect to their origins and to further investigate the source-identifying biomarker ions belonging to each bacterial group. The cluster analysis demonstrated that six out of eleven isolates were grouped correctly to their sources. Our results revealed a total of ten source-identifying biomarker ions, which exhibited statistically significant differences in peak intensity between average environmental and clinical mass spectra using ClinProTools software. Six out of ten mass ions were assigned as environmental-identifying biomarker ions (EIBIs), including, m/z 4,056, 4,214, 5,814, 7,545, 7,895, and 8,112, whereas the remaining four mass ions were defined as clinical-identifying biomarker ions (CIBIs) consisting of m/z 3,658, 6,322, 7,035, and 7,984. Hence, our findings represented, for the first time, the source-specific biomarkers of environmental and clinical B. pseudomallei. PMID:24914956

  8. Identification of a tachykinin-related neuropeptide from the honeybee brain using direct MALDI-TOF MS and its gene expression in worker, queen and drone heads.

    PubMed

    Takeuchi, H; Yasuda, A; Yasuda-Kamatani, Y; Kubo, T; Nakajima, T

    2003-06-01

    Using a combination of MALDI-TOF and on-line capillary HPLC/Q-Tof mass spectroscopy, we identified and determined the amino acid sequence of a novel neuropeptide in the brain of the honeybee Apis mellifera L., termed AmTRP peptide (Apis mellifera tachykinin-related peptide), related to insect tachykinin. A cDNA for a prepro-protein (prepro-AmTRP) of AmTRP was isolated and determined to encode seven AmTRPs 1-7. Northern blot analysis indicated that the prepro-AmTRP gene is expressed differentially in the nurse bee, forager, queen and drone heads. Strong expression was detected in the queen and forager heads, while weak and almost no significant expression was detected in the nurse and drone heads, respectively. These results suggest that AmTRP peptide functions as a neuromodulator and/or hormone, associated with sex-specific or age/division of labour-selective behaviour and/or physiology of the honeybees. PMID:12752663

  9. 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. PMID:25702991

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. MALDI-TOF mass spectrometry - a rapid method for the identification of dermatophyte species.

    PubMed

    Nenoff, Pietro; Erhard, Marcel; Simon, Jan C; Muylowa, Grace K; Herrmann, Jürgen; Rataj, Waldemar; Gräser, Yvonne

    2013-01-01

    Altogether 285 dermatophyte isolates of 21 different species - including both Trichophyton rubrum and T. interdigitale, but also eight additional Trichophyton species, Microsporum canis and seven other Microsporum species, as well as Epidermophyton floccosum and Arthroderma spp. - were analyzed using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and the AnagnosTec 'SARAMIS' (Spectral Archiving and Microbial Identification System) software. In addition, sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA was performed for a high number of the tested strains. Sufficient agreement was found between the results obtained with standard identification methods and those with the MALDI-TOF MS for species identification of dermatophytes. A mass spectra database was constructed which contained the species identifications of all 285 isolates. The results were confirmed for 164 of the isolates by sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA. Statistical analysis of all 285 dermatophyte strains showed that conventional identification matched the results of MALDI-TOF MS for 78.2% of the isolates tested. In the case of the 164 isolates for which the identifications were confirmed by PCR, the results of their conventional diagnosis and MALDI-TOF MS were in agreement for only 68.9 % (113 of 164 strains) of the test isolates. In contrast, there was agreement of 99.3 % or 98.8 % in the identifications obtained with PCR and MALDI-TOF MS techniques (283/285 or 162/164). The two exceptions were isolates that proved to be T. violaceum which could not be identified by the MALDI-TOF MS technique. In conclusion, the MALDI-TOF mass spectroscopy represents a fast and very specific method for species differentiation of dermatophytes grown in culture. PMID:22574631

  12. MALDI-TOF Mass Spectrometry Discriminates Known Species and Marine Environmental Isolates of Pseudoalteromonas

    PubMed Central

    Emami, Kaveh; Nelson, Andrew; Hack, Ethan; Zhang, Jinwei; Green, David H.; Caldwell, Gary S.; Mesbahi, Ehsan

    2016-01-01

    The genus Pseudoalteromonas constitutes an ecologically significant group of marine Gammaproteobacteria with potential biotechnological value as producers of bioactive compounds and of enzymes. Understanding their roles in the environment and bioprospecting for novel products depend on efficient ways of identifying environmental isolates. Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) biotyping has promise as a rapid and reliable method of identifying and distinguishing between different types of bacteria, but has had relatively limited application to marine bacteria and has not been applied systematically to Pseudoalteromonas. Therefore, we constructed a MALDI-TOF MS database of 31 known Pseudoalteromonas species, to which new isolates can be compared by MALDI-TOF biotyping. The ability of MALDI-TOF MS to distinguish between species was scrutinized by comparison with 16S rRNA gene sequencing. The patterns of similarity given by the two approaches were broadly but not completely consistent. In general, the resolution of MALDI-TOF MS was greater than that of 16S rRNA gene sequencing. The database was tested with 13 environmental Pseudoalteromonas isolates from UK waters. All of the test strains could be identified to genus level by MALDI-TOF MS biotyping, but most could not be definitely identified to species level. We conclude that several of these isolates, and possibly most, represent new species. Thus, further taxonomic investigation of Pseudoalteromonas is needed before MALDI-TOF MS biotyping can be used reliably for species identification. It is, however, a powerful tool for characterizing and distinguishing among environmental isolates and can make an important contribution to taxonomic studies. PMID:26903983

  13. MALDI-TOF Mass Spectrometry Discriminates Known Species and Marine Environmental Isolates of Pseudoalteromonas.

    PubMed

    Emami, Kaveh; Nelson, Andrew; Hack, Ethan; Zhang, Jinwei; Green, David H; Caldwell, Gary S; Mesbahi, Ehsan

