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Sample records for 2x3 tof ms

  1. Analysis of Combustion Chamber Deposits by ESI-TOF-MS and MALDI-TOF-MS

    SciTech Connect

    Reynolds, J G; Shields, S J; Roos, J W

    2001-06-14

    Combustion chamber deposits (CCDs) in internal combustion engines have been studied by various techniques to understand the relationship of performance degradation with deposit quantity and structure. XPS, XAS, NMR, and elemental analysis have offered insight into the bulk structure of C, H, N, O and metal components [1]. MS has offered some information about compound structure, but results are limited due to the insolubility and complexity of the materials. Recent advances in MS have opened new possibilities for analysis of CCDs. Here we report initial findings on the carbon structure of these deposits determined by ESI-TOF-MS and MADLI-TOF-MS.

  2. 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.

  3. Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.

    PubMed

    Pashkova, Anna; Chen, Hsuan-Shen; Rejtar, Tomas; Zang, Xin; Giese, Roger; Andreev, Victor; Moskovets, Eugene; Karger, Barry L

    2005-04-01

    The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.

  4. The application of MALDI TOF MS in biopharmaceutical research.

    PubMed

    Kafka, Alexandra P; Kleffmann, Torsten; Rades, Thomas; McDowell, Arlene

    2011-09-30

    The development and quality assessment of modern biopharmaceuticals, particularly protein and peptide drugs, requires an array of analytical techniques to assess the integrity of the bioactive molecule during formulation and administration. Mass spectrometry is one of these methods and is particularly suitable for determining chemical modifications of protein and peptide drugs. The emphasis of this review is the identification of covalent interactions between protein and peptide bioactives with polymeric pharmaceutical formulations using mass spectrometry with the main focus on matrix-assisted laser desorption/ionization (MALDI) coupled tandem time-of-flight (TOF/TOF) mass spectrometry (MS). The basics of MALDI TOF MS and collision-induced dissociation (CID)-based ion fragmentation will be explained and applications for qualitative characterization of protein and peptide drugs and their interactions with pharmaceutical polymers will be discussed using three case studies.

  5. MALDI-TOF MS analysis of plant proanthocyanidins.

    PubMed

    Monagas, María; Quintanilla-López, Jesús Eduardo; Gómez-Cordovés, Carmen; Bartolomé, Begoña; Lebrón-Aguilar, Rosa

    2010-01-20

    Proanthocyanidins or condensed tannins are among the most abundant polyphenols compounds in our diet and may play a key role in the prevention of cardiovascular and neurodegenerative diseases and cancer. These antioxidants are widely distributed in the plant kingdom both in food plants and in non-food plants. The biological activity of plant proanthocyanidins depends on their chemical structure and concentration. However, due to their structural diversity and complexity, the qualitative and quantitative analysis of proanthocyanidins is a difficult task. Mass spectrometry has enabled great advances in the characterization of plant proanthocyanidins. Among these techniques, MALDI-TOF MS has proved to be highly suited for the analysis of highly polydisperse and heterogeneous proanthocyanidins. The objective of the present paper was to assess the potential, limitations and future challenges of the analysis of plant proanthocyanidins by MALDI-TOF MS techniques. Firstly, the fundamental of this technique, including modes of operation, advantages and limitations, as well as quantitative and qualitative operations, have been summarized. Applications of MALDI-TOF analysis to plant proanthocyanidins reported in the last decade (1997-2008) have been extensively covered, including the sample preparation protocols and conditions used for proanthocyanidin analysis, as well as the main findings regarding the determination of the structural features of different plant proanthocyanidin types (procyanidins, propelargonidins, prodelphinidins, profisetinidins and prorobinetinidins). Finally, attempts in the assessment of the molecular weight distribution of proanthocyanidins by MALDI-TOF are described.

  6. Spontaneous-Desorption Ionizer for a TOF-MS

    NASA Technical Reports Server (NTRS)

    Schultz, J. Albert

    2006-01-01

    A time-of-flight mass spectrometer (TOF-MS) like the one mentioned in the immediately preceding article has been retrofitted with an ionizer based on a surface spontaneous-desorption process. This ionizer includes an electron multiplier in the form of a microchannel plate (MCP). Relative to an ionizer based on a hot-filament electron source, this ionizer offers advantages of less power consumption and greater mechanical ruggedness. The current density and stability characteristics of the electron emission of this ionizer are similar to those of a filament-based ionizer. In tests of various versions of this ionizer in the TOF-MS, electron currents up to 100 nA were registered. Currents of microamperes or more - great enough to satisfy requirements in most TOFMS applications - could be obtained by use of MCPs different from those used in the tests, albeit at the cost of greater bulk. One drawback of this ionizer is that the gain of the MCP decreases as a function of the charge extracted thus far; the total charge that can be extracted over the operational lifetime is about 1 coulomb. An MCP in the ion-detector portion of the TOF-MS is subject to the same limitation.

  7. Intraspecific variations in Conus purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-TripleTOF-MS.

    PubMed

    Rodriguez, Alena M; Dutertre, Sebastien; Lewis, Richard J; Marí, Frank

    2015-08-01

    The venom of cone snails is composed of highly modified peptides (conopeptides) that target a variety of ion channels and receptors. The venom of these marine gastropods represents a largely untapped resource of bioactive compounds of potential pharmaceutical value. Here, we use a combination of bioanalytical techniques to uncover the extent of venom expression variability in Conus purpurascens, a fish-hunting cone snail species. The injected venom of nine specimens of C. purpurascens was separated by reversed-phase high-performance liquid chromatography (RP-HPLC), and fractions were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in parallel with liquid chromatography-electrospray ionization (LC-ESI)-TripleTOF-MS to compare standard analytical protocols used in preparative bioassay-guided fractionations with a deeper peptidomic analysis. Here, we show that C. purpurascens exhibits pronounced intraspecific venom variability. RP-HPLC fractionation followed by MALDI-TOF-MS analysis of the injected venom of these nine specimens identified 463 distinct masses, with none common to all specimens. Using LC-ESI-TripleTOF-MS, the injected venom of these nine specimens yielded a total of 5517 unique masses. We also compare the injected venom of two specimens with their corresponding dissected venom. We found 2566 and 1990 unique masses for the dissected venom compared to 941 and 1959 masses in their corresponding injected venom. Of these, 742 and 1004 masses overlapped between the dissected and injected venom, respectively. The results indicate that larger conopeptide libraries can be assessed by studying multiple individuals of a given cone snail species. This expanded library of conopeptides enhances the opportunities for discovery of molecular modulators with direct relevance to human therapeutics. Graphical Abstract The venom of cone snails are extraordinarily complex mixtures of highly modified peptides. Venom

  8. Application of nano-LC-MALDI-TOF/TOF-MS for proteomic analysis of microvesicles.

    PubMed

    Kasprzyk, Joanna; Stępień, Ewa; Piekoszewski, Wojciech

    2017-03-01

    Activated platelets and platelet derived microvesicles (PMVs) emerged recently to be promising biomarkers. There is no universal procedure to carry out the proteomic analysis on microvesicles. In this study we proposed a nano-liquid chromatography (nano-LC) technique coupled off-line with a spectrometric measurement MALDI-TOF-MS/MS as a throughput and time-saving procedure. In this study we developed a simplified method to evaluate the protein composition of platelet organelles and PMVs. Platelet-rich plasma (PRP) was collected from healthy donors. PMVs were generated from washed and thrombin activated platelets. Activated platelets from every donor were used to compare the PMV proteome. Enzymatic digestion of protein lysate was carried out using Filter Aided Sample Preparation (FASP) method with trypsin as a proteolytic enzyme. Tryptic peptides derived from PMVs and activated platelets were analysed using nano-LC coupled off-line mode with a MALDI-TOF/TOF-MS. PMV and platelet protein identification was performed using the Mascot engine for searching against the Swiss-Prot human database. The precision tolerance was 100ppm for peptide masses and 0.7Da for fragment ion masses. Individual peptide matches with a score above 28 were considered statistically significant. In total, 446 proteins were identified in PMVs and 513 proteins in activated platelets. Among them 190 were specific for activated platelets and 123 were PMV specific. Cellular component analysis of identified proteins revealed that PMVs contained relatively more extracellular proteins than activated platelets (9.6 vs. 6.0 %) and unique synaptic proteins (0.3%). A new simplified bottom-up method for PMV proteome analysis allowed eliminating the drawbacks of the previously used protocols. This approach can be used in PMV proteome identification.

  9. The potential of combining ion trap/MS/MS and TOF/MS for identification of emerging contaminants

    USGS Publications Warehouse

    Ferrer, I.; Furlong, E.T.; Heine, C.E.; Thurman, E.M.

    2002-01-01

    The use of a method combining ion trap tandem mass spectrometry (MS/MS) and time of flight mass spectrometry (TOF/MS) for identification of emerging contaminates was discussed. The two tools together complemented each other in sensitivity, fragmentation and accurate mass determination. Liquid chromatography/electrospray ionization/ion-trap tandem mass spectrometry (LC/ESI/MS/MS), in positive ion mode of operation, was used to separate and identify specific compounds. Diagnostic fragment ions were obtained for a polyethyleneglycol(PEG) homolog by ion trap MS/MS, and fragments were measured by TOF/MS. It was observed that the combined method gave an exact mass measurement that differed from the calculated mass.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. 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...

  12. 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.

  13. Progress in proteomics for clinical microbiology: MALDI-TOF MS for microbial species identification and more.

    PubMed

    van Belkum, Alex; Chatellier, Sonia; Girard, Victoria; Pincus, David; Deol, Parampal; Dunne, Wm Michael

    2015-01-01

    Although classical proteomic approaches are still used regularly in routine clinical diagnostic procedures, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) MS has recently moved into diagnostic microbiology laboratories. MALDI-TOF MS is currently replacing phenotypic microbial identification. Many laboratories now use MALDI-TOF MS for its high efficiency, both from a diagnostic and a cost-per-analysis point of view. The US FDA has now cleared two of the commercially available systems for in vitro diagnostics. This will further spark development of MS applications in antimicrobial susceptibility testing and epidemiology. This review summarizes the state of affairs of MALDI-TOF MS in clinical microbiology; however, this is an active field of research subject to rapid evolution. We emphasize assessment of the clinical relevance and studies focusing on data obtained through comparative analyses of different MALDI-TOF MS instrumentation and multicenter validation studies. The future of MALDI-TOF MS, including antimicrobial susceptibility testing and epidemiological typing, is also highlighted.

  14. Toward an In Situ Organic and Atomic Microprobe with Laser TOF-MS

    NASA Technical Reports Server (NTRS)

    Brinckerhoff, W. B.; Cornish, T. J.; McEntire, R. W.; Cheng, A. F.; Benson, R. C.

    2000-01-01

    We present details of a new miniature laser time-of-flight mass spectrometer (TOF-MS) with improved resolution and sensitivity, for in situ analysis of elemental, isotopic, and organic/molecular composition.

  15. MALDI-TOF MS meets WGS in a VRE outbreak investigation.

    PubMed

    Schlebusch, S; Price, G R; Gallagher, R L; Horton-Szar, V; Elbourne, L D H; Griffin, P; Venter, D J; Jensen, S O; Van Hal, S J

    2017-03-01

    The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization-time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology-oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.

  16. Identification of microorganisms grown on chromogenic media by MALDI-TOF MS.

    PubMed

    Lüthje, Petra; Pranada, Arthur B; Carruthers-Lay, Duncan; Desjardins, Marc; Gaillot, Olivier; Wareham, David; Ciesielczuk, Holly; Özenci, Volkan

    2017-05-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and chromogenic media are widely used in clinical microbiology laboratories to facilitate the rapid selection and identification of pathogens. The aim of this study was to evaluate whether usage of chromogenic media limits the diagnostic performance of MALDI-TOF MS for microbial identification. A total of 386 microorganisms collected and analyzed at five laboratories were included. Isolates were cultured on relevant chromogenic media and non-selective agar plates in parallel and identified using the Bruker MALDI-TOF MS. Among the tested isolates, no misidentification was recorded and there was no medium-related difference in the identification level. However, score values were overall slightly but significantly lower for isolates grown on chromogenic media. In conclusion, the use of chromogenic culture media tested here had no relevant impact on MALDI-TOF MS performance for diagnostic purposes.

  17. [Discrimination of plasticizers and screening of phthalates in polyvinyl chloride using DART-TOF/MS].

    PubMed

    Abe, Yutaka; Yamaguchi, Miku; Mutsuga, Motoh; Hirahara, Yoshichika; Kawamura, Maiko; Kikura-Hanajiri, Ruri; Goda, Yukihiro; Kawamura, Yoko

    2010-01-01

    A technique using a direct analysis in real time (DART) ion source coupled with time of flight/mass spectrometry (TOF/MS) was developed to discriminate plasticizers and to screen phthalates in polyvinyl chloride (PVC). In DART-TOF/MS analysis of 40 plasticizers, the protonated molecular ion, [M+H](+), was detected for most plasticizers, and the molecular weight could be easily predicted. In the analysis of PVC sheets and toys, mass spectra of plasticizers were successfully detected, and accordingly, plasticizers in PVC were easily discriminated. PVC with a phthalates content in excess of 0.1% could be screened accurately according to the DART-TOF/MS ion intensity of phthalates corresponding to the limit of detection or a suitable criterion value. DART-TOF/MS analysis is a simple and rapid technique that is suitable for the discrimination of plasticizers and for screening of phthalates in PVC.

  18. Secondary metabolite profiling of Curcuma species grown at different locations using GC/TOF and UPLC/Q-TOF MS.

    PubMed

    Lee, Jueun; Jung, Youngae; Shin, Jeoung-Hwa; Kim, Ho Kyoung; Moon, Byeong Cheol; Ryu, Do Hyun; Hwang, Geum-Sook

    2014-07-04

    Curcuma, a genus of rhizomatous herbaceous species, has been used as a spice, traditional medicine, and natural dye. In this study, the metabolite profile of Curcuma extracts was determined using gas chromatography-time of flight mass spectrometry (GC/TOF MS) and ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) to characterize differences between Curcuma aromatica and Curcuma longa grown on the Jeju-do or Jin-do islands, South Korea. Previous studies have performed primary metabolite profiling of Curcuma species grown in different regions using NMR-based metabolomics. This study focused on profiling of secondary metabolites from the hexane extract of Curcuma species. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) plots showed significant differences between the C. aromatica and C. longa metabolite profiles, whereas geographical location had little effect. A t-test was performed to identify statistically significant metabolites, such as terpenoids. Additionally, targeted profiling using UPLC/Q-TOF MS showed that the concentration of curcuminoids differed depending on the plant origin. Based on these results, a combination of GC- and LC-MS allowed us to analyze curcuminoids and terpenoids, the typical bioactive compounds of Curcuma, which can be used to discriminate Curcuma samples according to species or geographical origin.

  19. Utility of the MALDI-TOF MS method to identify nontuberculous mycobacteria.

    PubMed

    Kodana, Masahiro; Tarumoto, Norihito; Kawamura, Tohru; Saito, Taeko; Ohno, Hideaki; Maesaki, Shigefumi; Ikebuchi, Kenji

    2016-01-01

    In comparison to the conventional real-time polymerase chain reaction method (PCR method) or the DNA-DNA hybridization method (DDH method), the utility of NTM identification by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method has seldom been reported. In this study, 75 clinical NTM isolates from our hospital between April 2013 and July 2014 were identified and analyzed using PCR, DDH, and MALDI-TOF MS methods, and the results for the MALDI-TOF MS method were compared with the others. Identification at the species level was in agreement for 71 (94.5%) of the 75 isolates. For further details, identification was possible for 23 (95.8%) of 24 Mycobacterium avium, 11 (100%) of 11 Mycobacterium intracellulare, and 1 (50%) of 2 isolates mixed with M. avium and M. intracellulare. Mycobacterium ksansasii, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium gordonae, and Mycobacterium chelonae identified by DDH method were same result by MALDI-TOF MS. Additionally, Mycobacterium mucogenicum, which could not be identified by the DDH method, was identified by the MALDI-TOF MS method. However, two isolates identified as Mycobacterium terrae by DDH method could not be identified by the MALDI-TOF MS method and were determined to be Mycobacterium arupense by 16S ribosomal RNA (rRNA) sequence analysis. The present findings show that, for rare bacterial species, identification is sometimes not possible, but, in most cases, the results of identification by the MALDI-TOF MS method have a high concordance rate with the results of the PCR and DDH methods.

  20. The benefit of combining nLC-MALDI-Orbitrap MS data with nLC-MALDI-TOF/TOF data for proteomic analyses employing elastase.

    PubMed

    Rietschel, Benjamin; Baeumlisberger, Dominic; Arrey, Tabiwang N; Bornemann, Sandra; Rohmer, Marion; Schuerken, Malte; Karas, Michael; Meyer, Bjoern

    2009-11-01

    The recently established coupling of a MALDI-type ion source to a linear ion trap and an orbitrap mass analyzer offers high-accuracy mass measurements compared to common MALDI-TOF/TOF instruments. Contrary to MALDI-TOF/TOF, the fragmentation of peptides in the new hybrid mass spectrometer is less efficient due to the generation of predominantly singly charged ions by the MALDI process. Therefore, data from two MALDI instruments, TOF/TOF and Orbitrap, were combined into a single data set in order to obtain accurate precursor masses as well as superior MS/MS spectra. This study demonstrates that an accurate precursor mass is particularly important for the nLC-MS/MS analyses of less-specific proteolytic digests. A potential gain of approximately one-third additional peptides identifications was theoretically estimated from previously published MALDI-TOF/TOF data. These calculations were verified by the nLC-MS/MS analysis of two elastatically digested proteomes, one cytosolic (Corynebacterium glutamicum) and one membrane (Halobacterium salinarium). Thereby it was discovered that the error distribution of a MALDI-Orbitrap can be significantly improved by applying an easy recalibration strategy. In summary, this study represents an updated workflow for the analysis of less-specific digests using nLC-MALDI.

  1. Sternal wound infection caused by Gordonia bronchialis: identification by MALDI-TOF MS

    PubMed Central

    Rodriguez-Lozano, Jesús; Pérez-Llantada, Enrique; Agüero, Jesús; Rodríguez-Fernández, Ana; Ruiz de Alegria, Carlos; Martinez-Martinez, Luis

    2016-01-01

    Introduction: Gordonia spp. infections are uncommon. However, a few clinical cases have been reported in the literature, particularly those involving immunocompromised hosts. Advanced microbiology diagnosis techniques, such as matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), have been recently introduced in clinical microbiology laboratories in order to improve microbial identification, resulting in better patient management. Case presentation: Here, we present a new clinical case of persistent wound infection caused by Gordonia bronchialis in a 64-year-old woman after a mitral valve replacement, using two MALDI-TOF-based systems for identifying this micro-organism. Conclusion: Both MALDI-TOF systems were able to identify Gordonia spp.; thus, providing a useful tool that overcomes the current limitations of phenotypic identification associated with this micro-organism. Although the technique validation deserves additional verification, our study provides guidance about MALDI-TOF as a fast and easy method for Gordonia spp. identification. PMID:28348789

  2. Evaluation of ice-tea quality by DART-TOF/MS.

    PubMed

    Rajchl, Aleš; Prchalová, Jana; Kružík, Vojtěch; Ševčík, Rudolf; Čížková, Helena

    2015-11-01

    DART (Direct Analysis in Real Time) coupled with Time-of-Flight Mass Spectrometry (TOF/MS) has been used for analyses of ice-teas. The article focuses on quality and authenticity of ice-teas as one of the most important tea-based products on the market. Twenty-one samples of ice-teas (black and green) were analysed. Selected compounds of ice-teas were determined: theobromine, caffeine, total phenolic compounds, total soluble solids, total amino acid concentration, preservatives and saccharides were determined. Fingerprints of DART-TOF/MS spectra were used for comprehensive assessment of the ice-tea samples. The DART-TOF/MS method was used for monitoring the following compounds: citric acid, caffeine, saccharides, artificial sweeteners (saccharin, acesulphame K), and preservatives (sorbic and benzoic acid), phosphoric acid and phenolic compounds. The measured data were subjected to a principal components analysis. The HPLC and DART-TOF/MS methods were compared in terms of determination of selected compounds (caffeine, benzoic acid, sorbic acid and saccharides) in the ice-teas. The DART-TOF/MS technique seems to be a suitable method for fast screening, testing quality and authenticity of tea-based products.

  3. [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.

  4. [Identification of staphylococci directly from positive blood culture bottles by MALDI-TOF MS system].

    PubMed

    Kilic, Abdullah; Baysallar, Mehmet

    2014-07-01

    Bloodstream infections are substantial causes of morbidity and mortality worldwide. Staphylococcus species are the most commonly isolated microorganisms from blood cultures in clinical microbiology laboratories. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time- of- Flight Mass Spectrometry) system allows the identification of microorganisms directly from positive blood culture bottles. The aim of this study was to evaluate the MALDI-TOF MS system for the identification of staphylococci directly from the positive blood culture bottles which revealed the presence of gram-positive cocci by staining. A total of 96 positive blood culture bottles that yielded gram-positive cocci by Gram stain were evaluated. These blood cultures were obtained from 69 patients between December 2013-February 2014. Conventional methods and BD Phoenix™ automated bacterial identification system (Becton Dickinson, USA) were used for routine identification. The strains were also identified by real-time Taqman PCR (qPCR) which was considered as the reference method. In MALDI-TOF MS method, MALDI Sepsityper™ Kit was used for the bacterial identification and all measurements were carried out by using Microflex LT instrument and FlexControl 3.0 software (Bruker Daltonics, USA). Of 96 culture bottles positive for gram-positive cocci, 90 were correctly identified as staphylococci at genus level with all the three study methods (qPCR, BD Phoenix, Bruker MALDI-TOF MS). The other six samples were identified as Enterococcus faecium (n= 4) and Streptococcus pyogenes (n= 2) by both Phoenix and the MALDI-TOF systems. Of the 90 samples, 87 were identified at the species level (15 S.aureus, 33 S.epidermidis, 29 S.hominis, 10 S.haemolyticus) and three at the genus level by the reference qPCR method. When comparing the results obtained by qPCR and Bruker MALDI-TOF MS, incompatibility was detected for three isolates. Those isolates were identified as S.hominis by qPCR, however two of them were

  5. Ni speciation in tea infusions by monolithic chromatography--ICP-MS and Q-TOF-MS.

    PubMed

    Ščančar, Janez; Zuliani, Tea; Žigon, Dušan; Milačič, Radmila

    2013-02-01

    For humans, Ni is not considered to be an essential trace element. Its compounds, at levels present in foodstuffs and drinks, are generally considered to be safe for consumption, but for individuals who already suffer from contact allergy to Ni and may be subject to develop systemic reactions from its dietary ingestion, dietary exposure to Ni must be kept under control. Being the second most popular beverage, tea is a potential source of dietary Ni. Present knowledge on its speciation in tea infusions is poor. Therefore, complete speciation analysis, consisting of separation by liquid chromatography using a weak CIM DEAE-1 monolithic column, "on-line" detection by inductively coupled plasma mass spectrometry (ICP-MS) and "off-line" identification of ligands by hybrid quadrupole time-of-flight mass spectrometry (Q-TOF MS), was implemented for the first time to study Ni speciation in tea infusions. Total concentrations of Ni in dry leaves of white, green, oolong and black tea (Camellia sinensis) and flowers of herbal chamomile (Matricaria chamomilla) and hibiscus (Hibiscus sabdariffa) tea were determined after microwave digestion by ICP-MS. They lay between 1.21 and 14.4 mg kg(-1). Good agreement between the determined and the certified values of the Ni content in the standard reference material SRM 1573a tomato leaves confirmed the accuracy of the total Ni determination. During the infusion process, up to 85 % of Ni was extracted from tea leaves or flowers. Separation of Ni species was completed in 10 min by applying aqueous linear gradient elution with 0.6 mol L(-1) NH(4)NO(3). Ni was found to be present in the chromatographic fraction in which quinic acid was identified by Q-TOF in all the tea infusions analysed, which had pH values between 5.6 and 6.0. The only exception was the infusion of hibiscus tea with a pH of 2.7, where results of speciation analysis showed that Ni is present in its divalent ionic form.

  6. MALDI-TOF MS in clinical parasitology: applications, constraints and prospects.

    PubMed

    Singhal, Neelja; Kumar, Manish; Virdi, Jugsharan Singh

    2016-10-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is currently being used for rapid and reproducible identification of bacteria, viruses and fungi in clinical microbiological laboratories. However, some studies have also reported the use of MALDI-TOF MS for identification of parasites, like Leishmania, Giardia, Cryptosporidium, Entamoeba, ticks and fleas. The present review collates all the information available on the use of this technique for parasites, in an effort to assess its applicability and the constraints for identification/diagnosis of parasites and diseases caused by them. Though MALDI-TOF MS-based identification of parasites is currently done by reference laboratories only, in future, this promising technology might surely replace/augment molecular methods in clinical parasitology laboratories.

  7. [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.

  8. MALDI-TOF MS: an upcoming tool for rapid detection of antibiotic resistance in microorganisms.

    PubMed

    Kostrzewa, Markus; Sparbier, Katrin; Maier, Thomas; Schubert, Sören

    2013-12-01

    MALDI-TOF MS profiling for microorganism detection has already been demonstrated in the 1990s, but has evolved to the first-line identification method in many laboratories just during the past five years. While this application of MALDI-TOF MS has proven its broad applicability, accuracy, robustness, and cost-effectiveness it is of particular interest to expand the capabilities of the mass spectrometric platform. Resistance detection is the most desirable further application of MALDI-TOF MS in microbiology, but maybe also the most challenging. Different approaches have been published regarding diverse antibiotic drugs and distinct microorganism classes. The current review shall give an overview about the developments of the recent years and their potential to get transformed in clinical useful assays in the future.

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

    PubMed

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

    2015-01-01

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

  10. 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

  11. GC/TOF-MS as a new method for halocarbon observation in the atmosphere

    NASA Astrophysics Data System (ADS)

    Obersteiner, Florian; Boenisch, Harald; Hoker, Jesica; Engel, Andreas

    2015-04-01

    The need for halocarbon measurements in the atmosphere arose with the anthropogenic emission of CFCs beginning in the 1950s and the discovery of their ozone depleting potential in the 1980s. CFCs were replaced by HCFCs and are nowadays replaced by HFCs, with new compounds continuously being developed and introduced to the atmosphere. While not being harmful to the ozone layer, HFCs are still greenhouse gases and many tend to be hazardous to human health at high concentration. They can also serve as tracers to study atmospheric transport at low concentration, making high precision measurement interesting to atmospheric studies. Gas chromatography coupled with time-of-flight mass spectrometry (GC/TOF-MS) is still a new method in the field of atmospheric halocarbon measurement compared to the well-established GC/QP(quadrupole)-MS. The QP-MS is indeed a very stable and easy-to-operate instrument but also limited by mass resolution and either mass range or sensitivity. We will present the general applicability of GC/TOF-MS to regular halocarbon observation by a time series of halocarbon measurements from the Taunus Observatory (Kleiner Feldberg, Germany) and the implementation of a second, high-resolution (max. R=4000) TOF-MS system. Both GC/TOF-MS systems are characterized with respect to reproducibility, non-linearity and limits of detection (LOD). Furthermore, the advantages of a higher mass resolution are demonstrated with respect to LOD, substance identification and substance quantification.

  12. [Study on chemical constituents in stems of Nelumbo nucifera by UPLC-ESI/Q-TOF-MS/MS].

    PubMed

    Shan, Feng; Yuan, Yuan; Kang, Li-ping; Huang, Lu-qi

    2015-08-01

    This paper employed UPLC-Electrospray Ionization /Quadrupole-Time of Flight-Mass /Mass Spectrometry( UPLC-ESI/Q-TOF-MS/MS) to analyze the chemical constituents in the stems of Nelumbo nucifera. The stems of N. nucifera were extracted with 75% methanol, and we applied an Agilent Zorbax SB-Aq column (2.1 mm x 100 mm, 1.8 μm) to UPLC analysis with water methanol-water( containing 0.05% formic acid) in gradient as mobile phase. The eluates were then detected by ESI-Q-TOF-MS/MS. Results indicated that 22 benzylisoquinoline alkaloids were indendified. Among them, one alkaloid may be a new compound and a component was found in the Lotus for the first time. We fully identify the composition of the Lotus stems for the first time, Which could provides theoretical foundation for further study and utilization of the medicinal resources.

  13. Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology.

    PubMed

    Angeletti, Silvia

    2016-09-06

    The microbiological management of patients with suspected bacterial infection includes the identification of the pathogen and the determination of the antibiotic susceptibility. These traditional approaches, based on the pure culture of the microorganism, require at least 36-48h. A new method, Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), has been recently developed to profile bacterial proteins from whole cell extracts and obtain a bacterial fingerprint able to discriminate microorganisms from different genera and species. By whole cell-mass spectrometry, microbial identification can be achieved within minutes from cultured isolate, rather than traditional phenotypic or genotypic characterizations. From the year 2009 an explosion of applications of this technology has been observed with promising results. Several studies have been performed and showed that MALDI-TOF represents a reliable alternative method for rapid bacteria and fungi identification in clinical setting. A future area of expansion is represented by the application of MALDI-TOF technology to the antibiotic susceptibility test. In conclusion, the revision of the literature available up to date demonstrated that MALDI-TOF MS represents an innovative technology for the rapid and accurate identification of bacterial and fungal isolates in clinical settings. By an earlier microbiological diagnosis, MALDI-TOF MS contributes to a reduced mortality and hospitalization time of the patients and consequently has a significant impact on cost savings and public health.

  14. Identification of in vitro and in vivo metabolites of alantolactone by UPLC-TOF-MS/MS.

    PubMed

    Yao, Donggui; Li, Zhe; Huo, Changhong; Wang, Yufang; Wu, Yibing; Zhang, Manli; Li, Ligeng; Shi, Qingwen; Kiyota, Hiromasa; Shi, Xiaowei

    2016-10-15

    Alantolactone (AL), an active sesquiterpene originating from Inula helenium, is a potential anticancer and anti-inflammatory agent. However so far, studies on AL metabolism have not been reported. In the present study, we have investigated for the first time the in vivo and in vitro metabolites of AL using ultra performance liquid chromatography combined with time of flight mass spectrometry (UPLC-TOF-MS/MS). A unique on-line information-dependent acquisition (IDA) method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was applied to trace all of the probable metabolites of AL. Five MMDF templates were set according to the core structure of AL and the general metabolite biotransformation patterns, and other five sulfur-containing dimer filter templates were first established on the basis of structural elucidation of AL metabolites obtained from rat intestinal bacteria biotransformation. As a result, 44 metabolites were characterized: 41 metabolites from rat urine, bile and feces after oral administration of AL, and 13 metabolites from AL biotransformation by rat intestinal bacteria. Particularly, 26 metabolites were identified as novel sulfur-containing products. The results indicated that addition of double bond at Δ((11,13)) and oxidization were the main metabolic reactions of AL. A new metabolism pathway to produce addition products of H2S to AL and further generate a series of sulfur-containing dimers of AL was revealed. This study significantly enriched our knowledge about AL metabolism, which will lead to a better understanding of the safety and efficacy of AL. At the same time, the established methodology can be widely applied for the structural determination of the metabolites of other sesquiterpene containing α-methylene-γ-lactone moiety.

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

    PubMed Central

    2011-01-01

    Background The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. Results In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. Conclusions MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library. PMID:22192890

  16. Serum biomarker screening for the diagnosis of early gastric cancer using SELDI-TOF-MS.

    PubMed

    Li, Ping; Zhang, Dianliang; Guo, Chunbao

    2012-06-01

    In this study, we performed a proteomic analysis of sera from stage I gastric cancer patients using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and established a diagnostic model for the early diagnosis of stage I gastric cancer. Serum samples from 169 gastric cancer patients and 83 age- and gender-matched healthy individuals were analyzed by SELDI-TOF-MS ProteinChip array technology. The SELDI-TOF-MS spectral data were analyzed using the Biomarker Wizard™ and Biomarker Patterns™ software to find differential proteins and develop a classification tree for gastric cancer. A total of 34 mass peaks were identified. Six peaks at a mass-to-charge ratio (m/z) of 2873, 3163, 4526, 5762, 6121 and 7778 were used to construct the diagnostic model. The model effectively distinguished gastric cancer samples from control samples, achieving a sensitivity and specificity of 93.49 and 91.57%, respectively. In addition, we identified 3 of the 6 protein peaks at 2873, 6121 and 7778 m/z, which distinguished between stage I and stage II/III/IV gastric cancer. The model had an accuracy of 88.89% for the identification of stage I gastric cancer. In conclusion, the diagnostic model for the detection of serum proteins by SELDI-TOF-MS ProteinChip array technology correctly distinguishes gastric cancer from healthy samples, and has the ability to screen and distinguish between early gastric cancer from advanced gastric cancer.

  17. MALDI-TOF MS distinctly differentiates nontypable Haemophilus influenzae from Haemophilus haemolyticus.

    PubMed

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

    2013-01-01

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

  18. Comparison of imipenem and meropenem antibiotics for the MALDI-TOF MS detection of carbapenemase activity.

    PubMed

    Rotova, Veronika; Papagiannitsis, Costas C; Skalova, Anna; Chudejova, Katerina; Hrabak, Jaroslav

    2017-04-05

    A comparison of carbapenem molecules for the detection of carbapenemase-producing bacteria by MALDI-TOF MS showed that imipenem exhibited higher sensitivity (97%) and specificity (100%) scores for Pseudomonas aeruginosa than meropenem. However, meropenem was more efficient (98% sensitivity and 100% specificity) against Enterobacteriaceae.

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

    PubMed

    Weesepoel, Yannick; Vincken, Jean-Paul; Pop, Raluca Maria; Liu, Kun; Gruppen, Harry

    2013-07-01

    The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono- and diesters. For rapid fingerprinting of these esters, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono- and diester palmitate standards in MALDI-TOF/TOF-MS showed that sodium adduct parent masses [M + Na](+) gave much simpler MS(2) spectra than radical / protonated [M](+●) / [M + H](+) parents. [M + Na](+) fragments yielded diagnostic polyene-specific eliminations and fatty acid neutral losses, whereas [M](+●) / [M + H](+) fragmentation resulted in a multitude of non-diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na](+) ionization by addition of sodium acetate, and best signal-to-noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI-TOF/TOF-MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono- and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all-trans esterified esters found in LC were identified with MALDI-TOF/TOF-MS, with the exception of two minor monoesters.

  20. 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

  1. Optimized conditions for a quantitative SELDI TOF MS protein assay.

    PubMed

    Lomas, Lee; Clarke, Charlotte H; Thulasiraman, Vanitha; Fung, Eric

    2012-01-01

    The development of peptide/protein analyte assays for the purpose of diagnostic tests is driven by multiple factors, including sample availability, required throughput, and quantitative reproducibility. Laser Desorption/ionization mass spectrometry methods (LDI-MS) are particularly well suited for both peptide and protein characterization, and combining chromatographic surfaces directly onto the MS probe in the form of surface enhanced laser desorption/ionization (SELDI)-biochips has improved the reproducibility of analyte detection and provided effective relative quantitation. Here, we provide methods for developing reproducible SELDI-based assays by providing a complex artificial protein matrix background within the sample to be analyzed that allows for a common and reproducible ionization background as well as internal normalization standards. Using this approach, quantitative assays can be developed with CVs typically less than 10% across assays and days. Although the method has been extensively and successfully implemented in association with a protein matrix from E. coli, any other source for the complex protein matrix can be considered as long as it adheres to a set of conditions including the following: (1) the protein matrix must not provide interferences with the analyte to be detected, (2) the protein matrix must be sufficiently complex such that a majority of ion current generated from the desorption of the sample comes from the complex protein matrix, and (3) specific and well-resolved protein matrix peaks must be present within the mass range of the analyte of interest for appropriate normalization.

  2. Species identification of Streptococcus bovis group isolates causing bacteremia: a comparison of two MALDI-TOF MS systems.

    PubMed

    Agergaard, Charlotte N; Knudsen, Elisa; Dargis, Rimtas; Nielsen, Xiaohui C; Christensen, Jens J; Justesen, Ulrik S

    2017-02-20

    This study compared two MALDI-TOF MS systems (Biotyper and VITEK MS) on clinical Streptococcus bovis group isolates (n=66). The VITEK MS gave fewer misidentifications and a higher rate of correct identifications than the Biotyper. Only the identification of S. lutetiensis by the VITEK MS was reliable. Additional optimization of the available system databases is needed.

  3. Structural Characterization of Ginsenosides from Flower Buds of Panax ginseng by RRLC-Q-TOF MS.

    PubMed

    Wu, Wei; Lu, Ziyan; Teng, Yaran; Guo, Yingying; Liu, Shuying

    2016-02-01

    Ginseng flower bud as a part of Panax ginseng has received much attention as a valuable functional food with medicinal potential. A few studies focused on systematic and comprehensive studies on its major ingredients. This study aims to rapidly characterize ginsenosides in ginseng flower buds and provide scientific basis for developing functional food, exploiting pharmaceutical effects and making full use of ginseng resources. A rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) method was developed for rapid qualitative and quantitative analysis of ginsenosides in ginseng flower buds. The compounds were identified by comparing retention time of the reference standards, accurate mass measurement and the fragment ions obtained from RRLC-Q-TOF-MS/MS analyses. A total of 14 kinds of ginsenosides were identified and 5 kinds of malonyl-ginsenosides were first tentatively identified in ginseng flower buds. Ten kinds of main ginsenosides were quantitatively analyzed. The developed RRLC-Q-TOF-MS method was demonstrated as an effective analytical means for rapid characterization of the ginsenosides in flower buds of P. ginseng. The research result is valuable for quality control, assessment of authenticity and stability evaluation of ginseng flower buds.

  4. 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.

  5. 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.

  6. Detection of in utero marijuana exposure by GC-MS, ultra-sensitive ELISA and LC-TOF-MS using umbilical cord tissue.

    PubMed

    Chittamma, A; Marin, S J; Williams, J A; Clark, C; McMillin, G A

    2013-09-01

    Smoking marijuana during pregnancy can cause health problems in the neonate. The detection of exposure can guide treatment to meet the short- and long-term medical and social needs. Umbilical cord tissue was analyzed for 11-nor-delta-9-carboxy-tetrahydrocannabinol (THC-COOH) by gas chromatography-mass spectrometry (GC-MS), and compared with ultra-sensitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS). Fortified extracts of drug-free cord tissue were used to determine the sensitivity and specificity of the LC-TOF-MS and ELISA assays, and 16 de-identified patient specimens previously analyzed by GC-MS were tested for THC-COOH by both methods. The cutoffs were 0.050 ng/g for the GC-MS assay, 0.1 ng/g for the ELISA assay and 1 ng/g for the LC-TOF-MS assay. Twelve specimens were negative by all three methods. Seven specimens were positive by GC-MS with concentrations from 0.066 to 6.095 ng/g. ELISA and LC-TOF-MS did not detect one specimen that was positive by GC-MS. LC-TOF-MS missed one specimen that was detected by GC-MS and ELISA. Five positive specimens were detected by all three methods. These results were consistent with the cutoff for each method. No false positives were detected by LC-TOF-MS or ELISA. Umbilical cord tissue is a viable specimen for the detection of in utero marijuana exposure. ELISA and GC-MS were more sensitive than LC-TOF-MS for the detection of THC-COOH in cord tissue, with the GC-MS method providing superior sensitivity.

  7. 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.

  8. A generic screening methodology for horse doping control by LC-TOF-MS, GC-HRMS and GC-MS.

    PubMed

    Kioussi, Maroula K; Lyris, Emmanouil M; Angelis, Yiannis S; Tsivou, Maria; Koupparis, Michael A; Georgakopoulos, Costas G

    2013-12-15

    In the present study a general screening protocol was developed to detect prohibited substances and metabolites for doping control purposes in equine sports. It was based on the establishment of a unified sample preparation and on the combined implementation of liquid and gas chromatographic MS analysis. The sample pretreatment began with two parallel procedures: enzymatic hydrolysis of sulfate and glucuronide conjugates, and methanolysis of the 17β-sulfate steroid conjugates. The extracts were treated for LC-TOF-MS, GC-HRMS and GC-MS assays. The majority of the prohibited substances were identified through a high mass accuracy technique, such as LC-TOF-MS, without prior derivatization. The sample preparation procedure included the formation of methylated and trimethylsilylated derivatives common in toxicological GC-MS libraries. The screening method was enhanced by post-run library searching using automated mass spectral deconvolution and identification system (AMDIS) combined with deconvolution reporting software (DRS). The current methodology is able to detect the presence of more than 350 target analytes in horse urine and may easily incorporate a lot of new substances without changes in chromatography. The full scan acquisition allows retrospective identification of prohibited substances in stored urine samples after reprocessing of the acquired data. Validation was performed for sixty representative compounds and included limit of detection, matrix interference - specificity, extraction recovery, precision, mass accuracy, matrix effect and carry over contamination. The suitability of the method was demonstrated with previously declared positive horse urine samples.

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

    PubMed

    Lauer, Franziska; Seifert, Stephan; Kneipp, Janina; Weidner, Steffen M

    2017-03-03

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

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

    PubMed Central

    Lauer, Franziska; Seifert, Stephan; Kneipp, Janina; Weidner, Steffen M.

    2017-01-01

    Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a well-implemented analytical technique for the investigation of complex biological samples. In MS, the sample preparation strategy is decisive for the success of the measurements. Here, sample preparation processes and target materials for the investigation of different pollen grains are compared. A reduced and optimized sample preparation process prior to MALDI-TOF measurement is presented using conductive carbon tape as target. The application of conductive tape yields in enhanced absolute signal intensities and mass spectral pattern information, which leads to a clear separation in subsequent pattern analysis. The results will be used to improve the taxonomic differentiation and identification, and might be useful for the development of a simple routine method to identify pollen based on mass spectrometry. PMID:28273807

  11. Surface preparation strategies for improved parallelization and reproducible MALDI-TOF MS ligand binding assays.

    PubMed

    Roth, Michael J; Maresh, Erica M; Plymire, Daniel A; Zhang, Junmei; Corbett, John R; Robbins, Roger; Patrie, Steven M

    2013-01-01

    Immunoassays are employed in academia and the healthcare and biotech industries for high-throughput, quantitative screens of biomolecules. We have developed monolayer-based immunoassays for MALDI-TOF MS. To improve parallelization, we adapted the workflow to photolithography-generated arrays. Our work shows Parylene-C coatings provide excellent "solvent pinning" for reagents and biofluids, enabling sensitive MS detection of immobilized components. With a unique MALDI-matrix crystallization technique we show routine interassay RSD <10% at picomolar concentrations and highlight platform compatibility for relative and label-free quantitation applications. Parylene-arrays provide high sample densities and promise screening throughputs exceeding 1000 samples/h with modern liquid-handlers and MALDI-TOF systems.

  12. Alternating current-assisted on-plate proteolysis for MALDI-TOF MS peptide mapping.

    PubMed

    Wang, Sheng; Wei, Bangguo; Yang, Pengyuan; Chen, Gang

    2008-11-01

    In this report, alternating current-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of a stainless steel MALDI plate with the assistance of low-voltage alternating current electricity. Alternating current (AC) was allowed to pass through the protein solutions via the MALDI plate and a platinum disc electrode. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of BSA and cytochrome c (Cyt-c). It was demonstrated that AC substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI-TOF MS with sequence coverages of 42% (BSA) and 77% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The present proteolysis strategy is simple and efficient, offering great promise for MALDI-TOF MS peptide mapping.

  13. Performance of a MALDI-TOF MS-based imipenem hydrolysis assay incorporating zinc sulfate.

    PubMed

    Knox, James; Palombo, Enzo

    2017-03-01

    A MALDI-TOF MS(1)-based imipenem hydrolysis assay was modified by adding ZnSO4. This improved detection of metallo-β-lactamase producing strains without compromising detection of other carbapenemase types. Using 129 genetically characterized Gram-negative bacilli, the sensitivity and specificity were 98.5% (95% confidence interval [CI]: 91.9-99.7%) and 100% (95% CI: 94.3-100%), respectively.

  14. 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.

  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.

  16. MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages.

    PubMed

    Wolters, Manuel; Rohde, Holger; Maier, Thomas; Belmar-Campos, Cristina; Franke, Gefion; Scherpe, Stefanie; Aepfelbacher, Martin; Christner, Martin

    2011-01-01

    Early detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and initiation of adequate infection control measures are important objectives in hospital hygiene. To reach these goals, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods like pulsed-field gel electrophoresis (PFGE), spa typing, or multilocus sequence typing (MLST) have a high discriminatory power, however, these methods are time consuming and cost intensive. The aim of this study was to investigate the potential of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for discrimination of major MRSA lineages. By analysis of mass spectra from 25 representative MRSA isolates belonging to the 5 major hospital-acquired (HA) MRSA clonal complexes (CC5, CC8, CC22, CC30, CC45; deduced from spa typing), reproducible spectrum differences were observed at 13 characteristic m/z values allowing robust discrimination of the clonal complexes. When 60 independent clinical MRSA isolates were tested for the presence or absence of the 13 characteristic MALDI-TOF MS peaks, 15 different profiles (MALDI types) could be detected. Hierarchical clustering of the MALDI types showed high concordance with the clonal complexes. Our results suggest that MALDI-TOF MS has the potential to become a valuable first-line tool for inexpensive and rapid typing of MRSA in infection control.

  17. Potential of MALDI-TOF MS as an alternative approach for capsular typing Streptococcus pneumoniae isolates

    PubMed Central

    Pinto, Tatiana C. A.; Costa, Natalia S.; Castro, Luciana F. S.; Ribeiro, Rachel L.; Botelho, Ana Caroline N.; Neves, Felipe P. G.; Peralta, Jose Mauro; Teixeira, Lucia M.

    2017-01-01

    Streptococcus pneumoniae can be classified in more than 90 capsular types, as traditionally determined by serological methods and more recently by PCR-based techniques. Such methods, however, can be expensive, laborious or unable to accurately discriminate among certain serotypes. Therefore, determination of capsular types, although extremely important for epidemiological purposes and for estimating the impact of pneumococcal conjugate vaccines, is mainly restricted to research laboratories, being rarely performed in the clinical setting. In the present study, MALDI-TOF MS was evaluated as an alternative tool to characterize 416 pneumococcal isolates belonging to serotypes 6A, 6B, 6C, 9N, 9V or 14. For MALDI-TOF MS analysis, each isolate was submitted to an extraction protocol using formic acid and acetonitrile. Measurements were performed with a Bruker Microflex LT mass spectrometer using default parameters and generating spectra in the range of 2,000–20,000 m/z. Spectra were analyzed with the BioNumerics software v7.6. Isolates were mainly distributed according to the capsular type in a Neighbor Joining tree and serotypes investigated were successfully discriminated by the presence/absence of 14 selected biomarkers. The results suggest that MALDI-TOF MS is a promising alternative for typing pneumococcal strains, highlighting its usefulness for rapid and cost-effective routine application in clinical laboratories. PMID:28349999

  18. Characterization of Dickeya and Pectobacterium species by capillary electrophoretic techniques and MALDI-TOF MS.

    PubMed

    Šalplachta, Jiří; Kubesová, Anna; Horký, Jaroslav; Matoušková, Hana; Tesařová, Marie; Horká, Marie

    2015-10-01

    Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.

  19. 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

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

    PubMed

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

    2009-12-01

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

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

    PubMed Central

    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

  2. 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.

  3. Applications of LC/ESI-MS/MS and UHPLC QqTOF MS for the determination of 148 pesticides in fruits and vegetables.

    PubMed

    Wang, Jian; Chow, Willis; Leung, Daniel

    2010-02-01

    This paper presented the applications of liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and ultra-high-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC QqTOF MS) for the determination of 148 pesticides in fruits and vegetables. Pesticides were extracted from fruits and vegetables using a buffered QuEChERS method. Quantification was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analog as internal standards in an analytical range from 5 to 500 microg/kg. The method performance parameters including overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a statistically designed experiment, i.e., a nested design. For LC/ESI-MS/MS, 95% of the pesticides had recoveries between 81% and 110%; 97% had an intermediate precision < or = 20%; and 95% (in fruits) or 93% (in vegetables) showed measurement uncertainty < or = 40%. Compared to LC/ESI-MS/MS, UHPLC QqTOF MS showed a relatively poor repeatability and large measurement uncertainty. About 93% (in fruits) or 94% (in vegetables) of the pesticides had recoveries between 81% and 110%; 86% (in fruits) or 90% (in vegetables) had an intermediate precision < or = 20%; and 79% (in fruits) or 88% (in vegetables) showed measurement uncertainty < or = 40%. LC/ESI-MS/MS proved to be the first choice for quantification or pre-target analysis due to its superior sensitivity and good repeatability. UHPLC QqTOF MS provided accurate mass measurement and isotopic patterns, and was an ideal tool for post-target screening and confirmation.

  4. 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.

  5. Species identification within Acinetobacter calcoaceticus-baumannii complex using MALDI-TOF MS.

    PubMed

    Toh, Benjamin E W; Paterson, David L; Kamolvit, Witchuda; Zowawi, Hosam; Kvaskoff, David; Sidjabat, Hanna; Wailan, Alexander; Peleg, Anton Y; Huber, Charlotte A

    2015-11-01

    Acinetobacter baumannii, one of the more clinically relevant species in the Acinetobacter genus is well known to be multi-drug resistant and associated with bacteremia, urinary tract infection, pneumonia, wound infection and meningitis. However, it cannot be differentiated from closely related species such as Acinetobacter calcoaceticus, Acinetobacter pittii and Acinetobacter nosocomialis by most phenotypic tests and can only be differentiated by specific, time consuming genotypic tests with very limited use in clinical microbiological laboratories. As a result, these species are grouped into the A. calcoaceticus-A. baumannii (Acb) complex. Herein we investigated the mass spectra of 73 Acinetobacter spp., representing ten different species, using an AB SCIEX 5800 MALDI-TOF MS to differentiate members of the Acinetobacter genus, including the species of the Acb complex. RpoB gene sequencing, 16S rRNA sequencing, and gyrB multiplex PCR were also evaluated as orthogonal methods to identify the organisms used in this study. We found that whilst 16S rRNA and rpoB gene sequencing could not differentiate A. pittii or A. calcoaceticus, they can be differentiated using gyrB multiplex PCR and MALDI-TOF MS. All ten Acinetobacter species investigated could be differentiated by their MALDI-TOF mass spectra.

  6. MALDI-TOF MS analysis of lipids from cells, tissues and body fluids.

    PubMed

    Fuchs, Beate; Schiller, Jürgen

    2008-01-01

    Many diseases as atherosclerosis and metabolic dysfunctions are known to correlate with changes of the lipid profile of tissues and body fluids. Therefore, the importance of reliable methods of lipid analysis is obvious. Although matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was so far primarily used for protein analysis, this method has itself proven to be very useful in lipid analysis, too. This review provides an overview of applications of MALDI-TOF MS in lipid analysis and summarizes the specific advantages and drawbacks of this modern soft-ionization method. The focus will be on the analysis of body fluids and cells as well as the diagnostic potential of the method in the lipid field. It will be shown that MALDI-TOF mass spectra can be recorded in a very short time and provide important information on the lipid as well as the fatty acyl composition of the lipids of an unknown sample. However, it will also be shown that only selected lipid classes (in particular those with quaternary ammonia groups as phosphatidylcholine) are detected if crude mixtures are analyzed as they are more sensitively detectable than other ones. This review ends with a short outlook emphasizing current methodological developments.

  7. 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.

  8. 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.

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

    PubMed

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

    2016-11-01

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

  10. Analysis of stem cell lipids by offline HPTLC-MALDI-TOF MS.

    PubMed

    Fuchs, Beate; Schiller, Jürgen; Süss, Rosmarie; Zscharnack, Matthias; Bader, Augustinus; Müller, Peter; Schürenberg, Martin; Becker, Michael; Suckau, Detlev

    2008-11-01

    MALDI-TOF MS is traditionally used for "proteomics", but is also a useful tool for lipid analysis. Depending on the applied matrix, however, some lipid classes are more sensitively detected than other ones and this may even lead to suppression effects if complex mixtures are analyzed. Therefore, a previous separation into the individual lipid classes is necessary. Using artificial lipid mixtures or easily available tissue extracts, it has been already shown that HPTLC-(High Performance Thin-Layer Chromatography)-separated lipids can be conveniently analyzed by MALDI-TOF MS directly on the TLC plate. Here we present an initial TLC-MALDI study of the lipid composition of ovine mesenchymal stem cells. Due to the complex composition of these cells, data are also compared to lipids extracted from human erythrocytes. It will be shown that even very minor lipid classes can be easily detected and with much higher sensitivity than by common staining protocols. Additionally, MS images of the developed TLC plates will be shown and potential applications, new methods of data analysis as well as problems discussed.

  11. GEMMA and MALDI-TOF MS of reactive PEGs for pharmaceutical applications.

    PubMed

    Kemptner, Jasmin; Marchetti-Deschmann, Martina; Siekmann, Juergen; Turecek, Peter L; Schwarz, Hans Peter; Allmaier, Günter

    2010-08-01

    One of the most prominent polymer group applied for drug conjugation is poly(ethylene) glycol (PEG). Since drug production is subjected to strict restrictions on the part of the FDA and EMEA, also PEG has to be characterized accurately. Particularly its molecular mass distribution (MMD) and polydispersity can result in unrequested inhomogeneous final products. Therefore evaluation of PEG before applying it to drug conjugation is essential. In this study a new analytical method for size and molecular mass determination based on electrophoretic mobility called GEMMA is used to characterize linear PEGs with two differing terminating functional groups. To confirm the data acquired by GEMMA a second, well-established method for molecular weight determination, MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry), was applied. Utilizing these two analytical approaches four monomethoxylated PEG-succinimidyl succinate (mPEG-SS) derivatives were investigated in terms of polydispersity and MMD. Although based on differing principles, both analytical methods yield comparable results. All obtained MMD maxima for the mPEG-SS batches lie within the company stated specifications, MMD+/-10% (based on MALDI-TOF MS data). For mPEG-SS 2K a polydispersity of 1.02 and for mPEG-SS 5K, 10K and 20K a polydispersity of 1.01 were determined from GEMMA as well as from MALDI-TOF MS data and are in agreement with the company's data (based on GPC data), namely 1.05-1.10.

  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.

  13. Glycoprotein analysis by delayed extraction and post-source decay MALDI-TOF-MS

    NASA Astrophysics Data System (ADS)

    Tsarbopoulos, A.; Bahr, U.; Pramanik, B. N.; Karas, M.

    1997-12-01

    Matrix-assisted laser desorption-ionization (MALDI)-MS analysis of glycoprotein samples is usually affected by metastable fragmentation, particularly in reflectron instruments. Use of delayed extraction (DE) is shown to minimize the observed metastable fragmentation. In the case of the CHO IL-4, which contains complex-type N-linked glycans, the disialylated component gives the most abundant MALDI signal with a dramatically improved resolution and mass accuracy over the non-DE linear TOF analysis. This increased resolution is advantageous for the 30 kDa Sf9-derived IL-4 receptor, where it is possible to differentiate the individual glycans, but not for higher mass and more heterogeneous glycoproteins. It is also shown that MALDI analysis of proteins with labile functional groups is more superior and reliable with linear DE-TOF systems rather than reflectron TOF analyzers. Further structural information on the sequence and disulfide mapping, as well as glycopeptide identification can be obtained by post-source decay (PSD) analysis of peptide and glycopeptide isolated fractions. The PSD formation of a characteristic triplet of ions separated by 33 Da in mass can be used to identify disulfide-paired peptides, even from complex digest mixtures of proteins.