    2016-01-01

    The genus Pseudoalteromonas constitutes an ecologically significant group of marine Gammaproteobacteria with potential biotechnological value as producers of bioactive compounds and of enzymes. Understanding their roles in the environment and bioprospecting for novel products depend on efficient ways of identifying environmental isolates. Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) biotyping has promise as a rapid and reliable method of identifying and distinguishing between different types of bacteria, but has had relatively limited application to marine bacteria and has not been applied systematically to Pseudoalteromonas. Therefore, we constructed a MALDI-TOF MS database of 31 known Pseudoalteromonas species, to which new isolates can be compared by MALDI-TOF biotyping. The ability of MALDI-TOF MS to distinguish between species was scrutinized by comparison with 16S rRNA gene sequencing. The patterns of similarity given by the two approaches were broadly but not completely consistent. In general, the resolution of MALDI-TOF MS was greater than that of 16S rRNA gene sequencing. The database was tested with 13 environmental Pseudoalteromonas isolates from UK waters. All of the test strains could be identified to genus level by MALDI-TOF MS biotyping, but most could not be definitely identified to species level. We conclude that several of these isolates, and possibly most, represent new species. Thus, further taxonomic investigation of Pseudoalteromonas is needed before MALDI-TOF MS biotyping can be used reliably for species identification. It is, however, a powerful tool for characterizing and distinguishing among environmental isolates and can make an important contribution to taxonomic studies. PMID:26903983

  14. 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

  15. Diversity of Clonostachys species assessed by molecular phylogenetics and MALDI-TOF mass spectrometry.

    PubMed

    Abreu, Lucas M; Moreira, Gláucia M; Ferreira, Douglas; Rodrigues-Filho, Edson; Pfenning, Ludwig H

    2014-12-01

    We assessed the species diversity among 45 strains of Clonostachys from different substrates and localities in Brazil using molecular phylogenetics, and compared the results with the phenotypic classification of strains obtained from matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analyses were based on beta tubulin (Tub), ITS-LSU rDNA, and a combined Tub-ITS DNA dataset. MALDI-TOF MS analyses were performed using intact conidia and conidiophores of strains cultivated on oatmeal agar and 4% malt extract agar. Six known species were identified: Clonostachys byssicola, Clonostachys candelabrum, Clonostachys pseudochroleuca, Clonostachys rhizophaga, Clonostachys rogersoniana, and Clonostachys rosea. Two clades and two singleton lineages did not correspond to known species represented in the reference DNA dataset and were identified as Clonostachys sp. 1-4. Multivariate cluster analyses of MALDI-TOF MS data classified the strains into eight clusters and three singletons, corresponding to the ten identified species plus one additional cluster containing two strains of C. rogersoniana that split from the other co-specific strains. The consistent results of MALDI-TOF MS supported the identification of strains assigned to C. byssicola and C. pseudochroleuca, which did not form well supported clades in all phylogenetic analyses, but formed distinct clusters in the MALDI-TOF dendrograms. PMID:25457948

  16. 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.

  17. 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. PMID:23182036

  18. Methylobacterium Species Promoting Rice and Barley Growth and Interaction Specificity Revealed with Whole-Cell Matrix-Assisted Laser Desorption / Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) Analysis

    PubMed Central

    Tani, Akio; Sahin, Nurettin; Fujitani, Yoshiko; Kato, Akiko; Sato, Kazuhiro; Kimbara, Kazuhide

    2015-01-01

    Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant–microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization- time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of appropriate species that can persist on the plant is important to achieve growth promotion. PMID:26053875

  19. Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in preparation of chitosan oligosaccharides (COS) with degree of polymerization (DP) 5-12 containing well-distributed acetyl groups

    NASA Astrophysics Data System (ADS)

    Chen, Mian; Zhu, Xiqiang; Li, Zhiming; Guo, Xueping; Ling, Peixue

    2010-02-01

    COS have many biological activities, and have been widely used as a health food. Molecular size is considered as a key parameter for COS' activities. However, many criteria are used practically, and true qualities of COS from different producers may not be always comparable. This can partly explain the disagreement in COS' functional researches, as resulting in COS, even with astonish effects, have not been further developed as a drug for tumor patients. As anti-tumor activities have been studied based on DP in pharmacological researches, we employed MALDI-TOF-MS to monitor fine structure, including DP, in COS' preparation and comparison. Then one of the COS products was analyzed with the composition of DP 5-12, mainly 7-10. Moreover, that COS' product contains well-distributed acetyl groups, while typical Commercial COS sample nearly contains no acetyl groups. As fresh precise parameters, the DP and the number of acetyl groups matching with special DP can be introduced in COS' further study on structure-activity relationships (SARs) as a new drug.

  20. 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. PMID:26300239

  1. Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry.

    PubMed

    Schaumann, Reiner; Knoop, Nicolas; Genzel, Gelimer H; Losensky, Kevin; Rosenkranz, Christiane; Stîngu, Catalina S; Schellenberger, Wolfgang; Rodloff, Arne C; Eschrich, Klaus

    2013-01-01

    Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli. PMID:23919091

  2. Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry

    PubMed Central

    Schaumann, Reiner; Knoop, Nicolas; Genzel, Gelimer H; Losensky, Kevin; Rosenkranz, Christiane; Stîngu, Catalina S; Schellenberger, Wolfgang; Rodloff, Arne C; Eschrich, Klaus

    2013-01-01

    Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli. PMID:23919091

  3. 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. PMID:26971637

  4. Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

    NASA Astrophysics Data System (ADS)

    Fagerquist, Clifton K.; Zaragoza, William J.

    2015-05-01

    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-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.

  5. Beyond identification: emerging and future uses for MALDI-TOF mass spectrometry in the clinical microbiology laboratory.

    PubMed

    DeMarco, Mari L; Ford, Bradley A

    2013-09-01

    The routine use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized microorganism identification in the clinical microbiology laboratory. Building from these now common microorganism identification strategies, this review explores future clinical applications of MALDI-TOF MS. This includes practical approaches for laboratorians interested in implementing direct identification processing methods for MALDI-TOF detection of microbes in bloodstream infection (BSI) and urinary tract infection (UTI), as well as, post-analytical approaches for classifying MALDI-TOF spectral data to detect characteristics other and species-level identification (e.g. strain-level classification, typing, and resistance mechanisms). PMID:23931841

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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

  8. A MALDI-TOF MS analysis study of the binding of 4-(N,N-dimethylamino)pyridine to amine-bis(phenolate) chromium(III) chloride complexes: mechanistic insight into differences in catalytic activity for CO2/epoxide copolymerization.

    PubMed

    Kozak, Christopher M; Woods, April M; Bottaro, Christina S; Devaine-Pressing, Katalin; Ni, Kaijie

    2015-01-01

    Amine-bis(phenolato)chromium(III) chloride complexes, [LCrCl], are capable of catalyzing the copolymerization of cyclohexene oxide with carbon dioxide to give poly(cyclohexane) carbonate. When combined with 4-(N,N-dimethylamino)pyridine (DMAP) these catalyst systems yield low molecular weight polymers with moderately narrow polydispersities. The coordination chemistry of DMAP with five amine-bis(phenolato)chromium(III) chloride complexes was studied by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The amine-bis(phenolato) ligands were varied in the nature of their neutral pendant donor-group and include oxygen-containing tetrahydrofurfuryl and methoxyethyl moieties, or nitrogen-containing N,N-dimethylaminoethyl or 2-pyridyl moieties. The relative abundance of mono and bis(DMAP) adducts, as well as DMAP-free ions is compared under various DMAP : Cr complex ratios. The [LCr](+) cations show the ability to bind two DMAP molecules to form six-coordinate complex ions in all cases, except when the pendant group is N,N-dimethylaminoethyl (compound ). Even in the presence of a 4 : 1 ratio of DMAP to Cr, no ions corresponding to [L3Cr(DMAP)2](+) were observed for the complex containing the tertiary sp(3)-hybridized amino donor in the pendant arm. The difference in DMAP-binding ability of these compounds results in differences in catalytic activity for alternating copolymerization of CO2 and cyclohexene oxide. Kinetic investigations by infrared spectroscopy of compounds 2 and 3 show that polycarbonate formation by 3 is twice as fast as that of compound 2 and that no initiation time is observed. PMID:26388443

  9. MALDI-TOF Mass Spectrometry of Naturally-Occurring Mixtures of Mono- and Di-rhamnolipids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed for high-throughput screening of naturally-occurring mixtures of rhamnolipids from Pseudomonas spp. Mono- and di-rhamnolipids are readily distinguished by characteristic molecular adduct i...

  10. Determination of hidden hazelnut oil proteins in extra virgin olive oil by cold acetone precipitation followed by in-solution tryptic digestion and MALDI-TOF-MS analysis.

    PubMed

    De Ceglie, Cristina; Calvano, Cosima Damiana; Zambonin, Carlo Giorgio

    2014-10-01

    Adulteration of extra-virgin olive oil (EVOO) with hazelnut oil (HO) is an illegal practice that could have severe health consequences for consumers due to the possible exposure to hidden hazelnut allergens. Here, matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry (MS) was used as a rapid and sensitive technique for the detection of a low concentration of hazelnut proteins in oil samples. Different protocols were tested for protein extraction, and the most efficient (cold acetone) was applied to HO and EVOO adulterated with HO. The subsequent in-solution tryptic digestion of protein extracts and MALDI-MS analysis, using α-cyano-4-chlorocinnamic acid as matrix, allowed the detection of stable hazelnut peptide markers (i.e., the m/z ions 1002.52, 1356.71, 1394.70, 1440.81, 1453.85, 1555.76, 1629.83, 1363.73, and 1528.67) attributable to the main hazelnut proteins Cor a 9, Cor a 11, and Cor a 1. Thus, the approach might allow the direct detection of specific hazelnut allergens in EVOO at low concentration without time-consuming pretreatments. PMID:25209075

  11. Identification of yeast isolated from dermatological patients by MALDI-TOF mass spectrometry.

    PubMed

    Seyfarth, Florian; Wiegand, Cornelia; Erhard, Marcel; Gräser, Yvonne; Elsner, Peter; Hipler, Uta-Christina

    2012-05-01

    Species identification of yeasts is based on biochemical (e.g. API ID 32 C®, bioMérieux) and molecular biological approaches. As an alternative to DNA-dependent methods, mass spectral analysis based identification of micro-organisms has become increasingly recognized. In a number of studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the rapid classification and identification of micro-organisms. In this study, the applicability of MALDI-TOF MS for identifying yeasts isolated from dermatological patients was analysed and compared with the results from the API ID 32 C® system. Furthermore, sequencing the internal transcribed spacer (ITS) regions of the ribosomal DNA was employed as reference method. Candida (C.) albicans was isolated in 41.9% of all cases, C. parapsilosis in 20.3%, C. glabrata in 10.8%, and C. krusei in 6, 8.1%. Rarely isolated yeasts were Candida colliculosa, famata, guilliermondii, lusitaniae, and tropicalis as well as Geotrichum candidum, Rhodotorula mucilaginosa and Trichosporon mucoides. The MALDI TOF results were equal to the results gained by ITS sequence analysis in 94%, whereas API ID 32 C® provided the correct diagnosis in 84.3% (of all cases). This lower identification rate is mostly referable to frequent misidentifications of C. krusei as C. inconspicua/norvegensis,Candida tropicalis, or Geotrichum capitatum. In contrast, all C. krusei strains were correctly identified by MALDI TOF MS. In conclusion, species identification by MALDI-TOF MS was proven to be consistent with ITS sequence analysis; the technique has a resolving power comparatively as high as ITS sequence analysis. PMID:21848605

  12. MALDI-TOF MS imaging of metabolites with a N-(1-naphthyl) ethylenediamine dihydrochloride matrix and its application to colorectal cancer liver metastasis.