  14. Top-Down Proteomic Identification of Furin-Cleaved α-Subunit of Shiga Toxin 2 from Escherichia coli O157:H7 Using MALDI-TOF-TOF-MS/MS

    PubMed Central

    Fagerquist, Clifton K.; Sultan, Omar

    2010-01-01

    A method has been developed to identify the α-subunit of Shiga toxin 2 (α-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 developed in-house. Expression of Stx2 was induced by culturing E. coli O157:H7 on solid agar supplemented with an antibiotic that elicits the bacterial SOS-response. Bacterial cell lysates were incubated in the presence of furin, a human enzyme, that cleaves α-Stx2 into A1 (~28 kDa) and A2 (~5 kDa) protein fragments. A subsequent disulfide reduction step unlinked A1 from A2. MALDI-TOF-MS of the furin-digested/disulfide-reduced sample showed a peak at mass-to-charge (m/z) 5286 that corresponded to the A2 fragment. No peak was observed that corresponded to the A1 fragment although its presence was confirmed by bottom-up proteomics. The peak at m/z 5286 was definitively identified by MALDI-TOF-TOF-MS/MS and top-down proteomics as the A2 fragment of α-Stx2. PMID:21331368

  15. "DUST BUSTER" - A Single Photon Ionization TOF MS for Cometary Dusts

    NASA Technical Reports Server (NTRS)

    Chen, C.-Y.; Calaway, W. F.; Lee, Typhoon; Moore, J. F.; Pellin, M. J.; Veryovkin, I. V.

    2003-01-01

    It is hard to predict the properties and composition of dust that will be returned by STARDUST from WED- 2. The most interesting but challenging case would be grains, pg to fg in weight, each carrying its own isotopic signature characteristic of its source zones in a variety of stars. How do we extract the maximum amount of science from such grains? Clearly, the best that can be accomplished is to measure every atom in each grain.Academia Sinica and Argonne National Laboratory (ANL) have entered into a collaboration to develop a SPI TOF MS instrument for analysis of stardust grains. A new instrument will be built at Academia Sinica based on the new TOF mass spectrometer design developed, built and operating at ANL. The instrument is intended for SPI TOF MS analysis of elements from Ca to Cu plus Li after first using SIMS to measure H, C, N, 0, Si, and S. There are still technical challenges facing the technique. We will need to improve submicrometer sample handling, avoid the effects of space charge, and increase the Mamie range of the detector. The most difficult obstacle to overcome may be the fact that the flux density of present high repetition rate, WV lasers is below the level needed to ensure full ionization (saturation) in the source region, which must be several mm in size to achieve the high useful yield needed for analysis of small stardust grains. A potential breakthrough effort is to exploit the novel free electron laser being pioneered at ANL. In principle, this FEL can reach ionization saturation and is tunable up to photon energies of 25 eV, which is higher than the ionization potential of any element.

  16. Identification and typing of free-living Acanthamoeba spp. by MALDI-TOF MS Biotyper.

    PubMed

    Del Chierico, Federica; Di Cave, David; Accardi, Cristel; Santoro, Maristella; Masotti, Andrea; D'Alfonso, Rossella; Berrilli, Federica; Urbani, Andrea; Putignani, Lorenza

    2016-11-01

    Over the years, the potential pathogenicity of Acanthamoeba for humans and animals has gained increasing attention from the scientific community. More than 24 species belong to this genus, however only some of them are causative agents of keratitis and encephalitis in humans. Due to technical difficulties in diagnosis, these infections are likely to be under-detected. The introduction of 18S rDNA amplification for the identification of Acanthamoeba has dramatically enhanced diagnosis performances, but the attestation of genotyping requires supplementary sequencing-based procedures. In this study, 15 Acanthamoeba strains were collected and grown on nutrient agar media. Each strain was genotyped by end-point PCR assay for the amplification of the 18S rDNA gene and the genotype was assigned by sequencing analysis through neighbor joining phylogenetic tree. In order to optimize standardization of the MALDI-TOF MS assay, we established the collection time point at the cystic phase. Two strains of each genotype were randomly chosen to customize the biotyper database. For all strains, 24 spectral measurements were acquired and submitted to identification and cluster analysis of spectra. The obtained results highlighted the correct identification of Acanthamoeba strains and the overlapping of spectra dendrogram clusters to the 18S genotype assignations. In conclusion, the MALDI-TOF MS Biotyper revealed the capability to identify and genotype the Acanthamoeba strains, providing a new frontier in the diagnostic identification of amaebae and in taxonomic and phylogenetic studies.

  17. Identification of Borrelia species after creation of an in-house MALDI-TOF MS database.

    PubMed

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

    2014-01-01

    Lyme borreliosis (LB) is a multisystemic disease caused by Borrelia burgdorferi sensu lato (sl) complex transmitted to humans by Ixodes ticks. B. burgdorferi sl complex, currently comprising at least 19 genospecies, includes the main pathogenic species responsible for human disease in Europe: B. burgdorferi sensu stricto (ss), B. afzelii, and B. garinii. In this study, for the first time, MALDI-TOF MS was applied to Borrelia spp., supplementing the existing database, limited to the species B. burgdorferi ss, B . spielmanii and B. garinii, with the species B. afzelii, in order to enable the identification of all the species potentially implicated in LB in Europe. Moreover, we supplemented the database also with B. hermsii, which is the primary cause of tick-borne relapsing fever in western North America, B. japonica, circulating in Asia, and another reference strain of B. burgdorferi ss (B31 strain). The dendrogram obtained by analyzing the protein profiles of the different Borrelia species reflected Borrelia taxonomy, showing that all the species included in the Borrelia sl complex clustered in a unique branch, while Borrelia hermsii clustered separately. In conclusion, in this study MALDI-TOF MS proved a useful tool suitable for identification of Borrelia spp. both for diagnostic purpose and epidemiological surveillance.

  18. Hybrid Ion-Detector/Data-Acquisition System for a TOF-MS

    NASA Technical Reports Server (NTRS)

    Burton, William D., Jr.; Schultz, J. Albert; Vaughn, Valentine; McCully, Michael; Ulrich, Steven; Egan, Thomas F.

    2006-01-01

    A modified ion-detector/data-acquisition system has been devised to increase the dynamic range of a time-of-flight mass spectrometer (TOF-MS) that, previously, included a microchannel-plate detector and a data-acquisition system based on counting pulses and time-tagging them by use of a time-to-digital converter (TDC). The dynamic range of the TOF-MS was limited by saturation of the microchannel plate detector, which can handle no more than a few million counts per second. The modified system includes (1) a combined microchannel plate/discrete ion multiplier and (2) a hybrid data-acquisition system that simultaneously performs analog current or voltage measurements and multianode single-ion-pulse-counting time-of-flight measurements to extend the dynamic range of a TDC into the regime in which a mass peak comprises multiple ions arriving simultaneously at the detector. The multianode data are used to determine, in real time, whether the detector is saturated. When saturation is detected, the data-acquisition system selectively enables circuitry that simultaneously determines the ion-peak intensity by measuring the time profile of the analog current or voltage detector-output signal.

  19. 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.

  20. Enhanced detection of in-gel released N-glycans by MALDI-TOF-MS.

    PubMed

    Weiz, Stefan; Kamalakumar, Aryaline; Biskup, Karina; Blanchard, Véronique

    2015-05-01

    Many biologically relevant glycoproteins need to be separated on 1D- or 2D-gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in-gel digestions with MALDI-TOF-MS. In this technical report, we investigated how the detection of in-gel released N-glycans could be improved by MALDI-TOF-MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D-arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl-β-glucopyranoside, a nonionic detergent, was studied during in-gel peptide-N(4) -(acetyl-ß-glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl-β-glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N-glycans. A LOD of under 7 pmol glycoprotein could be achieved.

  1. Combinatorial approach of LC-MS/MS and LC-TOF-MS for uncovering in vivo kinetics and biotransformation of ochratoxin A in rat.

    PubMed

    Han, Zheng; Zhao, Zhiyong; Shi, Jianxin; Liao, Yucai; Zhao, Zhihui; Zhang, Dabing; Wu, Yongning; De Saeger, Sarah; Wu, Aibo

    2013-04-15

    A combinatorial platform of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography coupled with time of flight mass spectrometry (LC-TOF-MS) has been developed to investigate the in vivo kinetics and biotransformation of ochratoxin A (OTA) in rats. The stable isotope dilution LC-MS/MS method was first validated by determining the linearity (R(2)≥0.9990), sensitivity (lower limit of quantitation of 0.05 ng mL(-1)), accuracy (83.3-108.3), precision (RSD≤15.6%) and stability (≥75.0%), and was approved for the determination OTA in plasma, heart, liver, spleen, lung, kidney and brain with a run time of 7.0 min. Simultaneously, an LC-TOF-MS method could unambiguously identify the metabolites of OTA in a total run time of 14 min. The subsequent studies on kinetics and distribution after oral administration of 0.2 mg/kg b.w. OTA in rat indicated that OTA could reach a maximum value of 1932.4±124.9 ng mL(-1) within 5h due to its fast absorption, and then was slowly eliminated in plasma with a half-life time (t1/2) of 75.6±29.0 h. Results of tissue accumulation after a daily oral administration of 0.1 mg/kg b.w. OTA during 20 days showed that the highest concentration of OTA was observed in lung (95.9±13.7 ng g(-1)), followed by liver (76.0±9.7 ng g(-1)), heart (62.0±4.2 ng g(-1)) and kidney (55.7±4.7 ng g(-1)). Furthermore, three less toxic metabolites of OTA were clearly identified: Ochratoxin β (OTβ) and ochratoxin B (OTB) methyl ester were found in kidney and spleen, respectively, while phenylalanine was detected in heart and kidney. Thus, a possible metabolic pathway of OTA was proposed. The above achieved results justified that the application of combinatorial LC-MS/MS and LC-TOF-MS methods are valuable tools to uncover the kinetics and metabolism of OTA for the interpretation of toxicological findings in animals and extrapolation of the resulting data as reference to humans.

  2. 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...

  3. Antihepatotoxic Effect and Metabolite Profiling of Panicum turgidum Extract via UPLC-qTOF-MS

    PubMed Central

    Farag, Mohamed A.; El Fishawy, Ahlam M.; El-Toumy, Sayed A.; Amer, Khadiga F.; Mansour, Ahmed M.; Taha, Hala E.

    2016-01-01

    Background: Panicum turgidum, desert grass, has not reported any detailed phytochemical or biological study as yet Objective: To establish P. turgidum secondary metabolite profile and to assess its antihepatotoxic effect Materials and Methods: Ultra-performance liquid chromatography (UPLC) coupled to quadrupole high-resolution time of flight mass spectrometry (qTOF-MS) was used for large-scale secondary metabolites profiling in P. turgidum extract, alongside assessing median lethal dose (LD50) and hepatoprotective effect against carbon tetrachloride (CCl4) intoxication Results: A total of 39 metabolites were identified with flavonoids as the major class present as O/C-glycosides of luteolin, apigenin, isorhamnetin and naringenin, most of which are first time to be reported in Panicum sp. Antihepatotoxic effect of P. turgidum crude extract was revealed via improving several biochemical marker levels and mitigation against oxidative stress in the serum and liver tissues, compared with CCl4 intoxicated group and further confirmed by histopathological examination. Conclusion: This study reveals that P. turgidum, enriched in C-flavonoids, presents a novel source of safe antihepatotoxic agents and further demonstrates the efficacy of UPLC-MS metabolomics in the field of natural products drug discovery. SUMMARY UPLC coupled to qTOF-MS was used for large scale secondary metabolites profiling in P. turgidum.A total of 39 metabolites were identified with flavonoids amounting as the major metabolite class.Anti-hepatotoxic effect of P. turgidum extract was revealed via several biochemical markers and histopathological examination.This study reveals that P. turgidum, enriched in C-flavonoids, present a novel source of antihepatotoxic agents. Abbreviations used: UPLC: Ultra-performance liquid chromatography (UPLC), LD50: median lethal dose, MDA: malondialdehyde, GSH: glutathione reductase, CAT: catalase, SOD: superoxide dismutase, ALT: alanine aminotransferase, AST: aspartate

  4. Stain efficiency and MALDI-TOF MS compatibility of seven visible staining procedures.

    PubMed

    Lin, Jian-feng; Chen, Qing-xi; Tian, Hong-yu; Gao, Xia; Yu, Mei-lan; Xu, Gen-jun; Zhao, Fu-kun

    2008-04-01

    Visible stain is still the most popular protein staining method used in proteomic approaches. However, most published data have been derived from comparisons between visible dyes and fluorescent dyes. In this work, we have focused on seven widely used visible staining procedures--Neuhoff CCB, blue silver, and five silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN)--and studied their stain efficiencies and MALDI-TOF MS compatibilities on 1-D and 2-D PAGE. It was concluded that blue silver is slightly better in terms of stain efficiency than Neuhoff CCB, but it presented worse MS compatibility. Neuhoff CCB presented better MS compatibility and superior linearity but worse sensitivity than silver stains. Among the five silvering procedures, He SN showed the best MS compatibility and a reasonable staining efficiency; Yan SN lowered the chances of obtaining the protein identity by PMF but gave the best stain efficiency; Vorum SN gave a very clear background and a great contrast, while Blum SN was the worst in this respect. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.

  5. Stability-indicating HPLC method development and structural elucidation of novel degradation products in posaconazole injection by LC-TOF/MS, LC-MS/MS and NMR.

    PubMed

    Yang, Yidi; Zhu, Xi; Zhang, Fei; Li, Wei; Wu, Ying; Ding, Li

    2016-06-05

    Stress testing was carried out under acidic, alkaline, oxidative, thermal and photolytic conditions to evaluate the intrinsic stability of posaconazole injection. A total of four degradation products were detected and the drug was found to be susceptible to oxidative and thermal degradations. Three unknown degradants formed under oxidative stress condition were isolated by preparative HPLC and unambiguously elucidated by LC-TOF/MS, LC-MS/MS, (1)H NMR, (13)C NMR and 2D NMR techniques. Based on the spectrometric and spectroscopic information, these novel degradation products were unequivocally assigned as the N-oxides of posaconazole. Probable mechanisms for the formation of the degradants were proposed. A new and selective HPLC method was developed and validated to separate, detect and quantify all the degradants in posaconazole injection.

  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. 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

  8. Differentiation of environmental aquatic bacterial isolates by MALDI-TOF MS.

    PubMed

    Popović, Natalija Topić; Kazazić, Snježana P; Strunjak-Perović, Ivančica; Čož-Rakovac, Rozelindra

    2017-01-01

    Identification of bacteria in aquatic and environmental applications, for monitoring purposes and research, for health assessments and therapy considerations of farmed and free-living aquatic organisms, still relies on conventional phenotypic and biochemical protocols. Although molecular techniques based on DNA amplification and sequencing are finding ways into diagnostic laboratories, they are time-consuming, costly and difficult in the case of multiplex assays. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid and accurate proteomic method reliable for identification of unknown bacteria to the genus and species level. Upon extension of databases, it will certainly find its position in environmental sciences. The paper presents an overview of the principle of the method, its effectiveness in comparison with conventional and molecular identification procedures, and applicability on environmental and aquatic isolates, discussing its advantages and shortcomings, as well as possible future implementations.

  9. Characterization of degradation products of idarubicin through LC-UV, MS(n) and LC-MS-TOF studies.

    PubMed

    Kaushik, Dheeraj; Bansal, Gulshan

    2013-11-01

    Idarubicin was subjected to forced degradation under the ICH recommended conditions of hydrolysis, oxidation, dry heat and photolysis to characterize its possible impurities and/or degradation products. The drug was found unstable to acid hydrolysis at 85°C and to alkaline hydrolysis, and oxidation at room temperature. The hydrolytic and oxidative degradation products were resolved with gradient and isocratic elution, respectively on an Inertsil RP18 (250 mm × 4.6mm; 5 μ) column with HCOONH4 (20mM, pH 3.0) and acetonitrile. The drug degraded to two products (O-I and O-II) in oxidative condition, two products (K-I and K-II) in alkaline hydrolytic, and one product (A-I) in acidic hydrolytic conditions. The purity of each in the LC-UV chromatogram was ascertained through LC-PDA analysis. The products were characterized through +ESI-MS(n) studies on the drug and LC-MS-TOF studies on the degraded drug solutions. Based on the multistage mass fragmentation pattern of idarubicin and accurate mass analysis of the degradation products, the O-I, O-II and A-I were characterized as desacetylidarubicin hydroperoxide, desacetylidarubicin and deglucosaminylidarubicin, respectively. The products K-I and K-II were not characterized due to their low concentrations and/or extremely weak ionization. The mechanisms of degradation of idarubicin to these products were proposed and discussed.

  10. Metabolomic analysis of serum from obese adults with hyperlipemia by UHPLC-Q-TOF MS/MS.

    PubMed

    Wang, Yang; Liu, Desheng; Li, Yue; Guo, Lei; Cui, Yinghua; Zhang, Xin; Li, Enyou

    2016-01-01

    The prevalence of obesity has dramatically increased and poses a major threat to human health. Obesity often accompanies hyperlipemia, which is strongly related to the occurrence and development of obesity-related chronic diseases. Differences in metabolomic profiling of serum between obese (with hyperlipemia) and normal-weight men (n = 30 in each group) were investigated using ultrahigh-pressure liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF MS/MS) and partial least-squares-discriminant analysis (PLS-DA). Obese men showed higher levels of weight, body mass index, fat mass, systolic blood pressure, fasting plasma glucose, triglyeride, total cholesterol, insulin, HOMA-IR and high-sensitivity CRP. Obese and normal-weight groups were clearly discriminated from each other on a PLS-DA score plot and nine major metabolites contributing to the discrimination were assigned, including increased 2-octenoylcarnitine, eicosadienoic acid, 12-hydroperoxyeicosatetraenoic acid, 4-hydroxyestrone sulfate, lysoPE[18:1(11Z)/0:0], thromboxane B2 and pyridinoline and decreased vitamin D3 glucosiduronate and 9,10-DHOME. These metabolites were associated with lipid metabolism and obesity-related diseases, and reflected the metabolic differences between normal and obese men, which may be important for future clinical diagnosis, treatment and assessment of the therapeutic effect on obesity-related chronic disease.

  11. Highly selective biomarkers for pesticides developed in Eisenia fetida using SELDI-TOF MS.

    PubMed

    Park, Doo-San; Jeon, Hwang-Ju; Park, Eun-Sil; Bae, In Kyung; Kim, Yong-Eun; Lee, Sung-Eun

    2015-03-01

    The repeated use of pesticides, and their subsequent residues, has contributed to severe adverse effects on the environment, including risks to human health. Therefore, it is important to assess the quality of the environment to ensure it remains free from pesticide residues. The six pesticides tested in this study showed high mortality on Eisenia fetida with LC50 values ranging from 7.7 to 37.9 g L(-1). The strongest lethal effect resulted from the organochlorine insecticide endosulfan (LC50=7.7 g L(-1)). Following exposure to the carbamate pesticides, acetylcholinesterase activity in E. fetida decreased dramatically in comparison to the control. Carboxylesterase activity was only lowered in E. fetida exposed to propoxur, when compared to the control. The remaining five pesticides had no significant effect on carboxylesterase activity in E. fetida. In order to discover pesticide-specific biomarkers with differentially expressed proteins after exposure to pesticides, protein patterns of pesticide-treated E. fetida were analyzed using SELDI-TOF MS with Q10 ProteinChips. Protein patterns were compared with their intensities at the same mass-to-charge ratios (m/z). All 42 peaks had intensities with associated p-values less than 0.089, and 40 of these peaks had associated p-values of 0.05. Using SELDI-TOF MS technology, selective biomarkers for the six pesticides tested were found in E. fetida; four proteins with 5425, 5697, 9523, and 9868 m/z were consistently observed in the earthworms following exposure to the carbamates.

  12. Elemental, Isotopic, and Organic Analysis on Mars with Laser TOF-MS

    NASA Technical Reports Server (NTRS)

    Brinckerhoff, W. B.; Cornish, T. J.

    2000-01-01

    The in-depth landed exploration of Mars will require increasingly sophisticated robotic analytical tools for both in situ composition science [1] and reconnaissance for sample return [2]. Beyond dust, rock surfaces, and topsoil, samples must be accessed within rocks and ice, well below surface soil, and possibly in elevated deposit layers. A range of spatial scales will be studied, and for the most information-rich microscopic analyses, samples must be acquired, prepared, and positioned with high precision. In some cases samples must also be brought into a vacuum chamber. After expending such resources, it will be important to apply techniques that provide a wide range of information about the samples. Microscopy, mineralogy, and molecular/organic, elemental, and isotopic analyses are all needed, at a minimum, to begin to address the in situ goals at Mars. These techniques must work as an efficient suite to provide layers of data, each layer helping to determine if further analysis on a given sample is desired. In the spirit of broad-band and efficient data collection, we are developing miniature laser time-of-flight mass spectrometers (TOF-MS) for elemental, isotopic, and molecular/organic microanalysis of unprepared solid samples. Laser TOF-MS uses a pulsed laser to volatilize and ionize material from a small region on the sample. The laser energy and focus can be adjusted for atomic and molecular content, sampling area, and depth. Ions travel through the instrument and are detected at a sequence of times proportional to the square root of their mass-to- charge ratios. Thus, each laser pulse produces a complete mass spectrum (in less than 50 microseconds). These instruments can now be significantly miniaturized (potentially to the size of a soda can) without a loss in performance. This effort is reviewed here with an emphasis on applications to Mars exploration.

  13. MALDI TOF MS profiling of bacteria at the strain level: a review.

    PubMed

    Sandrin, Todd R; Goldstein, Jason E; Schumaker, Stephanie

    2013-01-01

    Since the advent of the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) as a tool for microbial characterization, efforts to increase the taxonomic resolution of the approach have been made. The rapidity and efficacy of the approach have suggested applications in counter-bioterrorism, prevention of food contamination, and monitoring the spread of antibiotic-resistant bacteria. Strain-level resolution has been reported with diverse bacteria, using library-based and bioinformatics-enabled approaches. Three types of characterization at the strain level have been reported: strain categorization, strain differentiation, and strain identification. Efforts to enhance the library-based approach have involved sample pre-treatment and data reduction strategies. Bioinformatics approaches have leveraged the ever-increasing amount of publicly available genomic and proteomic data to attain strain-level characterization. Bioinformatics-enabled strategies have facilitated strain characterization via intact biomarker identification, bottom-up, and top-down approaches. Rigorous quantitative and advanced statistical analyses have fostered success at the strain level with both approaches. Library-based approaches can be limited by effects of sample preparation and culture conditions on reproducibility, whereas bioinformatics-enabled approaches are typically limited to bacteria, for which genetic and/or proteomic data are available. Biological molecules other than proteins produced in strain-specific manners, including lipids and lipopeptides, might represent other avenues by which strain-level resolution might be attained. Immunological and lectin-based chemistries have shown promise to enhance sensitivity and specificity. Whereas the limits of the taxonomic resolution of MALDI TOF MS profiling of bacteria appears bacterium-specific, recent data suggest that these limits might not yet have been reached.

  14. Elemental, Isotopic, and Organic Analysis on Mars with Laser TOF-MS

    NASA Astrophysics Data System (ADS)

    Brinckerhoff, W. B.; Cornish, T. J.

    2000-07-01

    The in-depth landed exploration of Mars will require increasingly sophisticated robotic analytical tools for both in situ composition science [1] and reconnaissance for sample return [2]. Beyond dust, rock surfaces, and topsoil, samples must be accessed within rocks and ice, well below surface soil, and possibly in elevated deposit layers. A range of spatial scales will be studied, and for the most information-rich microscopic analyses, samples must be acquired, prepared, and positioned with high precision. In some cases samples must also be brought into a vacuum chamber. After expending such resources, it will be important to apply techniques that provide a wide range of information about the samples. Microscopy, mineralogy, and molecular/organic, elemental, and isotopic analyses are all needed, at a minimum, to begin to address the in situ goals at Mars. These techniques must work as an efficient suite to provide layers of data, each layer helping to determine if further analysis on a given sample is desired. In the spirit of broad-band and efficient data collection, we are developing miniature laser time-of-flight mass spectrometers (TOF-MS) for elemental, isotopic, and molecular/organic microanalysis of unprepared solid samples. Laser TOF-MS uses a pulsed laser to volatilize and ionize material from a small region on the sample. The laser energy and focus can be adjusted for atomic and molecular content, sampling area, and depth. Ions travel through the instrument and are detected at a sequence of times proportional to the square root of their mass-to- charge ratios. Thus, each laser pulse produces a complete mass spectrum (in less than 50 microseconds). These instruments can now be significantly miniaturized (potentially to the size of a soda can) without a loss in performance. This effort is reviewed here with an emphasis on applications to Mars exploration.

  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.

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

    PubMed

    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.

  17. UPLC-ESI-TOF MS-Based Metabolite Profiling of the Antioxidative Food Supplement Garcinia buchananii.

    PubMed

    Stark, Timo D; Lösch, Sofie; Wakamatsu, Junichiro; Balemba, Onesmo B; Frank, Oliver; Hofmann, Thomas

    2015-08-19

    Comparative antioxidative analyses of aqueous ethanolic extracts from leaf, root, and stem of Garcinia buchananii revealed high activity of all three organs. To investigate the metabolite composition of the different parts of G. buchananii, an untargeted metabolomics approach using UPLC-ESI-TOF MS with simultaneous acquisition of low- and high-collision energy mass spectra (MS(e)) was performed. Unsupervised statistics (PCA) highlighted clear differences in the metabolomes of the three organs. OPLS-DA revealed (2R,3S,2″R,3″R)-GB-1, (2R,3S)-morelloflavone, and (2R,3S)-volkensiflavone as the most decisive marker compounds discriminating leaf from root and stem extract. Leaves represent the best source to isolate GB-1, morelloflavone, and volkensiflavone. Root extract is the best organ to isolate xanthones and stem bark extract the best source to isolate (2R,3S,2″R,3″R)-manniflavanone; the identified polyisoprenylated benzophenones are characteristic compounds for the leaf organ. Morelloflavone, volkensiflavone, and garcicowin C were isolated for the first time from G. buchananii, identified via MS, NMR, and CD spectroscopy, and showed in H2O2 scavenging, H/L-TEAC, and H/L-ORAC assays moderate to strong in vitro antioxidative activities.

  18. Analysis of hydraulic fracturing additives by LC/Q-TOF-MS.

    PubMed

    Ferrer, Imma; Thurman, E Michael

    2015-08-01

    The chemical additives used in fracturing fluids can be used as tracers of water contamination caused by hydraulic fracturing operations. For this purpose, a complete chemical characterization is necessary using advanced analytical techniques. Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS) was used to identify chemical additives present in flowback and produced waters. Accurate mass measurements of main ions and fragments were used to characterize the major components of fracking fluids. Sodium adducts turned out to be the main molecular adduct ions detected for some additives due to oxygen-rich structures. Among the classes of chemical components analyzed by mass spectrometry include gels (guar gum), biocides (glutaraldehyde and alkyl dimethyl benzyl ammonium chloride), and surfactants (cocamidopropyl dimethylamines, cocamidopropyl hydroxysultaines, and cocamidopropyl derivatives). The capabilities of accurate mass and MS-MS fragmentation are explored for the unequivocal identification of these compounds. A special emphasis is given to the mass spectrometry elucidation approaches used to identify a major class of hydraulic fracturing compounds, surfactants.