    PubMed

    Wang, Jianing; Qiu, Shulan; Chen, Suming; Xiong, Caiqiao; Liu, Huihui; Wang, Jiyun; Zhang, Ning; Hou, Jian; He, Qing; Nie, Zongxiu

    2015-01-01

    Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a label-free technique for identifying multiplex metabolites and determining both their distribution and relative abundance in situ. Our previous study showed that N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) could act as a matrix for laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) detection of oligosaccharides in solution. In the present study, NEDC-assisted LDI-TOF MSI yielded many more endogenous compound peaks between m/z 60 and m/z 1600 than 9-aminoacridine (9-AA). Our results show that NEDC-assisted LDI-TOF MSI is especially well-suited for examining distributions of glycerophospholipids (GPs) in addition to low molecular weight metabolites below m/z 400. Particularly, NEDC matrix allowed the LDI-TOF MSI of glucose in animal tissue. Furthermore, NEDC-assisted LDI-TOF MSI was applied to a mouse model of colorectal cancer liver metastasis. We revealed the distinct spatio-molecular signatures of many detected compounds in tumor or tumor-bearing liver, and we found that taurine, glucose, and some GPs decreased in tumor-bearing liver as the tumor developed in liver. Importantly, we also found a glucose gradient in metastatic tumor foci for the first time, which further confirms the energy competition between tumors and liver remnant due to the Warburg effect. Our results suggest that NEDC-assisted LDI MSI provides an in situ label-free analysis of multiple glycerophospholipids and low molecular weight metabolites (including glucose) with abundant peaks and high spatial resolution. This will allow future application to in situ definition of biomarkers, signaling pathways, and disease mechanisms. PMID:25474421

  13. 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

  14. 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

  15. Bacterial species identification from MALDI-TOF mass spectra through data analysis and machine learning.

    PubMed

    De Bruyne, Katrien; Slabbinck, Bram; Waegeman, Willem; Vauterin, Paul; De Baets, Bernard; Vandamme, Peter

    2011-02-01

    At present, there is much variability between MALDI-TOF MS methodology for the characterization of bacteria through differences in e.g., sample preparation methods, matrix solutions, organic solvents, acquisition methods and data analysis methods. After evaluation of the existing methods, a standard protocol was developed to generate MALDI-TOF mass spectra obtained from a collection of reference strains belonging to the genera Leuconostoc, Fructobacillus and Lactococcus. Bacterial cells were harvested after 24h of growth at 28°C on the media MRS or TSA. Mass spectra were generated, using the CHCA matrix combined with a 50:48:2 acetonitrile:water:trifluoroacetic acid matrix solution, and analyzed by the cell smear method and the cell extract method. After a data preprocessing step, the resulting high quality data set was used for PCA, distance calculation and multi-dimensional scaling. Using these analyses, species-specific information in the MALDI-TOF mass spectra could be demonstrated. As a next step, the spectra, as well as the binary character set derived from these spectra, were successfully used for species identification within the genera Leuconostoc, Fructobacillus, and Lactococcus. Using MALDI-TOF MS identification libraries for Leuconostoc and Fructobacillus strains, 84% of the MALDI-TOF mass spectra were correctly identified at the species level. Similarly, the same analysis strategy within the genus Lactococcus resulted in 94% correct identifications, taking species and subspecies levels into consideration. Finally, two machine learning techniques were evaluated as alternative species identification tools. The two techniques, support vector machines and random forests, resulted in accuracies between 94% and 98% for the identification of Leuconostoc and Fructobacillus species, respectively. PMID:21295428

  16. Actinobaculum schaalii: identification with MALDI-TOF

    PubMed Central

    Tuuminen, T; Suomala, P; Harju, I

    2014-01-01

    Actinobaculum schaalii is an emerging uropathogen. So far, its identification has been performed with 16S rRNA gene sequencing or PCR. The diagnosis has often been delayed due to fastidious growth and identification problems. Eleven clinical isolates of A. schaalii from bloodstream infections that were initially identified with 16S rRNA sequencing analysis were recovered and later identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). We present a review of bacteriological data of these patients, an algorithm for fast laboratory work-up and advocate the use of sensitized culture of urine to allow better recovery of A. schaalii in susceptible patients. PMID:25356339

  17. 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

    2015-06-01

    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

  18. 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. PMID:24878140

  19. Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry.

    PubMed

    Lee, Kyunghee; Bae, Dongwon; Lim, Dongbin

    2002-04-30

    Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel. PMID:12018838

  20. 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

    2014-09-01

    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. PMID:25212071

  1. 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. PMID:25912807

  2. 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

  3. 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

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

    PubMed Central

    Emami, Kaveh; Askari, Vahid; Ullrich, Matthias; Mohinudeen, Khwajah; Anil, Arga Chandrashekar; Khandeparker, Lidita; Burgess, J. Grant; Mesbahi, Ehsan

    2012-01-01

    To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time. PMID:22685576

  5. 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). PMID:25487809

  6. 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. PMID:26300860

  7. Antagonistic effects of Bacillus subtilis subsp. subtilis and B. amyloliquefaciens against Macrophomina phaseolina: SEM study of fungal changes and UV-MALDI-TOF MS analysis of their bioactive compounds.

    PubMed

    Torres, M J; Brandan, C Pérez; Petroselli, G; Erra-Balsells, R; Audisio, M C

    2016-01-01

    The antifungal effect of Bacillus subtilis subsp. subtilis PGPMori7 and Bacillus amyloliquefaciens PGPBacCA1 was evaluated against Macrophomina phaseolina (Tassi) Goid. Cell suspension (CS), cell-free supernatant (CFS) and the lipopeptide fraction (LF) of PGPMori7 and PGPBacCA1 were screened against three different M. phaseolina strains. CS exhibited the highest inhibitory effect (around 50%) when compared to those of CFS and LF, regardless of the fungal strain studied. The synthesis of lipopeptides was studied by UV-MALDI TOF. Chemical analysis of Bacillus metabolite synthesis revealed that surfactin and iturin were mainly produced in liquid medium. Potential fengycin was also co-produced when both Bacillus were cultivated in solid medium. In co-culture assays, the bacterial colony-fungal mycelium interface at the inhibition zone was evaluated by both scanning electron microscopy (SEM) and UV-MALDI TOF, the former to determine the structural changes on M. phaseolina cells and the latter to identify the main bioactive molecules involved in the inhibitory effect. PGPBacCA1 produced surfactin, iturin and fengycin in the inhibition zone while PGPMori7 only produced these metabolites within its colony and not in the narrow inhibition zone. Interestingly, SEM revealed that PGPBacCA1 induced damage in M. phaseolina sclerotia, generating a fungicidal effect as no growth was observed when normal growth conditions were reestablished. In turn, PGPMori7 inhibited the growth of the Macrophomina mycelium without fungal injury, resulting only in a fungistatic activity. From these results, it was determined that the two bacilli significantly inhibited the growth of an important phytopathogenic fungus by at least two different mechanisms: lipopeptide synthesis and competition among microorganisms. PMID:26686611

  8. Size Characterization of Colloidal Platinum Nanoparticles by MALDI-TOF Mass Spectrometry

    SciTech Connect

    Navin, Jason K.; Grass, Michael E.; Somorjai, Gabor A.; Marsh, Anderson L.

    2009-08-15

    In this work, matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) has been utilized to characterize colloidal platinum nanoparticles synthesized in the 1-4 nm size range. The nanoparticles were prepared via a solution-based method in which the size could be controlled by varying reaction conditions, such as the alcohol used as the reductant. Poly(vinylpyrrolidone), or PVP, (MW = 29,000 g/mol) was employed as a capping agent to stabilize the synthesized nanoparticles in solution. A model for determining the size of the metallic nanoparticle core from MALDI-TOF mass spectra has been developed and verified through correlation with particle sizes from transmission electron microscopy (TEM) and X-ray diffraction (XRD) measurements. In this model it was assumed that 1.85 nm nanoparticles are capped by one PVP chain, which was verified through experiments performed with capped and uncapped nanoparticles. Larger nanoparticles are capped by either two (2.60 and 2.94 nm) or three (3.69 nm) PVP chains. These findings clearly indicate the usefulness of MALDI-TOF MS as a technique for fully characterizing nanoscale materials in order to elucidate structure-property relationships.

  9. Characterization of honeybee venom by MALDI-TOF and nanoESI-QqTOF mass spectrometry.

    PubMed

    Matysiak, Jan; Schmelzer, Christian E H; Neubert, Reinhard H H; Kokot, Zenon J

    2011-01-25

    The aim of the study was to comprehensively characterize different honeybee venom samples applying two complementary mass spectrometry methods. 41 honeybee venom samples of different bee strains, country of origin (Poland, Georgia, and Estonia), year and season of the venom collection were analyzed using MALDI-TOF and nanoESI-QqTOF-MS. It was possible to obtain semi-quantitative data for 12 different components in selected honeybee venom samples using MALDI-TOF method without further sophisticated and time consuming sample pretreatment. Statistical analysis (ANOVA) has shown that there are qualitative and quantitative differences in the composition between honeybee venom samples collected over different years. It has also been demonstrated that MALDI-TOF spectra can be used as a "protein fingerprint" of honeybee venom in order to confirm the identity of the product. NanoESI-QqTOF-MS was applied especially for identification purposes. Using this technique 16 peptide sequences were identified, including melittin (12 different breakdown products and precursors), apamine, mast cell degranulating peptide and secapin. Moreover, the significant achievement of this study is the fact that the new peptide (HTGAVLAGV+Amidated (C-term), M(r)=822.53Da) has been discovered in bee venom for the first time. PMID:20850943

  10. In Situ Identification of Plant-Invasive Bacteria with MALDI-TOF Mass Spectrometry

    PubMed Central

    Pflüger, Valentin; Saad, Maged; Vogel, Guido; Tonolla, Mauro; Perret, Xavier

    2012-01-01

    Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues. PMID:22615938

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. 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

  13. Identification of Brucella by MALDI-TOF Mass Spectrometry. Fast and Reliable Identification from Agar Plates and Blood Cultures

    PubMed Central

    Ferreira, Laura; Vega Castaño, Silvia; Sánchez-Juanes, Fernando; González-Cabrero, Sandra; Menegotto, Fabiola; Orduña-Domingo, Antonio

    2010-01-01

    Background MALDI-TOF mass spectrometry (MS) is a reliable method for bacteria identification. Some databases used for this purpose lack reference profiles for Brucella species, which is still an important pathogen in wide areas around the world. We report the creation of profiles for MALDI-TOF Biotyper 2.0 database (Bruker Daltonics, Germany) and their usefulness for identifying brucellae from culture plates and blood cultures. Methodology/Principal Findings We created MALDI Biotyper 2.0 profiles for type strains belonging to B. melitensis biotypes 1, 2 and 3; B. abortus biotypes 1, 2, 5 and 9; B. suis, B. canis, B ceti and B. pinnipedialis. Then, 131 clinical isolates grown on plate cultures were used in triplicate to check identification. Identification at genus level was always correct, although in most cases the three replicates reported different identification at species level. Simulated blood cultures were performed with type strains belonging to the main human pathogenic species (B. melitensis, B. abortus, B. suis and B. canis), and studied by MALDI-TOF MS in triplicate. Identification at genus level was always correct. Conclusions/Significance MALDI-TOF MS is reliable for Brucella identification to the genus level from culture plates and directly from blood culture bottles. PMID:21151913

  14. Proteomic Profiling and Protein Identification by MALDI-TOF Mass Spectrometry in Unsequenced Parasitic Nematodes

    PubMed Central

    Millares, Paul; LaCourse, E. James; Perally, Samirah; Ward, Deborah A.; Prescott, Mark C.; Hodgkinson, Jane E.; Brophy, Peter M.; Rees, Huw H.