  19. Analysis of phospholipids in microalga Nitzschia closterium by UPLC-Q-TOF-MS

    NASA Astrophysics Data System (ADS)

    Yan, Xiaojun; Li, Haiying; Xu, Jilin; Zhou, Chengxu

    2010-01-01

    Precise structural identification of phospholipids in the microalga Nitzschia closterium has been established using ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) for direct analysis of total lipid extracts. Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode. Phospholipid molecular species identification was based on the characteristic product ions and neutral loss yielded by different phospholipids under ESI-MS/MS mode. The molecular species were confirmed by the carboxylate anions produced by phospholipids in negative mode; the regiospecificity of the two acyl chains was determined from the ratio of sn-1 to sn-2 carboxylate anion abundances. As a result, 18 lipid molecular species were identified for the first time in this microalga, comprising seven phosphatidylcholines (PC), two phosphatidylethanolamines (PE), two phosphatidylinositols (PI), and seven phosphatidylglycerols (PG). Lipid standards of PC, PE, PI, and PG were added to the total lipids as internal standards for semiquantitative analysis, revealing concentrations of phospholipids in this species between 0.09 and 3.37 nmol/mg. This method can produce a full structural profile of intact phospholipid molecular species and can be used for study of the physiological and ecological functions of lipids by monitoring their individual changes over time.

  20. Qualitative and quantitative analyses of alkaloids in Uncaria species by UPLC-ESI-Q-TOF/MS.

    PubMed

    Wang, Hai-Bo; Qi, Wen; Zhang, Lin; Yuan, Dan

    2014-01-01

    An ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) method has been optimized and established for the rapid analysis of the alkaloids in 22 samples originating from five Uncaria (U.) species. The accurate mass measurement of all the protonated molecules and subsequent fragment ions offers higher quality structural information for the interpretation of fragmentation pathways of the various groups of alkaloids. A total of 19 oxindole alkaloids, 16 indole alkaloids and 1 flavone were identified by co-chromatography of the sample extract with authentic standards, comparison of the retention time, characteristic molecular ions and fragment ions, or were tentatively identified by MS/MS determination. Moreover, the method was validated for the simultaneous quantification of the 24 components within 10.5 min. The potential chemical markers were identified for classification of the U. species samples by principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA). The results demonstrate the similarity and differences in alkaloids among the five U. species, which is helpful for the standardization and quality control of the medical materials of the U. Ramulus Cum Unics (URCU). Furthermore, with multivariate statistical analysis, the determined markers are more definite and useful for chemotaxonomy of the U. genus.

  1. MALDI-TOF MS fingerprinting facilitates rapid discrimination of phylotypes I, II and III of Propionibacterium acnes.

    PubMed

    Nagy, Elisabeth; Urbán, Edit; Becker, Simone; Kostrzewa, Markus; Vörös, Andrea; Hunyadkürti, Judit; Nagy, István

    2013-04-01

    Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used today for species determination of bacteria and fungi in routine microbiological laboratories, and can also be used for subtyping of bacteria, such as Bacteroides fragilis. Propionibacterium acnes is frequently referred to as an anaerobic skin commensal of relatively low pathogenicity. In addition to its accepted pathogenic role in acne, P. acnes is now emerging as an important opportunistic pathogen in many other clinical situations, including late-stage prosthetic joint infections, osteomyelitis, endocarditis, endophthalmitis, post-neurosurgical infections and possibly prostate cancer. At the population genetic level, P. acnes can be differentiated into a number of distinct phylogroups, known as types IA1, IA2, IB, IC, II and III, which may be associated with different types of infections and clinical conditions. The aim of the present study was to evaluate MS-based typing for resolution of these genetic groups after routine identification by MALDI-TOF MS (Bruker MALDI Biotyper). The software package ClinProTools 2.2 was used to analyze the protein based mass spectra of reference strains belonging to types IA, IB, IC, II and III. Phylogroup-specific peaks and peak shifts were then identified visually. In addition, peak variations between the different types of P. acnes were investigated by using FlexAnalysis 3.3 software (Bruker). A differentiating library was created, which was used to type further 48 clinical isolates of P. acnes. Typing data obtained by MALDI-TOF MS were then compared with the results from Multilocus Sequence Typing (MLST). Most of the clinical isolates (n = 19) belonged to the type IA grouping according to MALDI-TOF MS. By MLST, all isolates were identified as type IA1. Twenty-one clinical isolates belonged to the type IB cluster based on both MALDI-TOF MS and MLST typing. Eight clinical isolates were identified as type II strains

  2. Rapid analysis of the main components of the total glycosides of Ranunculus japonicus by UPLC/Q-TOF-MS.

    PubMed

    Rui, Wen; Chen, Hongyuan; Tan, Yuzhi; Zhong, Yanmei; Feng, Yifan

    2010-05-01

    A rapid method for the analysis of the main components of the total glycosides of Ranunculus japonicus (TGOR) was developed using ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The separation analysis was performed on a Waters Acquity UPLC system and the accurate mass of molecules and their fragment ions were determined by Q-TOF MS. Twenty compounds, including lactone glycosides, flavonoid glycosides and flavonoid aglycones, were identified and tentatively deduced on the basis of their elemental compositions, MS/MS data and relevant literature. The results demonstrated that lactone glycosides and flavonoids were the main constituents of TGOR. Furthermore, an effective and rapid pattern was established allowing for the comprehensive and systematic characterization of the complex samples.

  3. 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...

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

    PubMed

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

    2016-11-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful biotyping tool increasingly used for high-throughput identification of clinical microbial isolates, however, in food fermentation research this approach is still not well established. This study examines the microbial biodiversity of cocoa bean fermentation based on the isolation of micro-organisms in cocoa-producing regions, followed by MALDI-TOF MS in Switzerland. A preceding 6-week storage test to mimic lengthy transport of microbial samples from cocoa-producing regions to Switzerland was performed with strains of Lactobacillus plantarum, Acetobacter pasteurianus and Saccharomyces cerevisiae. Weekly MALDI-TOF MS analysis was able to successfully identify microbiota to the species level after storing live cultures on slant agar at mild temperatures (7°C) and/or in 75% aqueous ethanol at differing temperatures (-20, 7 and 30°C). The efficacy of this method was confirmed by on-site recording of the microbial biodiversity in cocoa bean fermentation in Bolivia and Brazil, with a total of 1126 randomly selected isolates. MALDI-TOF MS analyses revealed known dominant cocoa bean fermentation species with Lact. plantarum and Lactobacillus fermentum in the lactic acid bacteria taxon, Hanseniaspora opuntiae and S. cerevisiae in the yeast taxon, and Acet. pasteurianus, Acetobacter fabarum, Acetobacter ghanensis and Acetobacter senegalensis in the acetic acid bacteria taxon.

  5. An advanced LC-MS (Q-TOF) technique for the detection of amino acids in atmospheric aerosols

    EPA Science Inventory

    Methodology for detection of native (underivitized) amino acids in atmospheric aerosols has been developed. This article describes the use of LC-MS (Q-TOF) and microwave-assisted gas phase hydrolysis for detection of free and combined amino acids in aerosols collected in a Southe...

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

    PubMed

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

    2014-06-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a tool for identifying clinically relevant anaerobes. We evaluated the analytical performance characteristics of the Bruker Microflex with Biotyper 3.0 software system for identification of anaerobes and examined the impact of direct formic acid (FA) treatment and other pre-analytical factors on MALDI-TOF MS performance. A collection of 101 anaerobic bacteria were evaluated, including Clostridium spp., Propionibacterium spp., Fusobacterium spp., Bacteroides spp., and other anaerobic bacterial of clinical relevance. The results of our study indicate that an on-target extraction with 100% FA improves the rate of accurate identification without introducing misidentification (P<0.05). In addition, we modify the reporting cutoffs for the Biotyper "score" yielding acceptable identification. We found that a score of ≥1.700 can maximize the rate of identification. Of interest, MALDI-TOF MS can correctly identify anaerobes grown in suboptimal conditions, such as on selective culture media and following oxygen exposure. In conclusion, we report on a number of simple and cost-effective pre- and post-analytical modifications could enhance MALDI-TOF MS identification for anaerobic bacteria.

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

    PubMed Central

    Jovanović, Marko; Tyldesley-Worster, Richard; Pohlentz, Gottfried; Peter-Katalinić, Jasna

    2014-01-01

    Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted. PMID:24743894

  8. 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.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2012-11-01

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

  11. Immobilized carbon nanotubes as matrix for MALDI-TOF-MS analysis: applications to neutral small carbohydrates.

    PubMed

    Ren, Shi-fang; Zhang, Li; Cheng, Zhi-hong; Guo, Yin-long

    2005-03-01

    In this work, we reported on the advantages of immobilized carbon nanotubes as a novel MALDI-matrix. Recently, carbon nanotubes have been reported to be an effective MALDI matrix for small molecules (Anal. Chem.2003, 75, 6191), as it can eliminate the interfering matrix peaks as well as form a web morphology to fully disperse the analyte and allow strong ultraviolet absorption for enhanced pulsed laser desorption and ionization. In our study, to overcome the problem that the carbon nanotube matrix may fly off from the target, a type of polyurethane adhesive, NIPPOLAN-DC-205, is introduced to immobilize carbon nanotubes on the target, which enables widespread application of carbon nanotubes as matrix for MALDI-MS analysis. At the same time, the properties of the carbon nanotubes as an efficient matrix remained after immobilization. The presence of NIPPOLAN-DC-205 increases the time for analysis at a particular desorption spot by minimizing the time-consuming search for "hot spots" and facilitating experiments such as post source decay (PSD) which need longer-lasting signals. Moreover, NIPPOLAN-DC-205 produces no interference peaks and can easily be cleaned with acetone. Fast evaporation technology may be used to enhance signal reproducibility in MALDI analysis using carbon nanotubes as matrix. Consequently, the applicability of the carbon nanotube as matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of low molecular mass analytes is highly improved. The feasibility of the method employing polyurethane is demonstrated by comparison of the results produced from the carbon nanotube matrix with and without immobilization. In addition, neutral small carbohydrates, which are difficult to be ionized normally, can be cationized with high efficiency by MALDI-TOF-MS using the immobilized carbon nanotube matrix. The method was further applied to analyze peptides and detect urine glucose successfully.

  12. Use-dependent inhibition of P2X3 receptors by nanomolar agonist.

    PubMed

    Pratt, Emily B; Brink, Thaddeus S; Bergson, Pamela; Voigt, Mark M; Cook, Sean P

    2005-08-10

    P2X3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (< 1 nM) of desensitized P2X3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X3 current. Sustained applications of 0.5 nM ATP inhibited > 50% of current to repetitive applications of P2X3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [32P]ATP from desensitized P2X3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC50 for inhibition and increased the rate of recovery.

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

    PubMed

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

    2015-10-01

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

  14. 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.

  15. 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.

  16. Powerful GC-TOF-MS Techniques for Screening, Identification and Quantification of Halogenated Natural Products.

    PubMed

    S Haglund, Peter; Löfstrand, Karin; Siek, Kevin; Asplund, Lillemor

    2013-01-01

    Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC×GC TOFMS) and gas chromatography/high-resolution time-of-flight mass spectrometry (GC-HRT) were used to detect and identify halogenated natural products (HNPs) in tissue homogenate, in this case brominated analytes present in a marine snail. Two classes of brominated anthropogenic compounds, polybrominated diphenyl ethers (PBDEs) and brominated dibenzofurans, were analyzed for comparison. Following conventional preparation, the sample was analyzed using GC×GC-TOF-MS. Isotope ratio scripts were used to compile a list of putatively brominated analytes from amongst the thousands of features resolved in the two-dimensional chromatogram. The structured nature of the chromatogram was exploited to propose identifications for several classes of brominated compounds, and include additional candidates that fell marginally outside the script tolerances. The sample was subsequently analyzed by GC-HRT. The high-resolution mass spectral data confirmed many formula assignments, facilitated confident assignment of an alternate formula when an original proposal did not hold, and enabled unknown identification. Identified HNPs include hydroxylated and methoxylated PBDE analogs, polybrominated dibenzo-p-dioxins (PBDDs) and hydroxyl-PBDDs, permitting the environmental occurrence and fate of such compounds to be studied.

  17. Acoustic trapping for bacteria identification in positive blood cultures with MALDI-TOF MS.

    PubMed

    Hammarström, Björn; Nilson, Bo; Laurell, Thomas; Nilsson, Johan; Ekström, Simon

    2014-11-04

    Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently changing the clinical routine for identification of microbial pathogens. One important application is the rapid identification of bacteria for the diagnosis of bloodstream infections (BSI). A novel approach based on acoustic trapping and an integrated selective enrichment target (ISET) microchip that improves the sample preparation step for this type of analysis is presented. The method is evaluated on clinically relevant samples in the form of Escherichia coli infected blood cultures. It is shown that noncontact acoustic trapping enables capture, enrichment, and washing of bacteria directly from the complex background of crude blood cultures. The technology replaces centrifugation-based separation with a faster and highly automated sample preparation method that minimizes manual handling of hazardous pathogens. The presented method includes a solid phase extraction step that was optimized for enrichment of the bacterial proteins and peptides that are used for bacterial identification. The acoustic trapping-based assay provided correct identification in 12 out 12 cases of E. coli positive blood cultures with an average score of 2.19 ± 0.09 compared to 1.98 ± 0.08 when using the standard assay. This new technology opens up the possibility to automate and speed up an important and widely used diagnostic assay for bloodstream infections.

  18. Analysis of protein expression profile of oral squamous cell carcinoma by MALDI-TOF-MS.

    PubMed

    Roman, Eric; Lunde, Mai Lill Suhr; Miron, Talia; Warnakulasauriya, Saman; Johannessen, Anne Christine; Vasstrand, Endre Normann; Ibrahim, Salah Osman

    2013-03-01

    In this study, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF-MS) technology was used to examine differentially expressed proteins in oral squamous cell carcinoma (OSCC) tissues from Norway (n=15) and the UK (n=45). Twenty-nine proteins were found to be significantly overexpressed in the OSCCs examined compared to the normal controls. Identified proteins included, family of annexin proteins that play important roles in signal transduction pathways and regulation of cellular growth, keratin-1, heat-shock proteins (HSP), squamous cell carcinoma antigen (SCC-Ag), cytoskeleton proteins, and proteins involved in mitochondrial and intracellular signalling pathways. The expression of four selected proteins (annexin II and V, HSP-27, and SCC-Ag) was verified using western blot analysis of 76 fresh tissue biopsy specimens in total, from Norway (n=53) and the UK (n=23). Proteomic analysis of OSCCs examined here demonstrated involvement of several proteins that might function as potential biomarkers and molecular targets for early cancer diagnostics, and may contribute to a novel approach to therapeutics and for predicting prognosis of OSCC.

  19. Characterization of native and denatured ricin using MALDI-TOF/MS.

    PubMed

    Sehgal, P; Rao, M K; Kumar, O; Vijayaraghavan, R

    2010-10-05

    Ricin is a toxic protein present in the seeds of castor bean plant. It can be inactivated by heat; therefore characterization of denatured ricin is essential to differentiate it from native ricin and to avoid any ambiguity in its identification. In this study, potential of mass spectrometry using MALDI—TOF/MS has been exploited to investigate the effects of heat treatment on ricin and spiked food matrices. The molecular weights of ricin, ricin A (A1 and A2) and B chain were found to be 62.8 kDa, 31.2 kDa, 32.5 kDa and 32 kDa respectively. The mass spectrum revealed a polypeptide chain of 11.1 kDa for denatured ricin. The peptide mass fingerprinting showed 24 peptides, six were common both in native and denatured ricin. The differentiating peptide at position 294—318 (m/z 934.533) was observed only in denatured ricin. The three selected marker peptides m/z 1013.6, 1310.7, 1728.9 are chosen for identification of ricin inactivated by heat in spiked apple juice and milk samples by immunocapture analysis. There is always a probability of denatured non— toxic ricin being confused with native (toxic) ricin to create unnecessary panic. Keeping this probability in mind, our study will be of immense value in minimising such risk.

  20. Partially oxidised organic components in urban aerosol using GCXGC-TOF/MS

    NASA Astrophysics Data System (ADS)

    Hamilton, J. F.; Webb, P. J.; Lewis, A. C.; Hopkins, J. R.; Smith, S.; Davy, P.

    2004-08-01

    Partially oxidised organic compounds associated with PM2.5 aerosol collected in London, England, have been analysed using direct thermal desorption coupled to comprehensive gas chromatography-time of flight mass spectrometry (GCXGC-TOF/MS). Over 10000 individual organic components were isolated from around 10µg of aerosol material in a single procedure and with no sample pre-treatment. Chemical functionalities observed using this analytical technique ranged from alkanes to poly-oxygenated species. The chemical band structures commonly used in GCXGC for group type identifications overlap for this sample type, and have required mass spectrometry as an additional level of instrument dimensionality. An investigation of oxygenated volatile organic compounds (o-VOC) contained within urban aerosol has been performed and in a typical sample around 130 o-VOCs were identified based on retention behaviour and spectral match. In excess of 100 other oxygenated species were also observed but lack of mass spectral library or pure components prevents positive identification. Many of the carbonyl species observed could be mechanistically linked to gas phase aromatic hydrocarbon oxidation and there is good agreement in terms of speciation between the urban samples analysed here and those degradation products observed in smog chamber experiments of aromatic oxidation. The presence of partially oxidised species such as linear chain aldehydes and ketones and cyclic products such as furanones suggests that species generated early in the oxidative process may undergo gas to particle partitioning despite their relatively high volatility.

  1. Identification of astilbin metabolites produced by human intestinal bacteria using UPLC-Q-TOF/MS.

    PubMed

    Zhao, Min; Xu, Jun; Qian, Dawei; Guo, Jianming; Jiang, Shu; Shang, Er-xin; Duan, Jin-ao

    2014-07-01

    Astilbin, mainly isolated from a commonly used herbal medicine, Smilax glabra Roxb (SGR), exhibits a variety of pharmacological activities and biological effects. It is metabolized by intestinal bacteria after oral administration which leads to the variation of ethnopharmacological profile of this traditional medicine. However, little is known on the interactions of this active compound with intestinal bacteria, which would be very helpful in unravelling how SGR works. In this study, different pure bacteria from human feces were isolated and were used to investigate their conversion capability of astilbin. Ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with Metabolynx(TM) software was used to analyze astilbin and its metabolites. The parent compound and two metabolites (quercetin and eriodictyol) were detected in the isolated bacterial samples compared with blank samples. Quercetin was present in Enterococcus sp. 8B, 8-2 and 9-2 samples. Eriodictyol was only identified in Enterococcus sp. 8B sample. The metabolic routes and metabolites of astilbin produced by the different intestinal bacteria are reported for the first time. This will be useful for the investigation of the pharmacokinetic study of astilbin in vivo and the role of different intestinal bacteria in the metabolism of natural compounds.

  2. 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.

  3. 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

  4. Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine.

    PubMed

    Lu, Zhiwei; Wang, Qing; Wang, Meiling; Fu, Shuang; Zhang, Qingqing; Zhang, Zhixin; Zhao, Huizhen; Liu, Yuehong; Huang, Zhenhai; Xie, Ziye; Yu, Honghong; Gao, Xiaoyan

    2017-03-12

    Modified nucleosides, metabolites of RNA, are potential biomarkers of cancer before the appearance of morphological abnormalities. It is of great significance to comprehensively detect and identify nucleosides in human urine for discovery of cancer biomarkers. However, the lower abundance, the greater polarity and the matrix effects make it difficult to detect urinary nucleosides. In this paper, an integrated method consisted of sample preparation followed by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS) detection and primary identification, then ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer (UHPLC Q-Trap/MS) further identification and validation were introduced. Firstly, to enrich the nucleosides and eliminate the urine matrix effects, different sorbent materials of solid phase extraction (SPE) and the elution conditions were screened. Secondly, UPLC Q-TOF/MS was used to acquire mass data in MS(E) mode. The structural formulas of nucleosides in urine sample were primarily identified according to retention time, accurate mass precursor ions and fragment ions from in-house database and online database. Thirdly, the preliminary identified nucleoside structures lacking of characteristic fragment ions were verified by UHPLC Q-Trap/MS in multiple reaction monitoring trigger enhanced product ion scan (MRM-EPI) and neutral loss scan (NL). At last, phenylboronic acid (PBA)-based SPE was utilized due to its higher MS signal and weaker matrix effects under optimized extraction conditions. Fifty-five nucleosides were primarily identified by UPLC Q-TOF/MS, among which 50 nucleosides were confirmed by UHPLC Q-Trap/MS. Five nucleosides, namely 4',5'-didehydro-5'-deoxyadenosine, 4',5'-didehydro-5'-deoxyinosine, isonicotinamide riboside, peroxywybutosine and hydroxywybutosine, were found from urine for the first time. The results will expand the Human

  5. Derivatized mesoporous silica beads for MALDI-TOF MS profiling of human plasma and urine.

    PubMed

    Terracciano, Rosa; Pasqua, Luigi; Casadonte, Francesca; Frascà, Stella; Preianò, Mariaimmacolata; Falcone, Daniela; Savino, Rocco

    2009-05-20

    Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for large-scale screening of body fluids for the early detection of human diseases. Proteins, peptides, and metabolites present in cells, tissues, or in body fluids constitute the molecular signatures of individuals. The design and generation of material-based platforms for capturing molecular signatures from body fluids has gained increasing interest in recent years. Highly selective materials are attractive candidates for a wide range of applications in biofluid proteomics. We have therefore developed a procedure based on mesoporous silica particles for the selective binding and enrichment of low molecular weight plasma/serum proteins by MALDI MS analysis ( Terracciano, R., Gaspari, M., Testa, F., Pasqua, L., Cuda G., Tagliaferri, P., Cheng, M. C., Nijdam, A. J., Petricoin, E. F., Liotta, L. A., Ferrari, M., and Venuta, S. ( 2006 ) Selective binding and enrichment for low-molecular weight biomarker molecules in human plasma after exposure to nanoporous silica particles . Proteomics 6, 3243-3250 ). Mesoporous silica beads (MSB) are able to harvest peptides from plasma and serum by means of nanosized porous channels with high surface area, while excluding large size proteins. Moreover, the absorption properties can be modified since the pore walls can be functionalized with different chemical species due to the high concentration of silanol groups at the surface. In this study, we performed derivatization of MSB with different functionalities, and we evaluated the derivatized materials for plasma and urine peptidomic profiling. Aminopropyl, N-(2-aminoethyl)-3-aminopropyl, and N,N,N' tris-carboxymethyl ethylene diamine, have been introduced onto the mesoporous silica surfaces in order to modulate selective peptide enrichment. We also explored various experimental conditions in order to optimize the performance of chemically modified MSB in the peptide

  6. Determination of Curcuminoids in Curcuma longa Linn. by UPLC/Q-TOF-MS: An Application in Turmeric Cultivation.

    PubMed

    Ashraf, Kamran; Mujeeb, Mohd; Ahmad, Altaf; Ahmad, Niyaz; Amir, Mohd

    2015-09-01

    Cucuma longa Linn. (Fam-Zingiberaceae) is a valued medicinal plant contains curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) as major bioactive constituents. Previously reported analytical methods for analysis of curcuminoids were found to suffer from low resolution, lower sensitivity and longer analytical times. In this study, a rapid, sensitive, selective high-throughput ultra high performance liquid chromatography-tandem mass spectrometry (UPLC/Q-TOF-MS) method was developed and validated for the quantification of curcuminoids with an aim to reduce analysis time and enhance efficiency. UPLC/Q-TOF-MS analysis showed large variation (1.408-5.027% w/w) of curcuminoids among different samples with respect to their occurrence of metabolite and their concentration. The results showed that Erode (south province) contains highest quantity of curcuminoids and concluded to be the superior varieties. The results obtained here could be valuable for devising strategies for cultivating this medicinal plant.

  7. Immunoaffinity sample purification and MALDI-TOF MS analysis of alpha-Solanine and alpha-chaconine in serum.

    PubMed

    Driedger, D R; Sporns, P

    2001-02-01

    A sample purification technique was developed for the detection of potato glycoalkaloids (GAs) in blood serum by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GAs were extracted from spiked serum (5 mL) using a C(18) solid-phase extraction cartridge. The GAs were then selectively captured on antibody-coated agarose beads. The agarose beads were washed with water and the GAs eluted with 25 microL of methanol. MALDI-TOF MS was used to detect the GAs in the methanol eluent. Immunoaffinity sample purification of the GAs effectively reduced the signal suppression observed during the analysis of unpurified samples. alpha-Chaconine and alpha-solanine were detected in serum spiked with 1 ng/mL of each GA.

  8. Development of a Direct Headspace Collection Method from Arabidopsis Seedlings Using HS-SPME-GC-TOF-MS Analysis

    PubMed Central

    Kusano, Miyako; Iizuka, Yumiko; Kobayashi, Makoto; Fukushima, Atsushi; Saito, Kazuki

    2013-01-01

    Plants produce various volatile organic compounds (VOCs), which are thought to be a crucial factor in their interactions with harmful insects, plants and animals. Composition of VOCs may differ when plants are grown under different nutrient conditions, i.e., macronutrient-deficient conditions. However, in plants, relationships between macronutrient assimilation and VOC composition remain unclear. In order to identify the kinds of VOCs that can be emitted when plants are grown under various environmental conditions, we established a conventional method for VOC profiling in Arabidopsis thaliana (Arabidopsis) involving headspace-solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (HS-SPME-GC-TOF-MS). We grew Arabidopsis seedlings in an HS vial to directly perform HS analysis. To maximize the analytical performance of VOCs, we optimized the extraction method and the analytical conditions of HP-SPME-GC-TOF-MS. Using the optimized method, we conducted VOC profiling of Arabidopsis seedlings, which were grown under two different nutrition conditions, nutrition-rich and nutrition-deficient conditions. The VOC profiles clearly showed a distinct pattern with respect to each condition. This study suggests that HS-SPME-GC-TOF-MS analysis has immense potential to detect changes in the levels of VOCs in not only Arabidopsis, but other plants grown under various environmental conditions. PMID:24957989

  9. Profiling LC-DAD-ESI-TOF MS method for the determination of phenolic metabolites from avocado (Persea americana).

    PubMed

    Hurtado-Fernández, Elena; Carrasco-Pancorbo, Alegría; Fernández-Gutiérrez, Alberto

    2011-03-23

    A powerful HPLC-DAD-ESI-TOF MS method was established for the efficient identification of the chemical constituents in the methanolic extracts of avocado (Persea americana). Separation and detection conditions were optimized by using a standard mix containing 39 compounds belonging to phenolic acids and different categories of flavonoids, analytes that could be potentially present in the avocado extracts. Optimum LC separation was achieved on a Zorbax Eclipse Plus C18 analytical column (4.6×150 mm, 1.8 μm particle size) by gradient elution with water+acetic acid (0.5%) and acetonitrile as mobile phases, at a flow rate of 1.6 mL/min. The detection was carried out by ultraviolet-visible absorption and ESI-TOF MS. The developed method was applied to the study of 3 different varieties of avocado, and 17 compounds were unequivocally identified with standards. Moreover, around 25 analytes were tentatively identified by taking into account the accuracy and isotopic information provided by TOF MS.

  10. Validation of LC–TOF-MS Screening for Drugs, Metabolites, and Collateral Compounds in Forensic Toxicology Specimens

    PubMed Central

    Guale, Fessessework; Shahreza, Shahriar; Walterscheid, Jeffrey P.; Chen, Hsin-Hung; Arndt, Crystal; Kelly, Anna T.; Mozayani, Ashraf

    2013-01-01

    Liquid chromatography time-of-flight mass spectrometry (LC–TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic “Spice/K2” cannabinoids and cathinone “bath salt” designer drugs. The extract was applied to LC–TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC–TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework. PMID:23118149

  11. Effects of Extracellular Polymeric Substance Composition on Bacteria Disinfection by Monochloramine: Application of MALDI-TOF/TOF-MS and Multivariate Analysis.

    PubMed

    Coburn, Kimberly M; Wang, Qinzhe; Rediske, Dustin; Viola, Ronald E; Hanson, B Leif; Xue, Zheng; Seo, Youngwoo

    2016-09-06

    In our previous study, we reported that the transport of monochloramine is affected by the extracellular polymeric substance (EPS) composition, which in turn affects the cell viability of both biofilm and detached clusters.11 However, although the transport and reaction of monochloramine in biofilm could be observed, the specific biomolecules reacting with the disinfectant and the mechanism of disinfection remains elusive. In this study, the impact of EPS composition on bacteria disinfection by monochloramine was qualitatively determined using both wild-type and isogenic mutant Pseudomonas strains with different EPS-secretion capacity and composition. To evaluate their EPS reactivity and contribution to susceptibility to monochloramine, we investigated the bacteria disinfection process using Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption-ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Canonical correlation analysis and partial least-squares regression modeling were employed to explore the changes that EPS underwent during the monochloramine disinfection process. The analyses results suggested significant reactions of the monochloramine with peptide fragments of proteins that are associated with carbohydrate utilization. Selected enzymes also showed different levels of inhibition by monochloramine when tested.

  12. Biochips that Sequentially Capture and Focus Antigens for Immunoaffinity MALDI-TOF MS: A New Tool for Biomarker Verification

    PubMed Central

    Brauer, Heather Ann; Lampe, Paul D.; Yasui, Yutaka Y.; Hamajima, Nobuyuki; Stolowitz, Mark L.

    2011-01-01

    A novel approach to immunoaffinity MS is described wherein antibodies are appended to a patterned gold Biochip surface. The Biochip surface is patterned with an array of concentric immunocapture zones comprised of highly hydrophilic central zones surrounded by moderately hydrophilic zones that reside on a non-wetting background, with protein attachment via electrochemically cleavable linkers. After linker cleavage, matrix application forms a discrete spot suitable for MALDI-TOF-MS. Use of the Biochip to purify Transthyretin from human serum allowed distinct resolution of four disulfide conjugates and one truncated form isoforms with good mass resolution and sensitivity. PMID:20957758

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

    PubMed

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

    2015-02-01

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

  14. 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

  15. Identification and characterization of fluvastatin metabolites in rats by UHPLC/Q-TOF/MS/MS and in silico toxicological screening of the metabolites.

    PubMed

    Chavan, Balasaheb B; Kalariya, Pradipbhai D; Nimbalkar, Rakesh D; Garg, Prabha; Srinivas, R; Talluri, M V N Kumar

    2017-03-11

    The present study reports the in vivo and in vitro identification and characterization of metabolites of fluvastatin (FLU), the HMG CoA reductase inhibitor, using liquid chromatography-mass spectrometry (LC-MS). In vitro studies were conducted by incubating the drug with human liver microsomes (HLM) and rat liver microsomes (RLM). In vivo studies were carried out by administration of the drug in the form of suspension to the Sprague-Dawley (SD) rats followed by collection of urine, faeces and blood at different time points upto 24 h. Further, samples were prepared by optimized sample preparation method, which includes freeze liquid extraction (FLE), protein precipitation (PP) and solid phase extraction (SPE). The extracted and concentrated samples were analyzed using ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF/MS/MS). A total of 15 metabolites were observed in urine, which includes hydroxyl, sulphated, desisopropyl, dehydrogenated, dehydroxylated and glucuronide metabolites. A few of the metabolites were also present in faeces and plasma samples. In in vitro studies a few metabolites were observed which were also present in in vivo samples. All the metabolites were characterized using UHPLC/Q-TOF/MS/MS in combination with accurate mass measurement. Finally, in silico toxicity studies indicated that some of the metabolites show or possess carcinogenicity and skin sensitization. Several metabolites that were identified in rats are proposed to have toxicological significance based on in silico evaluation. However, these metabolites are of no human relevance.

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

    PubMed

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

    2016-12-01

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

  17. Gene analysis using mass spectrometric cleaved amplified polymorphic sequence (MS-CAPS) with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

    PubMed

    Kajiwara, Hideyuki

    2015-01-01

    Mass spectrometric cleaved amplified polymorphic sequence (MS-CAPS) is a method for detecting genes using a combination of short PCR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MS-CAPS can identify a single nucleotide polymorphism (SNP) in less than one hour and is suitable for plants, animals, bacteria, and food.

  18. Polydopamine-coated magnetic nanoparticles for enrichment and direct detection of small molecule pollutants coupled with MALDI-TOF-MS.

    PubMed

    Ma, Yu-rong; Zhang, Xiao-le; Zeng, Tao; Cao, Dong; Zhou, Zhen; Li, Wen-hui; Niu, Hongyun; Cai, Ya-qi

    2013-02-01

    Polydopamine-coated Fe(3)O(4) nanoparticles (Fe(3)O(4)@PDA NPs) were synthesized and applied as matrix for the detection of pollutants by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The synthesis of Fe(3)O(4)@PDA NPs was accomplished in 30 min by in situ polymerization of dopamine without any toxic reagent. Using Fe(3)O(4)@PDA NPs as matrix of MALDI-TOF, eleven small molecule pollutants (molecular weight from 251.6 to 499.3), including Benzo(a)pyrene (BaP), three perfluorinated compounds (PFCs), and seven antibiotics, were successfully detected in either positive or negative reflection mode without background interference. Furthermore, the Fe(3)O(4)@PDA NPs can also enrich trace amounts of hydrophobic target, such as BaP, from solution to nanoparticles surface. Then the Fe(3)O(4)@PDA-BaP can be isolated through magnetic sedimentation step and directly spotted on the stainless steel plate for MALDI measurement. With Fe(3)O(4)@PDA NPs as adsorbent and matrix, we also realized the analysis of BaP in tap water and lake water samples. Thus, a magnetic solid-phase extraction (MSPE)-MALDI-TOF-MS method was established for the measurement of BaP.

  19. Identification of phenolic compounds in aqueous and ethanolic rooibos extracts (Aspalathus linearis) by HPLC-ESI-MS (TOF/IT).

    PubMed

    Iswaldi, Ihsan; Arráez-Román, David; Rodríguez-Medina, Inmaculada; Beltrán-Debón, Raúl; Joven, Jorge; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

    2011-07-01

    Rooibos (Aspalathus linearis) is a rich source of polyphenols and used to make a mild-tasting tea containing no caffeine, is low in tannins compared to green or black teas, and has antioxidant and antimutagenic/antitumoral properties. In vivo results show that rooibos has beneficial effects upon the lipid profile by decreasing serum triglycerides and cholesterol. In this sense, we have developed a simple and rapid method to separate and characterize simultaneously the polyphenolic compounds in aqueous and ethanolic rooibos extracts using high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and ion trap multiple mass spectrometry (HPLC-ESI-IT-MS(2)). The phenolic compounds were separated on a C(18) column (4.6 × 150 mm, 1.8 μm) with 1% formic acid in water/acetonitrile 90:10 v/v and acetonitrile as mobile phases. The accuracy mass data generated by TOF-MS together with the fragmentation pattern obtained by IT-MS(2) experiments confirmed the presence of 25 and 30 phenolic compounds in the aqueous and ethanolic extracts, respectively.

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

    PubMed

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

    2013-05-15

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

  1. Indirect identification of antioxidants in Polygalae Radix through their reaction with 2,2-diphenyl-1-picrylhydrazyl and subsequent HPLC-ESI-Q-TOF-MS/MS.

    PubMed

    Shi, Qiyuan; Chen, Jiale; Zhou, Qinfen; Lei, Houliang; Luan, Lianjun; Liu, Xuesong; Wu, Yongjiang

    2015-11-01

    A rapid and efficient method for the identification of antioxidants in the traditional Chinese medicine Polygalae Radix (PR) by HPLC-ESI-Q-TOF-MS/MS is described. The method is based on the hypothesis that upon reaction of antioxidants with 1,1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas of compounds with potential antioxidant activities in the HPLC chromatogram will be significantly reduced in comparison to the untreated sample. The identity confirmation was achieved by Q-TOF-MS/MS technique. With this method, eight components were proposed possessing potent antioxidant activity. They were identified as sibiricose A5, sibiricose A6, sucrose monoester, polygalaxanthone III, tenuifoliside B, 3',6-disinapoylsucrose (DISS), sucrose diester, tenuifoliside C, respectively. DISS was proposed to be the most potent one. The antioxidant activity of DISS was evaluated by DPPH, ABTS radical scavenging assay and ferric-reducing antioxidant power (FRAP) assay in vitro. Vitamin C (Vc) was used as positive control substance. DISS showed moderate DPPH (DISS's IC50 value was 1024.17 μg/mL, Vc's was 294.68 μg/mL) and ABTS (IC50 324.13 μg/mL, Vc's was 117.50 μg/mL) free radical scavenging capacity and ferric-reducing antioxidant power. DISS can be used as a new source of natural antioxidant in foods and cosmetics.

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

    PubMed

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

    2014-02-01

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

  3. Rapid Identification and Assignation of the Active Ingredients in Fufang Banbianlian Injection Using HPLC-DAD-ESI-IT-TOF-MS.

    PubMed

    Li, Sensen; Lin, Zongtao; Jiang, Haixiu; Tong, Lingkun; Wang, Hong; Chen, Shizhong

    2016-08-01

    Fufang Banbianlian Injection (FBI) is a well-known traditional Chinese medicine formula composed of three herbal medicines. However, the systematic investigation on its chemical components has not been reported yet. In this study, a high-performance liquid chromatography combined with diode-array detector, and coupled to an electrospray ionization with ion-trap time-of-flight mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method, was established for the identification of chemical profile in FBI. Sixty-six major constituents (14 phenolic acids, 14 iridoids, 20 flavonoids, 2 benzylideneacetone compounds, 3 phenylethanoid glycosides, 1 coumarin, 1 lignan, 3 nucleosides, 1 amino acids, 1 monosaccharides, 2 oligosaccharides, 3 alduronic acids and citric acid) were identified or tentatively characterized by comparing their retention times and MS spectra with those of standards or literature data. Finally, all constituents were further assigned in the individual herbs (InHs), although some of them were from multiple InHs. As a result, 11 compounds were from Lobelia chinensis Lour, 33 compounds were from Scutellaria barbata D. Don and 38 compounds were from Hedyotis diffusa Willd. In conclusion, the developed HPLC-DAD-ESI-IT-TOF-MS method is a rapid and efficient technique for analysis of FBI sample, and could be a valuable method for the further study on the quality control of the FBI.

  4. 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.

  5. [Analysis on component difference in Citrus reticulata before and after being processed with salt by UPLC-Q-TOF/MS].

    PubMed

    Zeng, Rui; Fu, Juan; Wu, La-Bin; Huang, Lin-Fang

    2013-07-01

    To analyze components of Citrus reticulata and salt-processed C. reticulata by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS), and compared the changes in components before and after being processed with salt. Principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA) were adopted to analyze the difference in fingerprint between crude and processed C. reticulata, showing increased content of eriocitrin, limonin, nomilin and obacunone increase in salt-processed C. reticulata. Potential chemical markers were identified as limonin, obacunone and nomilin, which could be used for distinguishing index components of crude and processed C. reticulata.

  6. Coupled TLC and MALDI-TOF/MS Analyses of the Lipid Extract of the Hyperthermophilic Archaeon Pyrococcus furiosus

    PubMed Central

    Lobasso, Simona; Lopalco, Patrizia; Angelini, Roberto; Vitale, Rita; Huber, Harald; Müller, Volker; Corcelli, Angela

    2012-01-01

    The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea. PMID:23193375

  7. 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

  8. 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

  9. Separation and characterization of nitrated variants of the major birch pollen allergen by CZE-ESI-μTOF MS

    PubMed Central

    Gusenkov, Sergey; Ackaert, Chloé; Stutz, Hanno

    2013-01-01

    A CZE-ESI-TOF MS method has been optimized for the separation and identification of nitrated variants of the major birch pollen allergen from Betula verrucosa, isoform 1a (Bet v 1a). In-house nitration of recombinant Bet v 1a was done by peroxynitrite. As a BGE, 10 mmol/L ammonium bicarbonate with pH 7.50 provided best resolution. Nebulizer gas pressure and sheath liquid flow rate of 0.4 bar and 6 μL/min, respectively, maintained CZE selectivity and constituted stable electrospray conditions. A sheath liquid composition of 75% v/v methanol with 0.1% v/v formic acid in ultrapure water resulted in highest signal intensities. Alternatively, methanol could be replaced by 50% v/v isopropanol. Two modified allergen products derived from reaction mixtures that contained different amounts of the nitration reagent were compared by the elaborated CZE-ESI-TOF MS method. Up to twelve different Bet v 1a variants with one- to sixfold nitration could be distinguished. Several allergen fractions of equivalent nitration grade were resolved. Their different migration times indicate site-specific nitration with concomitant differences in pI and maybe also in hydrodynamic radius. The method allows for a characterization of in-house nitrated allergen samples that are intended for testing the postulated enhanced allergenicity of nitrated Bet v 1a variants. PMID:23857337

  10. 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.

  11. 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.

  12. GC-TOF/MS-based metabolomics approach to study the cellular immunotoxicity of deoxynivalenol on murine macrophage ANA-1 cells.

    PubMed

    Ji, Jian; Sun, Jiadi; Pi, Fuwei; Zhang, Shuang; Sun, Chao; Wang, Xiumei; Zhang, Yinzhi; Sun, Xiulan

    2016-08-25

    Gas chromatography-time of fly/mass spectrum (GC-TOF/MS) based complete murine macrophage ANA-1 cell metabolome strategy, including the endo-metabolome and the exo-metabolome, ANA-1 cell viability assays and apoptosis induced by diverse concentrations of DON were evaluated for selection of an optimized dose for in-depth metabolomic research. Using the optimized chromatography and mass spectrometry parameters, the metabolites detected by GC-TOF/MS were identified and processed with multivariate statistical analysis, including principal componentanalysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) analysis. The data sets were screened with a t-test (P) value < 0.05, VIP value > 1, similarity value > 500, leaving 16 exo-metabolite variables and 11 endo-metabolite variables for further pathway analysis. Implementing the integration of key metabolic pathways, the metabolism pathways were categorized into two dominating types, metabolism of amino acid and glycometabolism. Glycine, serine and threonine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis and phenylalanine metabolism were the significant amino acids affected by the metabolic pathways, indicating statistically significant fold changes including pyruvate, serine, glycine, lactate and threonine. Glycolysis or gluconeogenesis, starch and sucrose metabolism, and galactose metabolism, belonging to glycometabolism, were the pathways that were found to be primarily affected, resulting in abnormal metabolites such as glucose-1P, Glucose, gluconic acid, myo-inositol, sorbitol and glycerol.

  13. 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.

  14. Antigen digestion on the target plate of MALDI-TOF MS after isolation using an immunoaffinity membrane.

    PubMed

    Shimazaki, Youji; Nishimura, Yuri; Saito, Masaki

    2013-09-01

    A combination of methods is required to achieve separation of intact proteins and subsequently perform structure analysis to examine their unstable or external structures. The aim of this study was to develop a method of structure analysis in intact proteins after purification. Transferrin from human plasma was trapped by membrane-immobilized anti-transferrin antibody, which was produced by non-denaturing two-dimensional electrophoresis (2-DE), and transferred to a polyvinylidene fluoride (PVDF) membrane and stained with Ponceau S. The antigen transferrin was eluted by rinsing the membrane with trifluoroacetic acid (TFA) or aspartic acid. In addition, a method was established by which the purified human transferrin was enzymatically digested on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate. Thus, after purification of the human transferrin antigen from tens of microlitres of human plasma using an immunoaffinity membrane, transferrin polypeptide fragments were obtained on the plate following digestion with pepsin in the presence of 0.1% TFA or endoproteinase Lys-C or Lys-C/trypsin with 0.001% sodium dodecyl sulphate (SDS). The results indicated that the combined methods of isolation using an immunoaffinity membrane and enzymatic digestion on a MALDI-TOF MS plate could be applied to the purification and microanalysis of antigens. This approach would be particularly applicable to the analysis of the primary structure and the less stable and highly accessible regions of antigens from limited sample volumes.

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

    PubMed

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

    2016-12-01

    Information regarding the use of MALDI-TOF MS as an alternative to conventional laboratory methods for the rapid and reliable identification of bacterial isolates is still limited. In this study, MALDI-TOF MS was evaluated on 295 anaerobic isolates previously identified by 16S rRNA gene sequencing and with biochemical tests (Rapid ID 32A system, BioMérieux). In total, 85.8% of the isolates were identified by MALDI-TOF MS at the species level vs 49.8% using the Rapid ID 32A system (p < 0.0001). None of the isolates was discordantly identified at the genus level using MALDI-TOF MS and only 9 of them could not be identified using the method. Thus, our results show that MALDI-TOF MS is a robust and reliable tool for the identification of anaerobic isolates in the microbiology laboratory. Its implementation will reduce the turnaround time for a final identification and the number of isolates that require 16S rRNA sequencing.

  16. Direct identification of trypanosomatids by matrix-assisted laser desorption ionization-time of flight mass spectrometry (DIT MALDI-TOF MS).

    PubMed

    Avila, C C; Almeida, F G; Palmisano, G

    2016-08-01

    Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI-TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI-TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI-TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software-assisted identification at the strain level. Overall, this study shows the importance of MALDI-TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry-based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.

  17. A comparative analysis of chemical compositions in Camellia sinensis var. puanensis Kurihara, a novel Chinese tea, by HPLC and UFLC-Q-TOF-MS/MS.

    PubMed

    Li, Yi-Fang; Ouyang, Shu-Hua; Chang, Yi-Qun; Wang, Ting-Mei; Li, Wei-Xi; Tian, Hai-Yan; Cao, Hong; Kurihara, Hiroshi; He, Rong-Rong

    2017-02-01

    Camellia sinensis var. puanensis Kurihara (Puan tea) is a kind of ancient tea plant newly found in Jiangxipo and the surrounding areas of Puan County (Guizhou, China). People there always believe that drinking Puan tea is beneficial to the promotion of health and prevention of diseases. However, detailed information on its compositions has not been reported. Therefore, in this study, the varieties and contents of purine alkaloids and polyphenols in Puan tea were identified and determined by HPLC and UFLC-Q-TOF-MS/MS. Our results showed that theacrine, but not caffeine, was the dominated purine alkaloid detected in Puan tea. Meanwhile, Puan tea contained B-type procyanidin dimer, trimer and dimer monogallate, which were not detected in Camellia sinensis, Camellia ptilophylla and Camellia assamica var. kucha. The obtained results could support the local uses of Puan tea in health and nutrition and contribute to the research of tea variety.

  18. Serum pharmacochemistry for tracking bioactive components by UPLC-Q-TOF-MS/MS combined chromatographic fingerprint for quality assessment of Sanziguben Granule.

    PubMed

    Zhang, Chenxue; Lian, Ruixin; Mahmoodurrahman, Mohammed; Lai, Sisi; Zhao, Zhongxiang; Yu, Yang

    2016-09-01

    To more reasonably and effectively control the quality of Sanziguben Granule, chromatographic fingerprinting and serum pharmacochemistry of this traditional Chinese medicine compound were performed. A comprehensive comparison and evaluation of 15 batches of Sanziguben Granule was successfully conducted by using high performance liquid chromatography (HPLC) fingerprint analysis. After administering a set amount of Sanziguben Granule orally to rats, blood samples were collected and tested 4 times at intervals of 30min, 1h, 2h, and 4h using UPLC-Q-TOF-MS/MS. The blood showed presence of gallic acid and corilagin indicating the pharmacological significance of these two chemical compounds. According to the result, above mentional chemical compounds were designated biomarkers for quality control of Sanziguben Granule. Therefore, a purposeful and efficient method for quality control of Sanziguben Granule was established in the present study.

  19. 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

    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 (54

  20. 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

  1. Identification of in vivo and in vitro metabolites of 4,5-dimethoxycanthin-6-one by HPLC-Q-TOF-MS/MS.

    PubMed

    Miao, Xiaolei; Wang, Junjun; Chen, Liang; Peng, Zhihong; Chen, Yong

    2016-05-01

    4,5-Dimthexycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the main active ingredients of Picrasma quassioides, which is a widely used herbal medicine for the treatment of gastroenteritis, snakebite, infection and hypertension in China. In the present study, the in vitro metabolites of 4,5-dimethoxycanthin-6-one in rat, mouse, dog and human liver microsomes, as well as the in vivo metabolites in rat plasma and urine following a single oral dose of 4,5-dimethoxycanthin-6-one, were identified by high-performance liquid chromatography combined with triple TOF mass spectrometry (HPLC-TOF/MS/MS). The metabolites were elucidated based on an accurate mass measurement, the MS/MS fragmentation patterns, the retention times of the parent drug and its metabolites, and the relevant drug biotransformation rules. After incubation in liver mcrosomes for 50 min, 4,5-dimethoxycanthin-6-one produced 8 phase I metabolites including 2 mono-demethylated metabolites (M1, M2), 3 mono-hydroxylated metabolites (M3-M5), and 3 mono-demethylated and mono-hydroxylated metabolites (M6-M8) in rat and mouse liver microsomes, 7 phase I metabolites (without M7) in dog and human liver microsomes. After a single oral administration of 4,5-dimethoxycanthin-6-one to rats, there were 3 phase I metabolites (M1, M2 and M5) detected in rat plasma and 5 phase I metabolites (M1-M5) in rat urine. Phase II metabolites were not detected in rat plasma and urine. Among these metabolites, mono-demethylated metabolites (M1 and M2) were the major metabolites of 4,5-dimethoxycanthin-6-one, mono-hydroxylated metabolites (M3-M5) were the minor metabolites of 4,5-dimethoxycanthin-6-one.

  2. Micro-scale strategy to detect spermine and spermidine by MALDI-TOF MS in foods and identification of apoptosis-related proteins by nano-flow UPLC-MS/MS after treatment with spermine and spermidine.

    PubMed

    Su, Huai-Hsin; Chuang, Lea-Yea; Tseng, Wei-Lung; Lu, Chi-Yu

    2015-01-26

    Spermine and spermidine are multiple-nitrogen compounds found in many foods. Both compounds are essential for cell growth and human health. This study established a simple and fast method of detecting spermine and spermidine in food samples by matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry (MALDI-TOF MS). After a simple sample preparation procedure, spermine and spermidine were directly detected by MALDI-TOF MS with no additional purification procedure. The calibration curves for spermine and spermidine ranged from 0.1 to 10 μg/mL. In intra- and inter-batch assays of three different concentrations of spermine and spermidine, all relative standard deviations and relative errors were below 18.9%. These experimental results confirmed the practicability and effectiveness of the proposed MALDI-TOF MS method for fast determination of spermine and spermidine in food samples. Furthermore, since spermine and spermidine have important roles in apoptosis, up-regulation and down-regulation of spermine and spermidine during apoptosis were analyzed. After treating NRK-52E cells with spermine and spermidine, the cells were lysed, and cell proteins were collected, and digested. Apoptosis-related proteins were then identified by tandem MS.

  3. Comparison and optimization of two MALDI-TOF MS platforms for the identification of medically relevant yeast species.

    PubMed

    Pence, M A; McElvania TeKippe, E; Wallace, M A; Burnham, C-A D

    2014-10-01

    The rapid identification of yeast is essential for the optimization of antifungal therapy. The objective of our study was to evaluate two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms, the bioMérieux VITEK MS (IVD Knowledgebase v.2.0) and Bruker Biotyper (software version 3.1), for the rapid identification of medically relevant yeast. One hundred and seventeen isolates, representing six genera and 18 species, were analyzed using multiple direct smear methods to optimize identification. Sequence analysis was the gold standard for comparison. Isolates were analyzed with VITEK MS using the direct smear method +/- a 25 % formic acid on-plate extraction. For Biotyper, isolates were analyzed using direct smear without formic acid, and with 25 % and 100 % formic acid on-plate extractions. When all methods were included, VITEK MS correctly identified 113 (96.6 %) isolates after 24 h with one misidentification, and Biotyper correctly identified 77 (65.8 %) isolates using a threshold of ≥2.0 with no misidentifications. Using a revised threshold of ≥1.7, Biotyper correctly identified 103 (88.0 %) isolates, with 3 (2.6 %) misidentifications. For both platforms, the number of identifications was significantly increased using a formic acid overlay (VITEK MS, p < 0.01; Biotyper, p < 0.001), and reducing the Biotyper threshold from ≥2.0 to ≥1.7 significantly increased the rate of identification (p < 0.001). The data in this study demonstrate that the direct smear method with on-plate formic acid extraction can be used for yeast identification on both MS platforms, and more isolates are identified using the VITEK MS system (p < 0.01).

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

    PubMed

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

    2015-11-30

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

  5. Identification of phospholipids classes and molecular species in different types of egg yolk by using UPLC-Q-TOF-MS.

    PubMed

    Ali, Abdelmoneim H; Zou, Xiaoqiang; Lu, Jian; Abed, Sherif M; Yao, Yunping; Tao, Guanjun; Jin, Qingzhe; Wang, Xingguo

    2017-04-15

    Egg phospholipids (PLs) are currently the products of greatest commercial interest with major area of importance in various fields. Therefore, in this study, duck, hen and quail egg yolk PLs were isolated by solvent extraction with chilled acetone precipitation, and subsequently separated and identified by using ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Egg PLs were separated on hydrophilic interaction liquid chromatography (HILIC) with ethylene bridged hybrid (BEH) column by gradient elution using acetonitrile/ammonium formate as a mobile phase, and detected by mass spectrometry (MS) under electrospray ionization in positive and negative ion mode. Structural characterizations of 57 molecular species of egg yolk PLs were identified based on MS/MS fragment ion information and elemental composition in MassLynx 4.1 software. The obtained results showed that phosphatidylcholine (16:0-18:1), phosphatidylethanolamine (18:0-20:4), phosphatidylinositol (18:0-18:2), phosphatidylserine (18:0-18:2), sphingomyelin (d18:1/16:0) and lysophosphatidylcholine (16:0) were the predominant species among the different classes of egg yolk phospholipids.

  6. Characterization and differentiation of high energy amine peroxides by direct analysis in real time TOF/MS

    NASA Astrophysics Data System (ADS)

    Peña-Quevedo, Alvaro J.; Cody, Robert; Mina-Camilde, Nairmen; Ramos, Mildred; Hernández-Rivera, Samuel P.

    2007-04-01

    Characterization of hexamethelene triperoxide diamine (HMTD), tetramethylene diperoxide dicarbamide (TMDD) and tetramethylene diperoxide acetamide (TMDA) has been carried out using Direct Analysis in Real Time/Time of Flight Mass Spectrometry (DART-TOF/MS). The study also centered in the detection of their precursors such as hexamine and formaldehyde. Analysis of the compounds by GC-MS was also conducted. HMTD shows a clear peak at 209 m/z that allowed its detection in standard solutions and lab made standards. TATP samples with deuterium enrichment are being analyzed to compare results that could differentiate from HMTD and similar substances. All samples were characterized by Raman and FT-IR to confirm the DART results. Some of the vibrations observed were in the ν(O-O), ν(N-C), ν(N-H), ν(C-O), δ(CH 3-C) and δ(C-O). Development methodology for trace detection was compared with GC/MS and HPLC-MS results previously presented for HMTD and TATP.