    2012-01-01

    Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This

  15. MALDI-TOF mass spectrometry fingerprinting: A diagnostic tool to differentiate dematiaceous fungi Stachybotrys chartarum and Stachybotrys chlorohalonata.

    PubMed

    Gruenwald, Maike; Rabenstein, Andreas; Remesch, Markko; Kuever, Jan

    2015-08-01

    Stachybotrys chartarum and Stachybotrys chlorohalonata are two closely related species. Unambiguous identification of these two species is a challenging task if relying solely on morphological criteria and therefore smarter and less labor-intensive approaches are needed. Here we show that even such closely related species of fungi as S. chartarum and S. chlorohalonata are unequivocally discriminated by their highly reproducible MALDI-TOF-MS fingerprints (matrix assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints). We examined 19 Stachybotrys and one Aspergillus isolate by MALDI-TOF-MS. All but one isolate produced melanin containing conidia on malt extract agar. Mass spectra were obtained in good quality from the analysis of hyaline and darkly pigmented conidia by circumventing the property of melanin which causes signal suppression. MALDI-TOF fingerprint analysis clearly discriminated not only the two morphologically similar species S. chartarum and S. chlorohalonata from each other but separated them precisely from Stachybotrys bisbyi and Aspergillus versicolor isolates. Furthermore, even S. chartarum chemotypes A and S could be differentiated into two distinct groups by their MALDI-TOF fingerprints. The chemotypes of S. chartarum isolates were identified by trichodiene synthase 5 (tri5) sequences prior to mass spectra analysis. Additionally, species identities of all isolates were verified by their 18S rRNA and tri5 gene sequences. PMID:26036596

  16. Identification of clinical Pasteurella isolates by MALDI-TOF -- a comparison with VITEK 2 and conventional microbiological methods.

    PubMed

    Zangenah, Salah; Güleryüz, Gülay; Boräng, Stina; Ullberg, Måns; Bergman, Peter; Ozenci, Volkan

    2013-10-01

    The aim of this study was to compare the performance of four methods that are widely used in the clinical microbiology laboratory for identification of Pasteurella species. The 4 methods evaluated were VITEK2, VITEK MS (BioMerieux), and Bruker Biotyper MS (Bruker) as well as traditional biochemical tests. Sequencing of the sodA gene was used as the reference method. Sixty-five isolates of Pasteurella spp. from 65 patients were analyzed. One Pasteurella multocida isolate from American Type Culture Collection (Manassas, VA, USA) was used as a reference. Traditional biochemical tests accurately identified 62/66 (94%) isolates. Both Bruker and Vitek matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identified 59/66 (89%) strains, but VITEK2 could only identify 32/66 (48.5%) isolates correctly. The mean time to identification using biochemical tests was 20 hours; VITEK2 took 6 hours and MALDI-TOF approximately 10 minutes. In conclusion, MALDI-TOF is a quick method, which accurately identified most isolates of Pasteurella to the species level. Thus, MALDI-TOF constitutes a valuable diagnostic tool in the clinical laboratory. PMID:23886788

  17. Analysis of the lipid composition of bull spermatozoa by MALDI-TOF mass spectrometry--a cautionary note.

    PubMed

    Schiller, Jürgen; Müller, Karin; Süss, Rosemarie; Arnhold, Jürgen; Gey, Claudia; Herrmann, Andreas; Lessig, Jacqueline; Arnold, Klaus; Müller, Peter

    2003-11-01

    In this study we demonstrated the combination of MALDI-TOF MS and TLC as a fast and powerful tool to investigate the phospholipid (PL) composition of organic extracts of bull spermatozoa. Since phosphatidylcholine (PC) is the dominant PL species, an adequate resolution of MALDI-TOF spectra for sphingomyelin (SM) or phosphatidylethanolamine (PE) was achieved only after previous PL separation by TLC. We found a poor diversity especially for PE and PC, mainly containing ether-linked fatty acids which were 1-palmityl-2-docosahexaenoyl-PL and the corresponding alkenyl-acyl compound (plasmalogen) 1-palmitenyl-2-docosahexaenoyl-PL. For PC, both lipids were quantified after phospholipase A2 digestion to represent 44.2 and 37.2%, respectively, of the total PC. In contrast, the diacyl-PC content of bull spermatozoa was comparatively low (18.6% of total PC). In the presence of trifluoroacetic acid (TFA), which is routinely added to the MALDI-TOF matrix to improve the signal to noise ratio, a high lysophospholipid (LPL) content was detected in the PL extracts of bull spermatozoa, whereas TLC did not reveal significant amounts of LPL. The TFA mediated hydrolysis of the acid-labile alkenyl-acyl PL to the corresponding LPL was shown to cause this discrepancy. This assumption was verified by analysing the PL composition by MALDI-TOF MS before and after (i) digestion of sperm cell lipids with phospholipase A2 and (ii) exposition of spermatozoa to HCl fumes. We conclude that the analysis of samples containing alkenyl-acyl-PL by MALDI-TOF has to be performed with great caution. PMID:14580713

  18. Influence of storage conditions on MALDI-TOF MS profiling of gingival crevicular fluid: Implications on the role of S100A8 and S100A9 for clinical and proteomic based diagnostic investigations.