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

    PubMed

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

    2016-09-01

    Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under-represented (fewer than five MSPs). This resulted in an increase from 53.6% (75/140) to 82.1% (115/140) of GPAC isolates that could be identified at the species level using MALDI-TOF MS. An improved log score was obtained for 51.4% (72/140) of the strains. For strains with a sequence similarity <98.7% with their closest relative (n = 5) or with an inconclusive sequence identity (n = 4), no identification was obtained by MALDI-TOF MS or in the latter case an identity with one of its relatives. For some species the MSP of the type strain was not part of the confined cluster of the corresponding clinical isolates. Also, not all species formed a homogeneous cluster. It emphasizes the necessity of adding sufficient MSPs of human clinical isolates.

  8. Detection of triazole resistance among Candida species by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS).

    PubMed

    Saracli, Mehmet A; Fothergill, Annette W; Sutton, Deanna A; Wiederhold, Nathan P

    2015-09-01

    MALDI-TOF MS can rapidly identify microorganisms to the species level and may be able to detect antimicrobial resistance. We evaluated the ability of this technology to detect triazole resistance in Candida species.35 C. albicans, 35 C. glabrata, and 37 C. tropicalis strains were exposed to fluconazole, voriconazole, or posaconazole at two different concentrations plus a drug-free control: a midrange concentration (CLSI clinical breakpoint or epidemiologic cut-off value), and a high concentration (fluconazole 64 μg/ml, voriconazole & posaconazole 16 μg/ml). The MALDI-TOF MS spectra at these concentrations were used to create the individual composite correlation index (CCI) matrices for each isolate. When the CCI of the midrange/highest concentration was lower than that of the midrange/null concentration, the strain was classified as resistant. These results were then compared to the classifications for susceptible or resistant obtained by measuring the MICs according to the CLSI M27-A3 antifungal susceptibility testing (AFST) method.The MALDI-TOF MS assay was able to classify triazole susceptibility against all strains. Overall, essential agreement between MALDI-TOF MS and AFST varied between 54% and 97%, and was highest for posaconazole against C. glabrata. The reproducibility of the MALDI-TOF MS assay varied between 54.3 and 82.9% and was best for fluconazole against C. albicans and posaconazole against C. glabrata. Reproducibility was also higher for C. glabrata isolates compared to C. albicans and C. tropicalis.These results demonstrate that MALDI-TOF MS may be used to simultaneously determine the Candida species and classification as susceptible or resistant to triazole antifungals. Further studies are needed to refine the methodology and improve the reproducibility of this assay.

  9. 2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation.

    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.

  10. Optimization of microwave-assisted extraction for the characterization of olive leaf phenolic compounds by using HPLC-ESI-TOF-MS/IT-MS(2).

    PubMed

    Taamalli, Amani; Arráez-Román, David; Ibañez, Elena; Zarrouk, Mokhtar; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

    2012-01-25

    In the present work, a simple and rapid method for the extraction of phenolic compounds from olive leaves, using microwave-assisted extraction (MAE) technique, has been developed. The experimental variables that affect the MAE process, such as the solvent type and composition, microwave temperature, and extraction time, were optimized using a univariate method. The obtained extracts were analyzed by using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)) to prove the MAE extraction efficiency. The optimal MAE conditions were methanol:water (80:20, v/v) as extracting solvent, at a temperature equal to 80 °C for 6 min. Under these conditions, several phenolic compounds could be characterized by HPLC-ESI-MS/MS(2). As compared to the conventional method, MAE can be used as an alternative extraction method for the characterization of phenolic compounds from olive leaves due to its efficiency and speed.

  11. Identifying sources of Pb pollution in urban soils by means of MC-ICP-MS and TOF-SIMS.

    PubMed

    Rodríguez-Seijo, Andrés; Arenas-Lago, Daniel; Andrade, María Luisa; Vega, Flora A

    2015-05-01

    Lead pollution was evaluated in 17 urban soils from parks and gardens in the city of Vigo (NW Spain). The Pb isotope ratios ((207)Pb/(206)Pb, (208)Pb/(204)Pb, (206)Pb/(204)Pb and (208)Pb/(206)Pb) were determined after being measured by MC-ICP-MS. The association of the isotopes ((204)Pb, (206)Pb, (207)Pb and (208)Pb) with the different components of the soil was studied using TOF-SIMS. The isotopic ranges obtained for the samples were between 1.116 and 1.203 ((206)Pb/(207)Pb), 2.044-2.143 ((208)Pb/(206)Pb), 37.206-38.608 ((208)Pb/(204)Pb), 15.5482-15.6569 ((207)Pb/(204)Pb) and 17.357-18.826 ((206)Pb/(204)Pb). The application of the three-end-member model indicates that the Pb derived from petrol is the main source of Pb in the soils (43.51% on average), followed by natural or geogenic Pb (39.12%) and industrial emissions (17.37%). The emissions derived from coal combustion do not appear to influence the content of Pb in the soil. TOF-SIMS images show that the Pb mainly interacts with organic matter. This technique contributes to the understanding of the association of anthropogenic Pb with the components of the soil, as well as the particle size of these associations, thus allowing the possible sources of Pb to be identified.

  12. P2X3 Receptors and Sensory Transduction

    NASA Astrophysics Data System (ADS)

    Kennedy, Charles

    It has been known for many years that exogenously administered adenosine 5 -triphosphate (ATP) evokes acute pain, but the physiological and pathophysiological roles of endogenous ATP in nociceptive signalling are only now becoming clear. ATP produces its effects through P2X and P2Y receptors, and the P2X3 receptor is of notable importance. It shows a selective expression, at high levels in nociceptive sensory neurons, where it forms functional receptors on its own and in combination with the P2X2 receptor. Recent studies have used gene knockout methods, antisense oligonucleotides, small interfering RNA technologies, and a novel selective P2X3 antagonist, A-317491, to show that P2X3 receptors play a prominent role in both chronic inflammatory and neuropathic pain. Several other P2X subunits also appear to be expressed in sensory neurons and there is evidence for functional P2X1/5 or P2X2/6 heteromers in some of these. These data indicate that P2X receptors, particularly the P2X3 subtype, could be targetted in the search for new, effective analgesics.

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

    PubMed

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

    2017-01-01

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

  14. Metabolomic study of two rice lines infected by Rhizoctonia solani in negative ion mode by CE/TOF-MS.

    PubMed

    Suharti, Woro Sri; Nose, Akihiro; Zheng, Shao-Hui

    2016-11-01

    Rhizoctonia solani is a fungal pathogen that causes sheath blight disease in rice plants. In this study, metabolomic analysis using CE/TOF-MS in negative ion mode was used to investigate the resistance response of resistant and susceptible rice lines (32R and 29S, respectively) due to R. solani infection. Two rice lines showed different responses to the infection of R. solani. In 32R, R. solani infection induced significant increases in adenosine diphosphate (ADP), glyceric acid, mucic acid and jasmonic acid. In 29S, inosine monophosphate (IMP) was involved in the plant response to R. solani infection. Phenol compounds showed an increase as a response of the rice lines to R. solani infection. The study suggests that R. solani infection effects in 32R are associated with the induction of plant metabolic processes such as respiration, photorespiration, pectin synthesis, and lignin accumulation. In 29S, the R. solani infection is suggested to correlate with nitrogen metabolism.

  15. Metabolomic analysis of avocado fruits by GC-APCI-TOF MS: effects of ripening degrees and fruit varieties.

    PubMed

    Hurtado-Fernández, E; Pacchiarotta, T; Mayboroda, O A; Fernández-Gutiérrez, A; Carrasco-Pancorbo, A

    2015-01-01

    In order to investigate avocado fruit ripening, nontargeted GC-APCI-TOF MS metabolic profiling analyses were carried out. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to explore the metabolic profiles from fruit samples of 13 varieties at two different ripening degrees. Mannoheptulose; pentadecylfuran; aspartic, malic, stearic, citric and pantothenic acids; mannitol; and β-sitosterol were some of the metabolites found as more influential for the PLS-DA model. The similarities among genetically related samples (putative mutants of "Hass") and their metabolic differences from the rest of the varieties under study have also been evaluated. The achieved results reveal new insights into avocado fruit composition and metabolite changes, demonstrating therefore the value of metabolomics as a functional genomics tool in characterizing the mechanism of fruit ripening development, a key developmental stage in most economically important fruit crops.

  16. Identification of isomeric dicaffeoylquinic acids from Eleutherococcus senticosus using HPLC-ESI/TOF/MS and 1H-NMR methods.

    PubMed

    Tolonen, Ari; Joutsamo, Topi; Mattlla, Sampo; Kämäräinen, Terttu; Jalonen, Jorma

    2002-01-01

    Liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI/TOF/MS) and a novel NMR technique, developed to maximise the sensitivity obtained from the standard NMR spectrometer, have been applied to the identification of the phenolic constituents of Eleutherococcus senticosus. In addition, molecular modelling and dihedral bond angle calculations based on the vicinal 3JHH-coupling constants have been used in the unambiguous assignment of signals in the 1H-NMR spectra. 5'-O-Caffeoylquinic acid and three isomeric compounds, 1',5'-O-dicaffeoylquinic acid, 3',5'-O-dicaffeoylquinic acid and 4',5'-O-dicaffeoylquinic acid, have been isolated and identified from a sample. The isolation and structure determination of the latter two compounds are reported for the first time from this plant.

  17. Comparative Analysis of Volatile Composition in Chinese Truffles via GC × GC/HR-TOF/MS and Electronic Nose

    PubMed Central

    Zhang, Ning; Chen, Haitao; Sun, Baoguo; Mao, Xueying; Zhang, Yuyu; Zhou, Ying

    2016-01-01

    To compare the volatile compounds of Chinese black truffle and white truffle from Yunnan province, this study presents the application of a direct solvent extraction/solvent-assisted flavor evaporation (DSE-SAFE) coupled with a comprehensive two-dimensional gas chromatography (GC × GC) high resolution time-of-flight mass spectrometry (HR-TOF/MS) and an electronic nose. Both of the analytical methods could distinguish the aroma profile of the two samples. In terms of the overall profile of truffle samples in this research, more kinds of acids were detected via the method of DSE-SAFE. Besides, compounds identified in black truffle (BT), but not in white truffle (WT), or vice versa, and those detected in both samples at different levels were considered to play an important role in differentiating the two samples. According to the analysis of electronic nose, the two samples could be separated, as well. PMID:27058524

  18. 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

  19. Biogenic volatile organic compound analyses by PTR-TOF-MS: Calibration, humidity effect and reduced electric field dependency.

    PubMed

    Pang, Xiaobing

    2015-06-01

    Green leaf volatiles (GLVs) emitted by plants after stress or damage induction are a major part of biogenic volatile organic compounds (BVOCs). Proton transfer reaction time-of-flight mass spectrometry (PTR-TOF-MS) is a high-resolution and sensitive technique for in situ GLV analyses, while its performance is dramatically influenced by humidity, electric field, etc. In this study the influence of gas humidity and the effect of reduced field (E/N) were examined in addition to measuring calibration curves for the GLVs. Calibration curves measured for seven of the GLVs in dry air were linear, with sensitivities ranging from 5 to 10 ncps/ppbv (normalized counts per second/parts per billion by volume). The sensitivities for most GLV analyses were found to increase by between 20% and 35% when the humidity of the sample gas was raised from 0% to 70% relative humidity (RH) at 21°C, with the exception of (E)-2-hexenol. Product ion branching ratios were also affected by humidity, with the relative abundance of the protonated molecular ions and higher mass fragment ions increasing with humidity. The effect of reduced field (E/N) on the fragmentation of GLVs was examined in the drift tube of the PTR-TOF-MS. The structurally similar GLVs are acutely susceptible to fragmentation following ionization and the fragmentation patterns are highly dependent on E/N. Overall the measured fragmentation patterns contain sufficient information to permit at least partial separation and identification of the isomeric GLVs by looking at differences in their fragmentation patterns at high and low E/N.

  20. SELDI-TOF MS-based discovery of a biomarker in Cucumis sativus seeds exposed to CuO nanoparticles.

    PubMed

    Moon, Young-Sun; Park, Eun-Sil; Kim, Tae-Oh; Lee, Hoi-Seon; Lee, Sung-Eun

    2014-11-01

    Metal oxide nanoparticles (NPs) can inhibit plant seed germination and root elongation via the release of metal ions. In the present study, two acute phytotoxicity tests, seed germination and root elongation tests, were conducted on cucumber seeds (Cucumis sativus) treated with bulk copper oxide (CuO) and CuO NPs. Two concentrations of bulk CuO and CuO NPs, 200 and 600ppm, were used to test the inhibition rate of root germination; both concentrations of bulk CuO weakly inhibited seed germination, whereas CuO NPs significantly inhibited germination, showing a low germination rate of 23.3% at 600ppm. Root elongation tests demonstrated that CuO NPs were much stronger inhibitors than bulk CuO. SELDI-TOF MS analysis showed that 34 proteins were differentially expressed in cucumber seeds after exposure to CuO NPs, with the expression patterns of at least 9 proteins highly differing from those in seeds treated with bulk CuO and in control plants. Therefore, these 9 proteins were used to identify CuO NP-specific biomarkers in cucumber plants exposed to CuO NPs. A 5977-m/z protein was the most distinguishable biomarker for determining phytotoxicity by CuO NPs. Principal component analysis (PCA) of the SELDI-TOF MS results showed variability in the modes of inhibitory action on cucumber seeds and roots. To our knowledge, this is the first study to demonstrate that the phytotoxic effect of metal oxide NPs on plants is not caused by the same mode of action as other toxins.

  1. 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

  2. LC-MS-(TOF) analysis method for benzodiazepines in urine samples from alleged drug-facilitated sexual assault victims.

    PubMed

    ElSohly, Mahmoud A; Gul, Waseem; ElSohly, Kareem M; Avula, Bharathi; Khan, Ikhlas A

    2006-10-01

    In recent years there has been an increase in the number of reports in the U.S. of the use of drugs to commit sexual assault. In 1994, a nationwide urine testing program was developed to assess the incidence of the use of drugs to facilitate sexual assault and provide information for use in the investigation of these crimes. Urine samples were collected from victims of suspected drug-induced sexual assault by law enforcement agencies, emergency rooms, and rape crisis centers. The most implicated drug class was benzodiazepines, either alone or in combination with alcohol. In this report, a procedure was developed for the screening of 22 benzodiazepines in human urine by liquid chromatography-time of flight-mass spectrometry [LC-MS-(TOF)]. The limit of quantitation for all benzodiazepines ranged from 2 to 10 ng/mL, and the limit of detection was 0.5 to 3.0 ng/mL. These results suggest that the method sensitivity is suitable to screen for all 22 benzodiazepines in human urine at low levels. The method was used to analyze samples previously reported to have screened positive for benzodiazepines by immunoassay at 50 ng/mL cut off but failed to confirm by a gas chromatography-MS method. The results of reanalysis of these samples using this LC-MS method are reported.

  3. On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis.

    PubMed

    Li, Yan; Yan, Bo; Deng, Chunhui; Tang, Jia; Liu, Junyan; Zhang, Xiangmin

    2007-10-01

    In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.

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

    PubMed

    Wattal, C; Oberoi, J K

    2016-01-01

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

  5. Identification of Iridoids in Edible Honeysuckle Berries (Lonicera caerulea L. var. kamtschatica Sevast.) by UPLC-ESI-qTOF-MS/MS.

    PubMed

    Kucharska, Alicja Z; Fecka, Izabela

    2016-09-01

    Iridoid profiles of honeysuckle berry were studied. Compounds were identified by ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry UPLC-ESI-qTOF-MS/MS in positive and negative ions mode. The MS fragmentation pathways of detected iridoid glycosides were also studied in both modes. In the negative ESI mass spectra, iridoids with a methyl ester or lactone structure have preferentially produced adduct [M + HCOOH - H](-) ions. However, protonated ions of molecular fragments, which were released by glycosidic bond cleavage and following fragmentation of aglycone rings, were more usable for iridoid structure analysis. In addition, the neutral losses of H₂O, CO, CO₂, CH₃OH, acetylene, ethenone and cyclopropynone have provided data confirming the presence of functional substituents in the aglycone. Among the 13 iridoids, 11 were identified in honeysuckle berries for the first time: pentosides of loganic acid (two isomers), pentosides of loganin (three isomers), pentosyl sweroside, and additionally 7-epi-loganic acid, 7-epi-loganin, sweroside, secologanin, and secoxyloganin. The five pentoside derivatives of loganic acid and loganin have not been previously detected in the analyzed species. Honeysuckle berries are a source of iridoids with different structures, compounds that are rarely present in fruits.

  6. 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

  7. Optimization of matrix assisted desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) for the characterization of Bacillus and Brevibacillus species

    PubMed Central

    AlMasoud, Najla; Xu, Yun; Nicolaou, Nicoletta; Goodacre, Royston

    2014-01-01

    Over the past few decades there has been an increased interest in using various analytical techniques for detecting and identifying microorganisms. More recently there has been an explosion in the application of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for bacterial characterization, and here we optimize this approach in order to generate reproducible MS data from bacteria belonging to the genera Bacillus and Brevibacillus. Unfortunately MALDI-TOF-MS generates large amounts of data and is prone to instrumental drift. To overcome these challenges we have developed a preprocessing pipeline that includes baseline correction, peak alignment followed by peak picking that in combination significantly reduces the dimensionality of the MS spectra and corrects for instrument drift. Following this two different prediction models were used which are based on support vector machines and these generated satisfactory prediction accuracies of approximately 90%. PMID:25086893

  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.

  9. High-resolution MALDI-TOF MS study on analysis of low-molecular-weight products from photo-oxidation of poly(3-hexylthiophene).

    PubMed

    Mizukado, Junji; Sato, Hiroaki; Chen, Liang; Suzuki, Yasumasa; Yamane, Shogo; Aoyama, Yoshinori; Suda, Hiroyuki

    2015-08-01

    High-resolution matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) was used for the analysis of the low-molecular-weight products from the photo-oxidation of poly(3-hexylthiophene) (P3HT) in solution and thin film. Eight new peak series were observed in the low-mass range of the mass spectra of the products degraded in solution, and the formulas of the eight components were determined from the accurate mass. From SEC/MALDI-TOF MS, two components were identified as the degraded products, and the other six components were derived from the fragmentation of the degraded products during the MALDI process. A mechanism for the formation of these components was proposed on the basis of the results of MALDI-TOF MS. For the thin film degradation, a part of products in the solution degradation were observed, which supports that the oxidation of P3HT in solution and thin film proceeded in the same mechanism. This study shows that high-resolution MALDI-TOF MS is effective for the analysis of the low-molecular-weight products from P3HT photo-oxidation and expected to be feasible for the degradation analyses of other polymers. Copyright © 2015 John Wiley & Sons, Ltd.

  10. A sensitive method for detection of sulfamethazine and N4-acetylsulfamethazine residues in environmental samples using solid phase immunoextraction coupled with MALDI-TOF MS.

    PubMed

    Grant, G A; Frison, S L; Sporns, P

    2003-08-27

    Sulfamethazine (SMT) and its major metabolite, N(4)-acetylsulfamethazine (NA-SMT), were each recovered from spiked water (0.1 ppb) and 10% (w/v) aqueous suspensions of soil (1 ppb) or composted manure (1 ppb), by using a three-stage solid phase immunoextraction (SPIE) system, followed by detection with matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sulfonamide recovery rates are reported for separate stages of the SPIE system and for trace-level sulfonamide SPIE extraction from the environmental samples. SPIE MALDI-TOF MS is a rapid and definitive technique with potentially better efficiency relative to other established trace-level sulfonamide analytical methods. SPIE MALDI-TOF MS required 1.5 h per batch (8-24 samples/batch) for sample enrichment, 5 min per batch for probe preparation, and 5 min per sample to acquire and process the spectrum. This is the first time MALDI-TOF MS has been reported as a potential means of detecting trace-level drug residues in complex environmental samples.

  11. Identification of different respiratory viruses, after a cell culture step, by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS)

    PubMed Central

    Calderaro, Adriana; Arcangeletti, Maria Cristina; Rodighiero, Isabella; Buttrini, Mirko; Montecchini, Sara; Vasile Simone, Rosita; Medici, Maria Cristina; Chezzi, Carlo; De Conto, Flora

    2016-01-01

    In this study matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a reliable identification method for the diagnosis of bacterial and fungal infections, is presented as an innovative tool to investigate the protein profile of cell cultures infected by the most common viruses causing respiratory tract infections in humans. MALDI-TOF MS was applied to the identification of influenza A and B viruses, adenovirus C species, parainfluenza virus types 1, 2 and 3, respiratory syncytial virus, echovirus, cytomegalovirus and metapneumovirus. In this study MALDI-TOF MS was proposed as a model to be applied to the identification of cultivable respiratory viruses using cell culture as a viral proteins enrichment method to the proteome profiling of virus infected and uninfected cell cultures. The reference virus strains and 58 viruses identified from respiratory samples of subjects with respiratory diseases positive for one of the above mentioned viral agents by cell culture were used for the in vitro infection of suitable cell cultures. The isolated viral particles, concentrated by ultracentrifugation, were used for subsequent protein extraction and their spectra profiles were generated by MALDI-TOF MS analysis. The newly created library allowed us to discriminate between uninfected and respiratory virus infected cell cultures. PMID:27786297

  12. 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…

  13. 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

  14. 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.

  15. Investigation of serum protein profiles in scrapie infected sheep by means of SELDI-TOF-MS and multivariate data analysis

    PubMed Central

    2013-01-01

    Background Classical scrapie in sheep is a fatal neurodegenerative disease associated with the conversion PrPC to PrPSc. Much is known about genetic susceptibility, uptake and dissemination of PrPSc in the body, but many aspects of prion diseases are still unknown. Different proteomic techniques have been used during the last decade to investigate differences in protein profiles between affected animals and healthy controls. We have investigated the protein profiles in serum of sheep with scrapie and healthy controls by SELDI-TOF-MS and LC-MS/MS. Latent Variable methods such as Principal Component Analysis, Partial Least Squares-Discriminant Analysis and Target Projection methods were used to describe the MS data. Results The serum proteomic profiles showed variable differences between the groups both throughout the incubation period and at the clinical end stage of scrapie. At the end stage, the target projection model separated the two groups with a sensitivity of 97.8%, and serum amyloid A was identified as one of the protein peaks that differed significantly between the groups. Conclusions At the clinical end stage of classical scrapie, ten SELDI peaks significantly discriminated the scrapie group from the healthy controls. During the non-clinical incubation period, individual SELDI peaks were differently expressed between the groups at different time points. Investigations of differences in -omic profiles can contribute to new insights into the underlying disease processes and pathways, and advance our understanding of prion diseases, but comparison and validation across laboratories is difficult and challenging. PMID:24229425

  16. Identification of Proanthocyanidins from Litchi (Litchi chinensis Sonn.) Pulp by LC-ESI-Q-TOF-MS and Their Antioxidant Activity

    PubMed Central

    Lv, Qiang; Luo, Fenglei; Zhao, Xiaoyong; Liu, Yu; Hu, Guibing; Sun, Chongde; Li, Xian; Chen, Kunsong

    2015-01-01

    Content of total proanthocyanidins as well as total phenolics, flavonoids, antioxidant activities were evaluated for litchi (Litchi chinensis Sonn.) pulp of 32 cultivars. One cultivar, Hemaoli, showed the highest total proanthocyanidins and total phenolics, and DPPH or ABTS radical scavenging activities. ESI-MS and NMR analysis of the Hemaoli pulp crude extracts (HPCE) showed that procyandins composed of (epi)catechin unites with degree of polymerization (DP) of 2–6 were dominant proanthocyanidins in HPCE. After the HPCE was fractionated by a Sephadex LH-20 column, 32 procyanidins were identified by LC-ESI-Q-TOF-MS in litchi pulp for the first time. Quantification of individual procyanidin in HPCE indicated that epicatechin, procyanidin B2, procyanidin C1 and A-type procyanidin trimer were the main procyanidins. The radical scavenging activities of different fractions of HPCE as well as six procyanidins standards were evaluated by both DPPH and ABTS assays. HPCE fractions showed similar antioxidant activities with those of Vc and six individual procyanidins, the IC50 of which ranged from 1.88 ± 0.01 to 2.82 ± 0.10 μg/ml for DPPH assay, and from 1.52 ± 0.17 to 2.71 ± 0.15 μg/ml for ABTS assay. Such results indicate that litchi cultivars rich in proanthocyanidins are good resources of dietary antioxidants and have the potential to contribute to human health. PMID:25793378

  17. Metabolome classification of commercial Hypericum perforatum (St. John's Wort) preparations via UPLC-qTOF-MS and chemometrics.

    PubMed

    Farag, Mohamed A; Wessjohann, Ludger A

    2012-03-01

    The growing interest in the efficacy of phytomedicines and herbal supplements but also the increase in legal requirements for safety and reliable contents of active principles drive the development of analytical methods for the quality control of complex, multicomponent mixtures as found in plant extracts of value for the pharmaceutical industry. Here, we describe an ultra-performance liquid chromatography method (UPLC) coupled with quadrupole time of flight mass spectrometry (qTOF-MS) measurements for the large scale analysis of H. perforatum plant material and its commercial preparations. Under optimized conditions, we were able to simultaneously quantify and identify 21 metabolites including 4 hyperforins, 3 catechins, 3 naphthodianthrones, 5 flavonoids, 3 fatty acids, and a phenolic acid. Principal component analysis (PCA) was used to ensure good analytical rigorousness and define both similarities and differences among Hypericum samples. A selection of batches from 9 commercially available H. perforatum products available on the German and Egyptian markets showed variable quality, particularly in hyperforins and fatty acid content. PCA analysis was able to discriminate between various preparations according to their global composition, including differentiation between various batches from the same supplier. To the best of our knowledge, this study provides the first approach utilizing UPLC-MS-based metabolic fingerprinting to reveal secondary metabolite compositional differences in Hypericum extract.

  18. Metabolomics driven analysis of artichoke leaf and its commercial products via UHPLC-q-TOF-MS and chemometrics.

    PubMed

    Farag, Mohamed A; El-Ahmady, Sherweit H; Elian, Fatma S; Wessjohann, Ludger A

    2013-11-01

    The demand to develop efficient and reliable analytical methods for the quality control of herbal medicines and nutraceuticals is on the rise, together with an increase in the legal requirements for safe and consistent levels of active principles. Here, we describe an ultra-high performance liquid chromatography method (UHPLC) coupled with quadrupole high resolution time of flight mass spectrometry (qTOF-MS) analysis for the comprehensive measurement of metabolites from three Cynara scolymus (artichoke) cultivars: American Green Globe, French Hyrious, and Egyptian Baladi. Under optimized conditions, 50 metabolites were simultaneously quantified and identified including: eight caffeic acid derivatives, six saponins, 12 flavonoids and 10 fatty acids. Principal component analysis (PCA) was used to define both similarities and differences among the three artichoke leaf cultivars. In addition, batches from seven commercially available artichoke market products were analysed and showed variable quality, particularly in caffeic acid derivatives, flavonoid and fatty acid contents. PCA analysis was able to discriminate between various preparations, including differentiation between various batches from the same supplier. To the best of our knowledge, this study provides the first approach utilizing UHPLC-MS based metabolite fingerprinting to reveal secondary metabolite compositional differences in artichoke leaf extracts.

  19. Study of tungstate-protein interaction in human serum by LC-ICP-MS and MALDI-TOF.

    PubMed

    Rodríguez-Fariñas, Nuria; Gomez-Gomez, M Milagros; Camara-Rica, Carmen

    2008-01-01

    Oral administration of sodium tungstate is an effective treatment for type 1 and 2 diabetes in animal models; it does not incur significant side effects, and it may constitute an alternative to insulin. However, the mechanism by which tungstate exerts its observed metabolic effects in vivo is still not completely understood. In this work, serum-containing proteins which bind tungstate have been characterized. Size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) with a Phenomenex Bio-Sep-S 2000 column and 20 mM HEPES and 150 mM NaCl at pH 7.4 as the mobile phase was chosen as the most appropriate methodology to screen for tungsten-protein complexes. When human serum was incubated with tungstate, three analytical peaks were observed, one related to tungstate-albumin binding, one to free tungstate, and one to an unknown protein binding (MW higher than 300 kDa). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of the tungsten-containing fractions collected from SEC-ICP-MS chromatograms, after desalting and preconcentration processes, confirmed the association of tungstate with albumin and the other unknown protein. [figure: see text

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

    PubMed

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

    2014-05-01

    The mutualisation of analytical platforms might be used to address rising healthcare costs. Our study aimed to evaluate the feasibility of networking a unique matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) system for common use in several university hospitals in Brussels, Belgium. During a one-month period, 1,055 successive bacterial isolates from the Brugmann University Hospital were identified on-site using conventional techniques; these same isolates were also identified using a MALDI-TOF MS system at the Porte de Hal Laboratory by sending target plates and identification projects via transportation and the INFECTIO_MALDI software (Infopartner, Nancy, France), respectively. The occurrence of transmission problems (<2 %) and human errors (<1 %) suggested that the system was sufficiently robust to be implemented in a network. With a median time-to-identification of 5 h and 11 min (78 min, min-max: 154-547), MALDI-TOF MS networking always provided a faster identification result than conventional techniques, except when chromogenic culture media and oxidase tests were used (p < 0.0001). However, the limited clinical benefits of the chromogenic culture media do not support their extra cost. Our financial analysis also suggested that MALDI-TOF MS networking could lead to substantial annual cost savings. MALDI-TOF MS networking presents many advantages, and few conventional techniques (optochin and oxidase tests) are required to ensure the same quality in patient care from the distant laboratory. Nevertheless, such networking should not be considered unless there is a reorganisation of workflow, efficient communication between teams, qualified technologists and a reliable IT department and helpdesk to manage potential connectivity problems.

  1. 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.

  2. Rapid identification of bacteria from positive blood culture bottles by MALDI-TOF MS following short-term incubation on solid media.

    PubMed

    Altun, Osman; Botero-Kleiven, Silvia; Carlsson, Sarah; Ullberg, Måns; Özenci, Volkan

    2015-11-01

    Rapid identification of bacteria from blood cultures enables early initiation of appropriate antibiotic treatment in patients with bloodstream infections (BSI). The objective of the present study was to evaluate the use of matrix-associated laser desorption ionization-time of flight (MALDI-TOF) MS after a short incubation on solid media for rapid identification of bacteria from positive blood culture bottles. MALDI-TOF MS was performed after 2.5 and 5.5 h plate incubation of samples from positive blood cultures. Identification scores with values ≥ 1.7 were accepted as successful identification if the results were confirmed by conventional methods. Conventional methods included MALDI-TOF MS, Vitek 2, and diverse biochemical and agglutination tests after overnight culture. In total, 515 positive blood cultures with monomicrobial bacterial growth representing one blood culture per patient were included in the study. There were 229/515 (44.5%) and 286/515 (55.5%) blood culture bottles with Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB), respectively. MALDI-TOF MS following short-term culture could accurately identify 300/515 (58.3%) isolates at 2.5 h, GNB being identified in greater proportion (180/229; 78.6%) than GPB (120/286; 42.0%). In an additional 124/515 bottles (24.1%), identification was successful at 5.5 h, leading to accurate identification of bacteria from 424/515 (82.3%) blood cultures after short-term culture. Interestingly, 11/24 of the isolated anaerobic bacteria could be identified after 5.5 h. The present study demonstrates, in a large number of clinical samples, that MALDI-TOF MS following short-term culture on solid medium is a reliable and rapid method for identification of bacteria from blood culture bottles with monomicrobial bacterial growth.

  3. Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS

    PubMed Central

    Liang, Boying; Ju, Yue; Joubert, James R.; Kaleta, Erin J.; Lopez, Rodrigo; Jones, Ian W.; Hall, Henry K.; Ratnayaka, Saliya N.; Wysocki, Vicki H.; Saavedra, S. Scott

    2015-01-01

    Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors, however, the matrices and high vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-SorbPC) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B-subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS based on differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin B as a model receptor-ligand pair, the minimal detectable concentration of toxin was estimated to be 4 nM. On-plate trypsin digestion of bound cholera toxin B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner. PMID:25694144

  4. Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS.

    PubMed

    Liang, Boying; Ju, Yue; Joubert, James R; Kaleta, Erin J; Lopez, Rodrigo; Jones, Ian W; Hall, Henry K; Ratnayaka, Saliya N; Wysocki, Vicki H; Saavedra, S Scott

    2015-04-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4 nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.

  5. 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

  6. Forced degradation, LC-UV, MS(n) and LC-MS-TOF studies on azilsartan: Identification of a known and three new degradation impurities.

    PubMed

    Kaushik, Dhiraj; Kaur, Jasmeen; Paul Kaur, Vaneet; Saini, Balraj; Bansal, Yogita; Bansal, Gulshan

    2016-02-20

    In the present study, Azilsartan (AZL) was subjected to ICH recommended forced degradation conditions of hydrolysis, oxidation, dry heat and photolysis. The drug degraded to four degradation products (I-IV) under acidic, alkaline and water hydrolysis and photolysis. All the four degradation products were resolved in a single run on a C-18 column (250mm×4.6mm; 5μ) with isocratic elution using mobile phase composed of ammonium formate (20mM, pH 3.0), methanol and acetonitrile (40:5:40% v/v), at a flow rate of 0.8mlmin(-1) at ambient temperature. The products were characterized through +ESI-MS(n) spectra of AZL and LC-MS-TOF studies as 2-ethoxy-3H-benzo-imidazole-4-carboxylic acid (I), 2-hydroxy-3-[2'-(5-oxo-4,5-dihydro-[1,2,4]oxadiazol-4-ylmethyl]-3H-benzoimidazole-4-carboxylic acid (II, deethylated AZL), 3-[2'-(1H-diazirin-3-yl)-biphenyl]-4-ylmethyl]-2-ethoxy-3H-benzoimidazole-4-carboxylic acid (III), and 3-[4'-(2-ethoxy-benzo-imidazol-1-ylmethyl)-biphenyl-2-yl]-4H-[1,2,4]oxadiazol-5-one (IV, decarboxylated AZL). Product I was found to be a known process related impurity whereas the products II-IV were identified as new degradation impurities. The most probable mechanisms for formation of these degradation products were proposed.

  7. Study on degradation kinetics of 2-(2-hydroxypropanamido) benzoic acid in aqueous solutions and identification of its major degradation product by UHPLC/TOF-MS/MS.

    PubMed

    Zhang, Qili; Guan, Jiao; Rong, Rong; Zhao, Yunli; Yu, Zhiguo

    2015-08-10

    A RP-HPLC method was developed and validated for the degradation kinetic study of 2-(2-hydroxypropanamido) benzoic acid (HPABA), a promising anti-inflammatory drug, which would provide a basis for further studies on HPABA. The effects of pH, temperature, buffer concentration and ionic strength on the degradation kinetics of HPABA were discussed. Experimental parameters such as degradation rate constants (k), activation energy (Ea), acid and alkali catalytic constants (k(ac), k(al)), shelf life (t1/2) and temperature coefficient (Q10) were calculated. The results indicated that degradation kinetics of HPABA followed zero-order reaction kinetics; degradation rate constants (k) of HPABA at different pH values demonstrated that HPABA was more stable in neutral and near-neutral conditions; the function of temperature on k obeyed the Arrhenius equation (r = 0.9933) and HPABA was more stable at lower temperature; with the increase of ionic strength and buffer concentration, the stability of HPABA was decreased. The major unknown degradation product of HPABA was identified by UHPLC/TOF-MS/MS with positive electrospray ionization. Results demonstrated that the hydrolysis product was the primary degradation product of HPABA and it was deduced as anthranilic acid.

  8. Immobilized fusion protein affinity chromatography combined with HPLC-ESI-Q-TOF-MS/MS for rapid screening of PPARγ ligands from natural products.

    PubMed

    Zhu, Junfeng; Yi, Xiaojiao; Liu, Wenhui; Xu, Yingchun; Chen, Shuqing; Wu, Yongjiang

    2017-04-01

    Screening agonists of peroxisome proliferator-activated receptor-γ (PPARγ) from natural products is particularly motivated by the need for effective anti-diabetic agents. However, method for direct identification of PPARγ ligands from a complex sample is rarely reported. Here we propose a novel immobilized fusion protein affinity chromatography (IFPAC) to achieve rapid multicomponent screening. First, functional human PPARγ ligand binding domain (hPPARγLBD) was bacterially produced by fusion to glutathione S-transferase (GST). The unpurified GST-hPPARγLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose. Due to the strong affinity between GST and glutathione, the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization, which was rapidly purified under non-denaturing conditions. Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds. The proposed approach was applied in the high-throughput screening of PPARγ ligands from natural products, followed by rapid characterization of active compounds using HPLC-ESI-Q-TOF-MS/MS. Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARγ ligand. Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.

  9. 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

  10. Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS).

    PubMed

    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.

  11. 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

  12. 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

    2015-09-09

    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.

  13. Identification and characterization of stress degradants of lacosamide by LC-MS and ESI-Q-TOF-MS/MS: development and validation of a stability indicating RP-HPLC method.

    PubMed

    Ramisetti, Nageswara Rao; Kuntamukkala, Ramakrishna; Lakshetti, Sridhar; Sripadi, Prabhakar

    2014-07-01

    The current study dealt with the degradation behavior of lacosamide (LAC) under ICH prescribed stress conditions. LAC was found to be labile under acid and base hydrolytic stress conditions, while it was stable to neutral hydrolytic, oxidative, photolytic and thermal stress. In total, seven degradation products (DPs) were formed, which were separated on a C18 column using a stability-indicating method. LC-MS analyses indicated that one of the DPs had the same molecular mass as that of the drug. Structural characterization of DPs was carried out using ESI-Q-TOF-MS/MS technique. The degradation pathways and mechanisms of degradation of the drug were delineated by carrying out the degradation in different co-solvents viz. methanol, deuterated methanol, ethanol, 1-propanol and acetonitrile. The developed LC method was validated for the determination of related substances and assay of LAC as per ICH guidelines. This study demonstrates a comprehensive approach of LAC degradation studies during its development phase.

  14. 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

  15. Undisturbed and disturbed above canopy ponderosa pine emissions: PTR-TOF-MS measurements and MEGAN 2.1 model results

    NASA Astrophysics Data System (ADS)

    Kaser, L.; Karl, T.; Guenther, A.; Graus, M.; Schnitzhofer, R.; Turnipseed, A.; Fischer, L.; Harley, P.; Madronich, M.; Gochis, D.; Keutsch, F. N.; Hansel, A.

    2013-12-01

    We present the first eddy covariance flux measurements of volatile organic compounds (VOCs) using a proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS) above a ponderosa pine forest in Colorado, USA. The high mass resolution of the PTR-TOF-MS enabled the identification of chemical sum formulas. During a 30 day measurement period in August and September 2010, 649 different ion mass peaks were detected in the ambient air mass spectrum (including primary ions and mass calibration compounds). Eddy covariance with the vertical wind speed was calculated for all ion mass peaks. On a typical day, 17 ion mass peaks, including protonated parent compounds, their fragments and isotopes as well as VOC-H+-water clusters, showed a significant flux with daytime average emissions above a reliable flux threshold of 0.1 mg compound m-2 h-1. These ion mass peaks could be assigned to seven compound classes. The main flux contributions during daytime (10:00-18:00 LT) are attributed to the sum of 2-methyl-3-buten-2-ol (MBO) and isoprene (50%), methanol (12%), the sum of acetic acid and glycolaldehyde (10%) and the sum of monoterpenes (10%). The total MBO + isoprene flux was composed of 10% isoprene and 90% MBO. There was good agreement between the light- and temperature dependency of the sum of MBO and isoprene observed for this work and those of earlier studies. The above canopy flux measurements of the sum of MBO and isoprene and the sum of monoterpenes were compared to emissions calculated using the Model of Emissions of Gases and Aerosols from Nature (MEGAN 2.1). The best agreement between MEGAN 2.1 and measurements was reached using emission factors determined from site-specific leaf cuvette measurements. While the modeled and measured MBO + isoprene fluxes agree well, the emissions of the sum of monoterpenes is underestimated by MEGAN 2.1. This is expected as some factors impacting monoterpene emissions, such as physical damage of needles and branches due to

  16. Undisturbed and disturbed above canopy ponderosa pine emissions: PTR-TOF-MS measurements and MEGAN 2.1 model results

    NASA Astrophysics Data System (ADS)

    Kaser, L.; Karl, T.; Guenther, A.; Graus, M.; Schnitzhofer, R.; Turnipseed, A.; Fischer, L.; Harley, P.; Madronich, M.; Gochis, D.; Keutsch, F. N.; Hansel, A.

    2013-06-01

    We present the first eddy covariance flux measurements of volatile organic compounds (VOCs) using a proton-transfer-reaction time-of-flight mass-spectrometer (PTR-TOF-MS) above a ponderosa pine forest in Colorado, USA. The high mass resolution of the PTR-TOF-MS enabled the identification of chemical sum formulas. During a 30 day measurement period in August and September 2010, 649 different ion mass peaks were detected in the ambient air mass spectrum (including primary ions and mass calibration compounds). Eddy covariance with the vertical wind speed was calculated for all ion mass peaks. On a typical day, 17 ion mass peaks including protonated parent compounds, their fragments and isotopes as well as VOC-H+-water clusters showed a significant flux with daytime average emissions above a reliable flux threshold of 0.1 mg compound m-2 h-1. These ion mass peaks could be assigned to seven compound classes. The main flux contributions during daytime (10:00-18:00 LT) are attributed to the sum of 2-methyl-3-buten-2-ol (MBO) and isoprene (50%), methanol (12%), the sum of acetic acid and glycolaldehyde (10%) and the sum of monoterpenes (10%). The total MBO + isoprene flux was composed of 10% isoprene and 90% MBO. There was good agreement between the light and temperature dependency of the sum of MBO and isoprene observed for this work and those of earlier studies. The above canopy flux measurements of the sum of MBO and isoprene and the sum of monoterpenes were compared to emissions calculated using the Model of Emissions of Gases and Aerosols from Nature (MEGAN 2.1). The best agreement between MEGAN 2.1 and measurements was reached using emission factors determined from site specific leaf cuvette measurements. While the modelled and measured MBO + isoprene fluxes agree well the emissions of the sum of monoterpenes is underestimated by MEGAN 2.1. This is expected as some factors impacting monoterpene emissions, such as physical damage of needles and branches due to storms

  17. Characterization of Printing Inks Using DART-Q-TOF-MS and Attenuated Total Reflectance (ATR) FTIR.

    PubMed

    Williamson, Rhett; Raeva, Anna; Almirall, Jose R

    2016-05-01

    The rise in improved and widely accessible printing technology has resulted in an interest to develop rapid and minimally destructive chemical analytical techniques that can characterize printing inks for forensic document analysis. Chemical characterization of printing inks allows for both discrimination of inks originating from different sources and the association of inks originating from the same source. Direct analysis in real-time mass spectrometry (DART-MS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) were used in tandem to analyze four different classes of printing inks: inkjets, toners, offset, and intaglio. A total of 319 samples or ~ 80 samples from each class were analyzed directly on a paper substrate using the two methods. DART-MS was found to characterize the semi-volatile polymeric vehicle components, while ATR-FTIR provided chemical information associated with the bulk components of these inks. Complimentary data results in improved discrimination when both techniques are used in succession resulting in >96% discrimination for all toners, 95% for all inkjets, >92% for all offset, and >54% for all intaglio inks.

  18. Generators for the elliptic curve y2 = x3-nx

    NASA Astrophysics Data System (ADS)

    Fujita, Yasutsugu; Terai, Nobuhiro

    2010-07-01

    Let E:y2 = x3-nx be an elliptic curve over the rationals with a positive integer n. Mordell's theorem asserts that the group of rational points on E is finitely generated. Our interest is in the generators for its free part. Duquesne (2007) showed that if n = (2k2-2k+1)(18k2+30k+17) is square-free, then certain two points of infinite order can always be in a system of generators. We generalize this result and show that the same is true for "infinitely many" infinite families n = n(k,l) with two variables.

  19. Antioxidant and metabolite profiling of North American and neotropical blueberries using LC-TOF-MS and multivariate analyses.

    PubMed

    Ma, Chunhui; Dastmalchi, Keyvan; Flores, Gema; Wu, Shi-Biao; Pedraza-Peñalosa, Paola; Long, Chunlin; Kennelly, Edward J

    2013-04-10

    There are many neotropical blueberries, and recent studies have shown that some have even stronger antioxidant activity than the well-known edible North American blueberries. Antioxidant marker compounds were predicted by applying multivariate statistics to data from LC-TOF-MS analysis and antioxidant assays of 3 North American blueberry species (Vaccinium corymbosum, Vaccinium angustifolium, and a defined mixture of Vaccinium virgatum with V. corymbosum) and 12 neotropical blueberry species (Anthopterus wardii, Cavendishia grandifolia, Cavendishia isernii, Ceratostema silvicola, Disterigma rimbachii, Macleania coccoloboides, Macleania cordifolia, Macleania rupestris, Satyria boliviana, Sphyrospermum buxifolium, Sphyrospermum cordifolium, and Sphyrospermum ellipticum). Fourteen antioxidant markers were detected, and 12 of these, including 7 anthocyanins, 3 flavonols, 1 hydroxycinnamic acid, and 1 iridoid glycoside, were identified. This application of multivariate analysis to bioactivity and mass data can be used for identification of pharmacologically active natural products and may help to determine which neotropical blueberry species will be prioritized for agricultural development. Also, the compositional differences between North American and neotropical blueberries were determined by chemometric analysis, and 44 marker compounds including 16 anthocyanins, 15 flavonoids, 7 hydroxycinnamic acid derivatives, 5 triterpene glycosides, and 1 iridoid glycoside were identified.

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

    PubMed Central

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

    2015-01-01

    Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of card­iolipin (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

  1. 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.

  2. Detection of 191 Taxifolin Metabolites and Their Distribution in Rats Using HPLC-ESI-IT-TOF-MS(n).

    PubMed

    Yang, Ping; Xu, Feng; Li, Hong-Fu; Wang, Yi; Li, Feng-Chun; Shang, Ming-Ying; Liu, Guang-Xue; Wang, Xuan; Cai, Shao-Qing

    2016-09-13

    Taxifolin is a ubiquitous bioactive constituent of foods and herbs. To thoroughly explore its metabolism in vivo, an HPLC-ESI-IT-TOF-MS(n) method combined with specific metabolite detection strategy was used to detect and identify the metabolites of taxifolin in rats. Of the 191 metabolites tentatively identified, 154 were new metabolites, 69 were new compounds and 32 were dimers. This is the first report of the in vivo biotransformation of a single compound into more than 100 metabolites. Furthermore, acetylamination and pyroglutamic acid conjugation were identified as new metabolic reactions. Seventeen metabolites were found to have various taxifolin-related bioactivities. The potential targets of taxifolin and 63 metabolites were predicted using PharmMapper, with results showing that more than 60 metabolites have the same five targets. Metabolites with the same fragment pattern may have the same pharmacophore. Thus these metabolites may exert the same pharmacological effects as taxifolin through an additive effect on the same drug targets. This observation indicates that taxifolin is bioactive not only in the parent form, but also through its metabolites. These findings enhance understanding of the metabolism and effective forms of taxifolin and may provide further insight of the beneficial effects of taxifolin and its derivatives.

  3. Derivatization of organophosphorus nerve agent degradation products for gas chromatography with ICPMS and TOF-MS detection.

    PubMed

    Richardson, Douglas D; Caruso, Joseph A

    2007-06-01

    Separation and detection of seven V-type (venomous) and G-type (German) organophosphorus nerve agent degradation products by gas chromatography with inductively coupled plasma mass spectrometry (GC-ICPMS) is described. The nonvolatile alkyl phosphonic acid degradation products of interest included ethyl methylphosphonic acid (EMPA, VX acid), isopropyl methylphosphonic acid (IMPA, GB acid), ethyl hydrogen dimethylamidophosphate sodium salt (EDPA, GA acid), isobutyl hydrogen methylphosphonate (IBMPA, RVX acid), as well as pinacolyl methylphosphonic acid (PMPA), methylphosphonic acid (MPA), and cyclohexyl methylphosphonic acid (CMPA, GF acid). N-(tert-Butyldimethylsilyl)-N-methyltrifluroacetamide with 1% TBDMSCl was utilized to form the volatile TBDMS derivatives of the nerve agent degradation products for separation by GC. Exact mass confirmation of the formation of six of the TBDMS derivatives was obtained by GC-time of flight mass spectrometry (TOF-MS). The method developed here allowed for the separation and detection of all seven TBDMS derivatives as well as phosphate in less than ten minutes. Detection limits for the developed method were less than 5 pg with retention times and peak area precisions of less than 0.01 and 6%, respectively. This method was successfully applied to river water and soil matrices. To date this is the first work describing the analysis of chemical warfare agent (CWA) degradation products by GC-ICPMS.

  4. UPLC-DAD/Q-TOF-MS Based Ingredients Identification and Vasorelaxant Effect of Ethanol Extract of Jasmine Flower.

    PubMed

    Yin, Yongqiang; Ying, Xuhui; Luan, Hairong; Zhao, Zhenying; Lou, Jianshi; Wang, Deli; Li, Hailin; Wu, Hong

    2014-01-01

    Chinese people commonly make jasmine tea for recreation and health care. Actually, its medicinal value needs more exploration. In this study, vasorelaxant effect of ethanol extract of jasmine flower (EEJ) on isolated rat thoracic aorta rings was investigated and [Ca(2+)] was determined in vascular smooth muscle cells by laser scanning confocal microscope (LSCM). The result of aorta rings showed that EEJ could cause concentration-dependent relaxation of endothelium-intact rings precontracted with phenylephrine or KCl which was attenuated after preincubation of the rings with L-NAME and three different K(+) channel inhibitors; however, indomethacin and glibenclamide did not affect the vasodilatation of EEJ. In addition, EEJ could inhibit contraction induced by PE on endothelium-denuded rings in Ca(2+)-free medium as well as by accumulation of Ca(2+) in Ca(2+)-free medium with high K(+). LSCM also showed that EEJ could lower the elevated level of [Ca(2+)] induced by KCl. These indicate that the vasodilation of EEJ is in part related to causing the release of nitric oxide, activation of K(+) channels, inhibition of influx of excalcium, and release of calcium from sarcoplasmic reticulum. A total of 20 main ingredients, were identified in EEJ by UPLC-DAD/Q-TOF-MS. The vasodilation activity should be attributed to the high content of flavonoid glycosides and iridoid glycosides found in EEJ.

  5. UPLC TOF MS for sensitive quantification of naturally occurring pyrrolizidine alkaloids in Petasites hybridus extract (Ze 339).

    PubMed

    Schenk, Alexander; Siewert, Beate; Toff, Stephan; Drewe, Jürgen

    2015-08-01

    Due to increasing regulatory awareness of their hepatotoxic, genotoxic and possibly carcinogenic potential, pyrrolizidine alkaloid (PA) content has to be thoroughly monitored in herbal medicinal preparations. Recently, new very low PA regulatory threshold concentrations have been requested by the authorities. Therefore, a highly sensitive and reproducible UPLC TOF MS method for the quantification of the PAs senkirkine, senecionine, seneciphylline, senecionine-N-oxide and seneciphylline-N-oxide in a CO2-extract of Petasites hybridus leaves (Ze 339) has been developed. The limit of quantification (LOQ) was 2ppb for all PAs. Recovery at the LOQ was between 88.9 and 141.9%, the repeatability precision between 3.5 and 13.6%. Linearity of the five PAs showed correlation coefficients between 0.9995 and 0.9998 and coefficients of variation between 7.44 and 8.56%. A working range between 2 ppb and 200 ppb could be fixed. In the tested batches of the P. hybridus extract Ze 339, the absence of PAs could be demonstrated. In conclusion, this assay allows to determine trace PA concentrations in P. hybridus extract Ze 339, making it suitable for analytical PA monitoring in accordance with regulatory requirements.

  6. Immunoadjuvant activity, toxicity assays, and determination by UPLC/Q-TOF-MS of triterpenic saponins from Chenopodium quinoa seeds.

    PubMed

    Verza, Simone G; Silveira, Fernando; Cibulski, Samuel; Kaiser, Samuel; Ferreira, Fernando; Gosmann, Grace; Roehe, Paulo M; Ortega, George G

    2012-03-28

    The adjuvant activity of Chenopodium quinoa (quinoa) saponins on the humoral and cellular immune responses of mice subcutaneously immunized with ovalbumin (OVA) was evaluated. Two quinoa saponin fractions were obtained, FQ70 and FQ90, and 10 saponins were determined by UPLC/Q-TOF-MS. Mice were immunized subcutaneously with OVA alone or adjuvanted with Quil A (adjuvant control), FQ70, or FQ90. FQ70 and FQ90 significantly enhanced the amount of anti-OVA-specific antibodies in serum (IgG, IgG1, and IgG2b) in immunized mice. The adjuvant effect of FQ70 was significantly greater than that of FQ90. However, delayed type hypersensitivity responses were higher in mice immunized with OVA adjuvanted with FQ90 than mice treated with FQ70. Concanavalin A (Con A)-, lipopolysaccharide-, and OVA-stimulated splenocyte proliferation were measured, and FQ90 significantly enhanced the Con A-induced splenocyte proliferation. The results suggested that the two quinoa saponin fractions enhanced significantly the production of humoral and cellular immune responses to OVA in mice.

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

    PubMed

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

    2015-09-22

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

  8. Characterization of stress degradation products of duloxetine hydrochloride employing LC-UV/PDA and LC-MS/TOF studies.

    PubMed

    Chadha, Renu; Bali, Alka; Bansal, Gulshan

    2016-03-20

    Duloxetine HCl was subjected to forced degradation under conditions of hydrolysis (neutral, acidic and alkaline), oxidation, photolysis and thermal stress, as suggested in the ICH guideline Q1A(R2). The drug showed significant degradation under acidic, alkaline and aqueous hydrolytic as well as photolytic conditions. The drug remained stable under thermal and oxidative stress conditions. In total, seventeen degradation products (I-XVII) were formed under varied conditions, which could be separated by chromatography of respective degraded solutions on C18 (250 mm×4.6 mm; 5 μ, Nulceodur) column using isocratic elution method. Detection wavelength was selected as 290 nm. MS/TOF accurate mass studies were carried out to establish the complete fragmentation pathway of the drug and degradation products, which, in turn, was utilized in characterization of the products. The degradation pathway of the drug leading to generation of fifteen products I-X, XII-XIII, XV-XVII was postulated and this has not been reported so far.

  9. 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

  10. 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.

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

    PubMed

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

    2013-12-01

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

  12. Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

    PubMed Central

    Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Huestis, Marilyn A

    2014-01-01

    Background Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. Conclusion For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods. PMID:25046048

  13. The comparative research on constituents of Radix Aconiti and its processing by HPLC quadrupole TOF-MS.

    PubMed

    Wu, Jian; Hong, Bo; Wang, Jia; Wang, Xi; Niu, Sijia; Zhao, Chunjie

    2012-11-01

    Based upon the regulations stipulated by the State Food and Drug Administration of China, only the processed, detoxified tubers and roots of Aconitum are allowed to be administered orally, used in clinical decoctions and adopted as raw materials for pharmaceutical manufacturing, so the processing principle of preparation of Radix Aconiti is important for ensuring the Radix Aconiti praeparata quality. A simple approach was described for HPLC-Q-TOF-MS screening and identification of many of the aconitine alkaloids present in unprocessed Radix Aconiti and Radix Aconiti praeparata. To compare their fingerprints, the processing principle of preparation of Radix Aconiti was developed. Twenty-nine compounds and 26 compounds were assigned to aconitine alkaloids and tentatively identified by comparing accurate mass and fragments information with that of the authentic standards or by mass spectrometry analysis and retrieving the reference literature. The nonester alkaloids were almost the same. The diester diterpene alkaloids were decreased, the monoester-diterpene alkaloids were increased and lipo-alkaloids decreased obviously in the processing of the preparation. These transformed components could be regarded as potential chemical markers that can be used to distinguish between raw and processed herbs.