    PubMed

    Preianò, Mariaimmacolata; Maggisano, Giuseppina; Lombardo, Nicola; Montalcini, Tiziana; Paduano, Sergio; Pelaia, Girolamo; Savino, Rocco; Terracciano, Rosa

    2016-03-01

    Gingival crevicular fluid (GCF) may be a source of diagnostic biomarkers of periodontitis/gingivitis. However, peptide fingerprints may change, depending on GCF collection, handling and storage. We evaluated how storage conditions affect the quality and the reproducibility of MALDI-TOF profiles of this fluid. GCF was collected on paper strips from four subjects with healthy gingiva. Our findings demonstrated that sample storage conditions significantly affect GCF peptide pattern over time. Specifically, the storage of GCF immediately extracted from paper strips generates less variations in molecular profiles compared to the extraction performed after the storage. Significant spectral changes were detected for GCF samples stored at -20°C directly on the paper strips and extracted after three months, in comparison to the freshly extracted control. Noteworthy, a significant decrease in the peak area of HNP-3, S100A8, full-length S100A9 and its truncated form were detected after 3 months at -80°C. The alterations found in the "stored GCF" profile not only may affect the pattern-based biomarker discovery but also make its use not adequate for in vitro diagnostic test targeting S100A8, S100A9 proposed as potential diagnostic biomarkers for periodontal disease. In summary, this study shows that the best preserved signatures were obtained for the GCF samples eluted in trifluoroacetic acid and then immediately stored at -80°C for 1 month. The wealth of information gained from our data on protein/patterns stability after storage might be helpful in defining new protocols which enable optimal preservation of GCF specimen. PMID:26711623

  19. Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation

    PubMed Central

    2014-01-01

    Background Leptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software. Results The spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data. Conclusions In conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for

  20. Application the mass spectrometry MALDI-TOF technique for detection of Babesia canis canis infection in dogs.

    PubMed

    Adaszek, Łukasz; Banach, Tomasz; Bartnicki, Michał; Winiarczyk, Dagmara; Łyp, Paweł; Winiarczyk, Stanisław

    2014-11-01

    The aim of this study was to use rapid mass spectrometry (MS)-based proteomics analyses for diagnosis of Babesia canis canis infections in dogs. The study was conducted on two groups of dogs--healthy dogs and dogs infected with B. canis canis which demonstrated symptoms of babesiosis. The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS technique revealed the presence of a protein fraction of 51-52 kDa in the blood serum of all the animals infected with the protozoa, which was not found in the serum of healthy dogs. The proteins are suspected to be disease markers, whereas the MALDI-TOF technique itself has high specificity and sensitivity and can be applied in analytical laboratories in the diagnosis of canine babesiosis. PMID:25238794

  1. MALDI-TOF mass spectrometry for the monitoring of she-donkey's milk contamination or adulteration.

    PubMed

    Cunsolo, Vincenzo; Muccilli, Vera; Saletti, Rosaria; Foti, Salvatore

    2013-02-01

    Donkey's milk (DM), representing a safe and alternative food in both IgE-mediated and non-IgE-mediated cow's milk protein allergy, can be categorized as precious pharma-food. Moreover, an economically relevant interest for the use of DM in cosmetology is also developing. The detection of adulterations and contaminations of DM is a matter of fundamental importance from both an economic and allergenic standpoint, and, to this aim, fast and efficient analytical approaches to assess the authenticity of this precious nutrient are desirable. Here, a rapid matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based method aimed to the detection of bovine or caprine milk in raw DM is reported. The presence of the extraneous milks was revealed by monitoring the protein profiles of the most abundant whey proteins, α-lactalbumin (α-LA) and β-lactoglobulin, used as molecular markers. The possibility of obtaining a quantitative analysis of the level of cow or goat milk in DM based on the MALDI-TOF peak areas of α-LAs was also explored. The results showed that the experimental quantitative values were in good agreement with the real composition of each mixture. As pretreatment of the milk samples is not required, and owing to the speed and the high sensitivity of MALDI-MS, the protocol here reported could represent a reliable method for routine analyses aimed to assess the absence of contamination in raw fresh DM samples. PMID:23378086

  2. High sensitivity identification of membrane proteins by MALDI TOF-MASS spectrometry using polystyrene beads.

    PubMed

    Bensalem, Noura; Masscheleyn, Sandrine; Mozo, Julien; Vallée, Benoit; Brouillard, Franck; Trudel, Stéphanie; Ricquier, Daniel; Edelman, Aleksander; Guerrera, Ida Chiara; Miroux, Bruno

    2007-04-01

    Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents. PMID:17355127

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

    PubMed

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

    2015-07-01

    Knowledge of the plasma lipid composition is essential to clarify the specific roles of different lipid species in various pathophysiological processes. In this study, we developed an analytical strategy combining high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and off-line coupling with matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF/MS) to determine the composition of plasma and major lipoproteins at two levels, lipid classes and lipid species. We confirmed the suitability of MALDI-TOF/MS as a quantitative measurement tool studying the linearity and repeatability for triglycerides (TG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, data obtained with this method were correlated with other lipid classes and species measurements using currently available technologies. To establish the potential utility of our approach, human plasma very low density- (VLDL), low density- (LDL) and high density- (HDL) lipoproteins from 10 healthy donors were separated using ultracentrifugation, and compositions of nine lipid classes, cholesteryl esters (CE), TG, free cholesterol (FC), PE, phosphatidylinositol (PI), sulfatides (S), PC, lysophosphatidylcholine (LPC) and sphingomyelin (SM), analyzed. In total, 157 lipid species in plasma, 182 in LDL, 171 in HDL, and 148 in VLDL were quantified. The lipidomic profile was consistent with known differences in lipid classes, but also revealed unexpected differences in lipid species distribution of lipoproteins, particularly for LPC and SM. In summary, the methodology developed in this study constitutes a valid approach to determine the lipidomic composition of plasma and lipoproteins. PMID:26004846

  4. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins.

    PubMed

    Koehbach, Johannes; Gruber, Christian W; Becker, Christian; Kreil, David P; Jilek, Alexander

    2016-05-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

  5. MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins

    PubMed Central

    2016-01-01

    Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods. PMID:26985971

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

    PubMed

    Lopes, Rogério Biaggioni; Faria, Marcos; Souza, Daniela Aguiar; Bloch, Carlos; Silva, Luciano P; Humber, Richard A

    2014-01-01

    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 phylogenetic classification of species in the Metarhizium anisopliae complex. Initially the phylogenetic analysis of 5' strains by sequencing of the 59' end of the TEF-1α gene region revealed seven species within M. anisopliae sensu lato and two varieties outside this complex. Because initial studies on MS profiles from different cell types showed that mycelial fragments or conidia produced on nutrient-poor medium may yield too much background noise, all subsequent spectrometric analyses were performed with acidhydrolyzed conidia from 10-12 d old PDA cultures. The initial MALDI-TOF reference library included protein spectral profiles from nine taxonomically distinct, molecularly identified isolates sharing high genetic homology with the ex-type or ex-epitype isolates of these taxa in Metarhizium. A second reference library added one isolate each for M. anisopliae sensu stricto and M. robertsii. The second, larger reference library (including 11 taxa) allowed nearly perfect MALDI-TOF matching of DNA-based species identification for the 40 remaining isolates molecularly recognized as M. anisopliae sensu stricto (n = 19), M. robertsii (n = 6), M. majus (n = 3), M. lepidiotae (n = 1), M. acridum (n = 3), M. flavoviride var. pemphigi (n = 1), plus seven unidentified strains (six of them phylogenetically close to M. anisopliae sensu stricto and one outside the Metarhizium pingshaense-anisopliae-robertsii-brunneum clade). Due to the increasing frequency of phylogenetically (genomically) based taxonomic revisions of fungi, this approach is especially useful for culture collections, because once the protein profiles of Metarhizium isolates are obtained taxonomic updating of MALDI-TOF

  7. Sequence-dependent enrichment of a model phosphopeptide: a combined MALDI-TOF and NMR study.

    PubMed

    Matheron, Lucrèce; Lucrèce, Matheron; Sachon, Emmanuelle; Emmanuelle, Sachon; Burlina, Fabienne; Fabienne, Burlina; Sagan, Sandrine; Sandrine, Sagan; Lequin, Olivier; Olivier, Lequin; Bolbach, Gèrard; Gèrard, Bolbach

    2011-04-15

    The goal of this study was to detect and quantify by MALDI-TOF MS the phosphorylation of a peptide containing the recognition motif of the Protein Kinase C (PKC). Such model peptide can be used as a phosphorylation probe to follow intracellular kinase/phosphatase activities. This study allowed us to establish relationships between sequence specificities and affinity for TiO(2) or IMAC media. The peptide has the sequence biotin-GGGGCFRTPSFLKK-NH(2) in which the serine residue can be phosphorylated. Enrichment of the corresponding phosphopeptide, by the dedicated IMAC and TiO(2) affinity chromatography methods, proved inefficient. By combining MALDI-TOF and NMR data, we first showed that the lack of affinity of the phosphopeptide for TiO(2) was partly related to the basic property of its peptide sequence. Furthermore, the peptide shows local structuration around the P(9)- S(10) segment, with formation of a salt bridge between the guanidinium group of the R(7) side chain and the phosphate moiety. In conjunction with an inadequate position of the {biotin-G(4)} N-terminal tag, this local structure could shield the phosphate group, preventing interaction with TiO(2). To improve TiO(2) affinity, the peptide sequence was modified accordingly. The new sequences retained the biological properties while their enrichment by IMAC or TiO(2) became possible. PMID:21428305

  8. Comparison of the Bruker MALDI-TOF Mass Spectrometry System and Conventional Phenotypic Methods for Identification of Gram-Positive Rods

    PubMed Central

    Barberis, Claudia; Almuzara, Marisa; Join-Lambert, Olivier; Ramírez, María Soledad; Famiglietti, Angela; Vay, Carlos

    2014-01-01

    In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥1,5 and ≥1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥1,5 for genus level and ≥1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories. PMID:25184254

  9. A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry

    PubMed Central

    Schaumann, Reiner; Knoop, Nicolas; Genzel, Gelimer H.; Losensky, Kevin; Rosenkranz, Christiane; Stîngu, Catalina S.; Schellenberger, Wolfgang; Rodloff, Arne C.; Eschrich, Klaus

    2012-01-01

    Summary Background Matrix-Assisted Laser-Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has already proven to be a powerful tool for species identification in microbiological laboratories. As adequate and rapid screening methods for antibiotic resistance are crucially needed, the present study investigated the discrimination potential of MALDI-TOF MS among extended-spectrum-beta-lactamase (ESBL) or metallo-beta-lactamases- (MBL) producing and the nonproducing strains of Escherichia coli (n=19), Klebsiella pneumoniae (n=19), and Pseudomonas aeruginosa (n=38), respectively. Material/Methods We used a MALDI-TOF MS protocol, usually applied for species identification, in order to integrate a screening method for beta-lactamases into the routine species identification workflow. The acquired spectra were analyzed by visual inspection, statistical similarity analysis and support vector machine (SVM) classification algorithms. Results Neither visual inspection nor mathematical similarity analysis allowed discrimination between spectra of beta-lactamase-producing and the nonproducing strains, but classification within a species by SVM-based algorithms could achieve a correct classification rate of up to 70%. Conclusions This shows that MALDI-TOF MS has definite potential to discriminate antibiotic-resistant strains due to ESBL and MBL production from nonproducing strains, but this performance is not yet sufficiently reliable for routine microbiological diagnostics. PMID:22936198

  10. 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