  14. Capillary and gel electromigration techniques and MALDI-TOF MS--suitable tools for identification of filamentous fungi.

    PubMed

    Horká, Marie; Kubesová, Anna; Salplachta, Jiří; Zapletalová, Eva; Horký, Jaroslav; Slais, Karel

    2012-02-24

    Microbial strains are now spreading out of their original geographical areas of incidence and previously adequate morphological identification methods often must be accompanied by a phenotypic characterization for the successful microbial identification. The fungal genus Monilinia represents a suitable example. Monilinia species represent important fruit pathogens responsible for major losses in fruit production. Four closely related spp. of Monilinia: Monilinia laxa, Monilinia fructigena, Monilinia fructicola and Monilia polystroma have been yet identified. However, the classical characterization methods are not sufficient for current requirements, especially for phytosanitary purposes. In this study, rapid and reproducible methods have been developed for the characterization of Monilinia spp. based on the utilization of five well-established analytical techniques: CZE, CIEF, gel IEF, SDS-PAGE and MALDI-TOF MS, respectively. The applicability of these techniques for the identification of unknown spores of Monilinia spp. collected from infected fruits was also evaluated. It was found that isoelectric points, migration velocities or the protein patterns can be used as the identification markers in the case of cultivated filamentous fungi. Moreover, the results obtained by capillary electromigration techniques are independent on the host origin of the spores. On the other hand, the host origin of the fungi can play an important role in the precise fungi identification by the other techniques.

  15. Nano-structured support materials, their characterisation and serum protein profiling through MALDI/TOF-MS.

    PubMed

    Najam-Ul-Haq, M; Rainer, M; Heigl, N; Szabo, Z; Vallant, R; Huck, C W; Engelhardt, H; Bischoff, K-D; Bonn, G K

    2008-02-01

    In the bioanalytical era, novel nano-materials for the selective extraction, pre-concentration and purification of biomolecules prior to analysis are vital. Their application as affinity binding in this regard is needed to be authentic. We report here the comparative application of derivatised materials and surfaces on the basis of nano-crystalline diamond, carbon nanotubes and fullerenes for the analysis of marker peptides and proteins by material enhanced laser desorption ionisation mass spectrometry MELDI-MS. In this particular work, the emphasis is placed on the derivatization, termed as immobilised metal affinity chromatography (IMAC), with three different support materials, to show the effectiveness of MELDI technique. For the physicochemical characterisation of the phases, near infrared reflectance spectroscopy (NIRS) is used, which is a well-established method within the analytical chemistry, covering a wide range of applications. NIRS enables differentiation between silica materials and different fullerenes derivatives, in a 3-dimensional factor-plot, depending on their derivatizations and physical characteristics. The method offers a physicochemical quantitative description in the nano-scale level of particle size, specific surface area, pore diameter, pore porosity, pore volume and total porosity with high linearity and improved precision. The measurement takes only a few seconds while high sample throughput is guaranteed.

  16. Evaluation of the quality of herbal teas by DART/TOF-MS.

    PubMed

    Prchalová, J; Kovařík, F; Rajchl, A

    2017-02-01

    The paper focuses on the optimization, settings and validation of direct analysis in real time coupled with time-of-flight detector when used for the evaluation of the quality of selected herbal teas (fennel, chamomile, nettle, linden, peppermint, thyme, lemon balm, marigold, sage, rose hip and St. John's wort). The ionization mode, the optimal ionization temperature and the type of solvent for sample extraction were optimized. The characteristic compounds of the analysed herbal teas (glycosides, flavonoids and phenolic and terpenic substances, such as chamazulene, anethole, menthol, thymol, salviol and hypericin) were detected. The obtained mass spectra were evaluated by multidimensional chemometric methods, such as cluster analysis, linear discriminate analysis and principal component analysis. The chemometric methods showed that the single variety herbal teas were grouped according to their taxonomic affiliation. The developed method is suitable for quick identification of herbs and can be potentially used for assessing the quality and authenticity of herbal teas. Direct analysis in real time/time-of-flight-MS is also suitable for the evaluation of selected substances contained in the mentioned herbs and herbal products. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Partially oxidised organic components in urban aerosol using GCXGC-TOF/MS

    NASA Astrophysics Data System (ADS)

    Hamilton, J.; Webb, P.; Lewis, A.; Hopkins, J.; Smith, S.; Davy, P.

    2004-03-01

    Partially oxidised organic compounds associated with PM2.5 aerosol collected in London, England, have been analysed using direct thermal desorption coupled to comprehensive gas chromatography-time of flight mass spectrometry (GCXGC-OF/MS). Over 10 000 individual organic components were isolated from around 10 μg of aerosol material in a single procedure and with no sample pre-treatment. Chemical functionalities observed using this analytical technique ranged from alkanes to poly-oxygenated species. The chemical band structures commonly used in GCXGC for group type identifications overlap for this sample type, and have required mass spectrometry as an additional level of instrument dimensionality. An investigation of oxygenated volatile organic compounds (o-VOC) contained within urban aerosol has been performed and in a typical sample around 130 o-VOCs were identified based on retention behaviour and spectral match. In excess of 100 other oxygenated species were also observed but lack of mass spectral library or pure components prevents positive identification. Many of the carbonyl species observed could be mechanistically linked to gas phase aromatic hydrocarbon oxidation and there is good agreement in terms of speciation between the urban samples analysed here and those degradation products observed in smog chamber experiments of aromatic oxidation. The presence of partially oxidised species such as linear chain aldehydes and ketones and cyclic products such as furanones suggests that species generated relatively early in the oxidative process may undergo gas to particle partitioning despite their relatively high volatility.

  18. Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis.

    PubMed

    Qiao, Liang; Roussel, Christophe; Wan, Jingjing; Yang, Pengyuan; Girault, Hubert H; Liu, Baohong

    2007-12-01

    An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.

  19. Forced degradation of fingolimod: effect of co-solvent and characterization of degradation products by UHPLC-Q-TOF-MS/MS and 1H NMR.

    PubMed

    Patel, Prinesh N; Kalariya, Pradipbhai D; Gananadhamu, S; Srinivas, R

    2015-11-10

    Fingolimod (FGL), an immunomodulator drug for treating multiple sclerosis, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per International Conference on Harmonization specified conditions. The drug showed extensive degradation under base hydrolysis, however, it was stable under all other conditions. A total of three degradation products (DPs) were observed. The chromatographic separation of the drug and its degradation products was achieved on a Fortis C18 (100×2.1mm, 1.7μm) column with a mobile phase composed of 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) in gradient mode. All the DPs were identified and characterized by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-Q-TOF-MS) in combination with accurate mass measurements. The major DP was isolated and characterized by Nuclear Magnetic resonance spectroscopy. This is a typical case of degradation where acetonitrile used as co-solvent in stress studies, reacts with FGL in base hydrolytic conditions to produce acetylated DPs. Hence, it can be suggested that acetonitrile is not preferable as a co-solvent for stress degradation of FGL. The developed UHPLC method was validated as per ICH guidelines.

  20. Characterisation and quantification of phenolic compounds of extra-virgin olive oils according to their geographical origin by a rapid and resolutive LC-ESI-TOF MS method.

    PubMed

    Ouni, Youssef; Taamalli, Ameni; Gómez-Caravaca, Ana Maria; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto; Zarrouk, Mokhtar

    2011-08-01

    The phenolic compounds present in seven samples of olive fruits were analysed by a rapid and resolutive LC-ESI-TOF MS method. All samples were collected during the normal picking period for olive oil production, in central and south Tunisia, and were obtained from the Oueslati variety cultivated in different olive growing areas. In the Tunisian samples, 22 compounds have been characterised by LC-ESI-TOF MS analysis. Results showed no qualitative differences in the phenolic fractions between virgin olive oils from different geographical region. However, significant quantitative differences were observed in a wide number of phenolic compounds. These results permit to use the phenolic fractions as an indicator of each region.

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

    PubMed

    Şamlı, Asuman; İlki, Arzu

    2016-10-01

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

  2. Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)

    PubMed Central

    2012-01-01

    Background In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. Results Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. Conclusions MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii

  3. Usefulness of MALDI-TOF MS as a Diagnostic Tool for the Identification of Streptococcus Species Recovered from Clinical Specimens of Pigs

    PubMed Central

    Pérez-Sancho, Marta; Vela, Ana I.; García-Seco, Teresa; González, Sergio; Domínguez, Lucas; Fernández-Garayzábal, Jose Francisco

    2017-01-01

    The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database (Bruker Daltoniks, Germany) was supplemented with the main spectrum profiles (MSP) of the reference strains of S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: S. porci (4113, 6133, 7975 and 8228 m/z Da), S. plurextorum (3979, 4078, 4665, 6164, 6491, 6812, 7959 and 9330 m/z Da) and S. porcorum (3385, 3954, 4190, 6772, 7908, and 8381 m/z Da). After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as S. suis, S. porcinus, S. dysgalactiae, S. hyovaginalis, S. porcorum, S. alactolyticus, S. hyointestinalis and S. orisratti. This is the first time that the latter three species have been reported from clinical specimens of pigs. Overall, there was good concordance (95.9%) between the results obtained from MALDI-TOF MS identification (best hint) and those from genotyping. Our results demonstrate the good performance of MALDI-TOF MS (100% sensitivity and specificity) for identifying most of the species of streptococci that can frequently be isolated from diseased pigs. However, conflicting results were observed in the correct identification of some isolates of S. dysgalactiae and S. alactolyticus. PMID:28125697

  4. Analysis of E. rutaecarpa Alkaloids Constituents In Vitro and In Vivo by UPLC-Q-TOF-MS Combined with Diagnostic Fragment

    PubMed Central

    Yang, Shenshen; Tian, Meng; Yuan, Lei; Deng, Haoyue; Wang, Lei; Li, Aizhu; Hou, Zhiguo; Li, Yubo

    2016-01-01

    Evodia rutaecarpa (Juss.) Benth. (Rutaceae) dried ripe fruit is used for dispelling colds, soothing liver, and analgesia. Pharmacological research has proved that alkaloids are the main active ingredients of E. rutaecarpa. This study aimed to rapidly classify and identify the alkaloids constituents of E. rutaecarpa by using UPLC-Q-TOF-MS coupled with diagnostic fragments. Furthermore, the effects of the material base of E. rutaecarpa bioactive ingredients in vivo were examined such that the transitional components in the blood of rats intragastrically given E. rutaecarpa were analyzed and identified. In this study, the type of alcohol extraction of E. rutaecarpa and the corresponding blood sample were used for the analysis by UPLC-Q-TOF-MS in positive ion mode. After reviewing much of the literature and collected information on the fragments, we obtained some diagnostic fragments of the alkaloids. Combining the diagnostic fragments with the technology of UPLC-Q-TOF-MS, we identified the compounds of E. rutaecarpa and blood samples and compared the ion fragment information with that of the alkaloids in E. rutaecarpa. A total of 17 alkaloids components and 6 blood components were identified. The proposed method was rapid, accurate, and sensitive. Therefore, this technique can reliably and practically analyze the chemical constituents in traditional Chinese medicine (TCM). PMID:27446630

  5. A fully validated GC-TOF-MS method for the quantification of fatty acids revealed alterations in the metabolic profile of fatty acids after smoking cessation.

    PubMed

    Goettel, Michael; Niessner, Reinhard; Pluym, Nikola; Scherer, Gerhard; Scherer, Max

    2017-01-15

    We developed and validated an efficient and robust method for the simultaneous quantification of 44 fatty acid species in human plasma via GC-TOF-MS. The method is characterized by its robustness, accuracy and precision covering a wide range of fatty acid species with various saturation degrees including short chain fatty acids (beginning with FA 4:0) and long chain fatty acids (up to FA 32:0). The fatty acids were methylated prior to analyses and subsequently detected as fatty acid methyl esters by means of GC-TOF-MS. A highly substituted polar column allowed the separation of geometrical and positional isomers of fatty acid species. The method was applied to plasma samples of a strictly diet controlled clinical smoking cessation study including 39 smokers followed over the course of three months after having quit. Statistical significant alterations within the fatty acid profile were observed when comparing the baseline (subjects still smoking) with one week, one month and three months of smoking cessation. After 3 months of smoking cessation, a partial recovery of alterations in the fatty acid profile evoked by smoking was observed. In conclusion, the developed fatty acid profiling method using GC-TOF-MS has proven as a reliable tool for the quantitative determination of 44 individual fatty acid species within clinical studies.

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

    PubMed

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

    2014-08-01

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

  7. 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.

  8. Molecular weight determination of high molecular mass (glyco)proteins using CGE-on-a-chip, planar SDS-PAGE and MALDI-TOF-MS.

    PubMed

    Müller, Roland; Marchetti-Deschmann, Martina; Elgass, Helmut; Breiteneder, Heimo; Kratzmeier, Martin; Allmaier, Günter

    2010-12-01

    The molecular weights (MW) of seven (glyco)proteins, of which five were plasma-derived, with MWs higher than 200 kDa were determined with three techniques: CGE-on-a-chip, SDS-PAGE and MALDI-TOF-MS. While the analysis of medium to high MW proteins with SDS-PAGE was an already well-established technique, the usefulness of MALDI-TOF-MS for the exact MW determination of high mass proteins was only partly described in literature so far. CGE-on-a-chip is the newest of all three applied techniques and was so far not applicable. Therefore, it was not evaluated for high MW (glyco)proteins. All proteins were analyzed under nonreducing as well as reducing conditions. In this work, it was demonstrated that all three described techniques were capable of determining the MW of all high molecular weight (glyco)proteins. The noncommercial CGE-on-a-chip assay allowed for the first time the electrophoretic separation of proteins in the MW range from 14 to 1000 kDa. MW assignment was limited to 500 kDa in the case of SDS-PAGE and 660 kDa in the case of the high MW CGE-on-a-chip assay. With the proper matrix and sample preparation, analysis with a standard MALDI-TOF-MS provided accurate MWs for all high MW proteins up to 1 MDa.

  9. Dihydrobenzoic acid modified nanoparticle as a MALDI-TOF MS matrix for soft ionization and structure determination of small molecules with diverse structures.

    PubMed

    Tseng, Mei-Chun; Obena, Rofeamor; Lu, Ying-Wei; Lin, Po-Chiao; Lin, Ping-Yu; Yen, Yung-Sheng; Lin, Jiann-Tsuen; Huang, Li-De; Lu, Kuang-Lieh; Lai, Long-Li; Lin, Chun-Cheng; Chen, Yu-Ju

    2010-11-01

    Efficient structural characterization is important for quality control when developing novel materials. In this study, we demonstrated the soft ionization capability of the hybrid of immobilized silica and 2,5-dihydrobenzoic acid (DHB) on iron oxide magnetic nanoparticles in MALDI-TOF MS with a clean background. The ratio between SiO(2) and DHB was examined and was found to affect the surface immobilization of DHB on the nanoparticle, critically controlling the ionization efficiency and interference background. Compared with commercial DHB, the functionalized nanoparticle-assisted MALDI-TOF MS provided superior soft ionization with production of strong molecular ions within 5 ppm mass accuracy on a variety of new types of synthetic materials used for solar cells, light emitting devices, dendrimers, and glycolipids, including analytes with either thermally labile structures or poor protonation tendencies. In addition, the enhancements of the molecular ion signal also provided high-quality product-ion spectra allowing structural characterization and unambiguous small molecule identification. Using this technique, the structural differences among the isomers were distinguished through their characteristic fragment ions and comprehensive fragmentation patterns. With the advantages of long-term stability and simple sample preparation by deposition on a regular sample plate, the use of DHB-functionalized nanoparticles combined with high-resolution MALDI-TOF MS provides a generic platform for rapid and unambiguous structure determination of small molecules.

  10. Development of the MAE/UHPLC-MS-TOF method for determination of benzodiazepines in human bio-fluids for toxicological analysis.

    PubMed

    Woźniakiewicz, Aneta; Wietecha-Posłuszny, Renata; Woźniakiewicz, Michał; Nowak, Julia; Kościelniak, Paweł

    2015-04-10

    A rapid method of microwave-assisted extraction (MAE) followed by ultrahigh performance liquid chromatography with mass spectrometry with time of flight detection (UHPLC-MS-TOF) was optimized and validated for the purpose of determination of five benzodiazepines in human serum and blood samples. Extraction parameters and conditions of the UHPLC-MS-TOF method were defined. Validation of the developed method was performed at three concentration levels: 10, 100 and 250 ng/mL of each drug for both serum and blood samples. For serum and blood the limit of detection was found in the ranges 0.46-2.58 ng/mL and 0.43-1.87 ng/mL, precision (RSD): 0.3-6.7% and 0.9-8.4%, accuracy of the assay (RE): -5.3 to +2.4% and -5.7 to +7.6%, recovery: 80.5-104.3% and 79.9-106.9%, matrix effects: 95.9-110.5% and 97.5-114.2%, respectively. Moreover, the optimized and validated MAE/UHPLC-MS-TOF method was applied to analysis of blood samples.

  11. Rapid Sputum Multiplex Detection of the M. tuberculosis Complex (MTBC) and Resistance Mutations for Eight Antibiotics by Nucleotide MALDI-TOF MS

    PubMed Central

    Su, Kang-Yi; Yan, Bo-Shiun; Chiu, Hao-Chieh; Yu, Chong-Jen; Chang, So-Yi; Jou, Ruwen; Liu, Jia-Long; Hsueh, Po-Ren; Yu, Sung-Liang

    2017-01-01

    The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) adds further urgency for rapid and multiplex molecular testing to identify the MTB complex and drug susceptibility directly from sputum for disease control. A nucleotide matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed to identify MTB (MTBID panel) and 45 chromosomal mutations for resistance to eight antibiotics (MTBDR panel). We conducted a 300 case trial from outpatients to evaluate this platform. An MTBID panel specifically identified MTB with as few as 10 chromosome DNA copies. The panel was 100% consistent with an acid-fast stain and culture for MTB, nontuberculous mycobacteria, and non-mycobacteria bacteria. The MTBDR panel was validated using 20 known MDR-MTB isolates. In a 64-case double-blind clinical isolates test, the sensitivity and specificity were 83% and 100%, respectively. In a 300-case raw sputum trial, the MTB identification sensitivity in smear-negative cases using MALDI-TOF MS was better than the COBAS assay (61.9% vs. 46.6%). Importantly, the failure rate of MALDI-TOF MS was better than COBAS (11.3% vs. 26.3%). To the best of our knowledge, the test described herein is the only multiplex test that predicts resistance for up to eight antibiotics with both sensitivity and flexibility. PMID:28134321

  12. Evaluation of ozone-based treatment processes for wastewater containing microcontaminants using LC-QTRAP-MS and LC-TOF/MS.

    PubMed

    Gómez, M J; Martínez Bueno, M J; Agüera, A; Hernando, M D; Fernández-Alba, A R; Mezcua, M

    2008-01-01

    This article describes the development of an enhanced liquid chromatography-mass spectrometry (LC-MS) method for the analysis of a selected group of 57 organic contaminants in wastewater. This group comprises 39 pharmaceuticals belonging to different therapeutical classes and 10 of their most frequent metabolites. Six pesticides and two disinfectants were also included. The LC-MS method was developed using a hybrid quadrupole/linear ion trap (Q TRAP) analyzer operating in selected reaction monitoring (SRM) mode (in both positive and negative electrospray ionization) in combination with a time-of flight (TOF) mass analyser. The application of both techniques provided very good results in terms of accurate quantification and unequivocal identification. Quantification was based on the use of a linearly accelerating (LINAC) high-pressure collision cell, which enable the analysis of a high number of compounds with enough acquisition data points for an optimal peak definition in SRM. Unequivocal identification was provided by the acquisition of at least two SRM transitions and by obtaining accurate mass measurements of the identified compounds with errors lower than 2 ppm. As an alternative for compounds where a second transition cannot be detected by Q-Trap-MS, the application of survey scans in enhanced product ion (EPI) was evaluated. The analytical performance of the method was evaluated in effluent wastewater samples. Linearity of response over three orders of magnitude was demonstrated (R2>0.99 for most compounds). Matrix effects resulting in suppression of the response were frequently observed, between 2-50% for most of compounds, except 4-DAA and 4-AA, which exhibit higher values (68%). Signal enhancement was also detected in 16 compounds. Method limits of detection (LOD) were between 0.1-50 ng L(-1). Finally, the methodology was successfully applied to the evaluation of the efficiency of two ozone-based treatments applied to the effluent from the secondary

  13. Comprehensive and simultaneous coverage of lipid and polar metabolites for endogenous cellular metabolomics using HILIC-TOF-MS.

    PubMed

    Fei, Fan; Bowdish, Dawn M E; McCarry, Brian E

    2014-06-01

    The comprehensive metabolomic analyses using eukaryotic and prokaryotic cells are an effective way to identify biomarkers or biochemical pathways which can then be used to characterize disease states, differences between cell lines or inducers of cellular stress responses. One of the most commonly used extraction methods for comprehensive metabolomics is the Bligh and Dyer method (BD) which separates the metabolome into polar and nonpolar fractions. These fractions are then typically analysed separately using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatography (LC), respectively. However, this method has low sample throughput and can also be biased to either polar or nonpolar metabolites. Here, we introduce a MeOH/EtOH/H2O extraction paired with HILIC-time-of-flight (TOF)-mass spectrometry (MS) for comprehensive and simultaneous detection of both polar and nonpolar metabolites that is compatible for a wide array of cellular species cultured in different growth media. This method has been shown to be capable of separating polar metabolites by a HILIC mechanism and classes of lipids by an adsorption-like mechanism. Furthermore, this method is scalable and offers a substantial increase in sample throughput compared to BD with comparable extraction efficiency. This method was able to cover 92.2% of the detectable metabolome of Gram-negative bacterium Sinorhizobium meliloti, as compared to 91.6% of the metabolome by a combination of BD polar (59.4%) and BD nonpolar (53.9%) fractions. This single-extraction HILIC approach was successfully used to characterize the endometabolism of Gram-negative and Gram-positive bacteria as well as mammalian macrophages.

  14. A UHPLC-TOF/MS method based metabonomic study of total ginsenosides effects on Alzheimer disease mouse model.

    PubMed

    Gong, Yingge; Liu, Ying; Zhou, Ling; Di, Xin; Li, Wei; Li, Qing; Bi, Kaishun

    2015-11-10

    A metabonomic method was established to find potential biomarkers and study the metabolism disturbance in Alzheimer disease animal model. Total ginsenosides, as potential agent in neuroprotection and anti-inflammation, was also studied to learn the regulation mechanism to plasma metabolites in model animals. In experiment, amyloid beta 1-42 was occupied to form Alzheimer disease animal model. After drug administration, animals were evaluated by Morris water maze behavior test and sacrificed. Plasma samples were then analyzed using UHPLC-TOF/MS method to determine the endogenous metabolites. Behavior test results revealed that the spatial learning and memory abilities were deficit in model mice, and total ginsenosides could improve cognition abilities in dose-dependent manners. Principal component analysis showed that model and sham were divided into two groups, which means the metabolic network of mice was disturbed after modeling. Accordingly, 19 biomarkers were found and identified. In model group, the levels of proline, valine, tryptophan, LPC (14:0), LPC (15:0), LPC (15:1), LPC (17:0), LPC (18:2), LPC (18:3) and LPC (20:4) were up-regulated, while the levels of acetylcarnitine, palmitoylcarnitine, vaccenylcarnitine, phytosphingosine, N-eicosanoylethanolamine, hexadecenoic acid, docosahexaenoic acid, docosapentaenoic acid and octadecadienoic acid were down-regulated. The levels of these metabolites were recovered in different degrees after total ginsenosides administration. Combining with behavior study results, total ginsenosides could ameliorate both cognition symptoms and metabolic changes in model animals. This metabonomic approach provided a feasible way to understand the endogenous alterations of AD and to study the pharmacodynamic activity of novel agents.

  15. Analysis of roasted and unroasted Pistacia terebinthus volatiles using direct thermal desorption-GCxGC-TOF/MS.

    PubMed

    Gogus, F; Ozel, M Z; Kocak, D; Hamilton, J F; Lewis, A C

    2011-12-01

    The objective of this study was to determine the effects of roasting time on volatile components of Pistacia terebinthus L., a fruit growing wild in Turkey. The whole fruit samples were pan roasted for 0, 5, 10, 15, 20 and 25min at 200°C. Volatile compounds were isolated and identified using the direct thermal desorption (DTD) method coupled with comprehensive gas chromatography - time of flight mass spectrometry (GCxGC-TOF/MS). The major components of the fresh hull of P. terebinthus were α-pinene (10.37%), limonene (8.93%), β-pinene (5.53%), 2-carene (4.47%) and γ-muurolene (4.29%). Eighty-three constituents were characterised from the volatiles of fresh whole P. terebinthus fruits obtained by direct thermal desorption with α-pinene (9.62%), limonene (5.54%), γ-cadinane (5.48%), β-pinene (5.46%), β-caryophyllene (5.24%) being the major constituents. The type and the number of constituents characterised were observed to change with differing roasting times. Limonene (5.56%), α-pinene (4.84%), 5-methylfurfural (4.78%), 5-hydroxymethylfurfural (5-HMF, 3.89%), dimethylmetoxyfuranone (3.67%) and 3-methyl-2(5H)furanone (3.12%) were identified as the major components among the 104 compounds characterised in the volatiles of P. terebinthus, roasted for 25min. In addition, volatiles of fully roasted P. terebinthus fruits contained furans and furanones (15.42%), pyridines (4.45%) and benzene derivatives (3.81%) as the major groups.

  16. Comprehensive quantitative analysis of Shuang-Huang-Lian oral liquid using UHPLC-Q-TOF-MS and HPLC-ELSD.

    PubMed

    Zhang, Tian-Bo; Yue, Rui-Qi; Xu, Jun; Ho, Hing-Man; Ma, Dik-Lung; Leung, Chung-Hang; Chau, Siu-Leung; Zhao, Zhong-Zhen; Chen, Hu-Biao; Han, Quan-Bin

    2015-01-01

    Shuang-Huang-Lian oral liquid (SHL) is a well-known Chinese patent drug containing three herbal medicines: Radix Scutellariae, Flos Lonicerae Japonicae and Fructus Forsythiae. It is usually used to treat acute upper respiratory tract infection caused by virus or bacteria. Although the licensing of botanical drug Veregen approved by FDA has indicated the importance of quantitative analysis in quality control of herbal medicines, quantitative evaluation of a Chinese patent drug like SHL remains a challenge due to the complex chemical profile. In this study, 15 small molecular components of SHL (four flavonoids, six quinic acid derivatives, three saponins and two phenylethanoid glycosides) were simultaneously determined using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The contents of the three major saccharides, namely fructose, glucose and sucrose were quantified using high performance liquid chromatography-evaporative light scattering detector on an amino column (HPLC-ELSD). The macromolecules were quantified by precipitating in 80% ethanol, drying the precipitate, and then weighing. The established methods were validated in terms of linearity, sensitivity, precision, accuracy and stability and then successfully applied to analyze 12 batches of commercial products of SHL produced by four different manufacturers. The results indicated that 57.52-78.11% (w/w) of SHL could be quantitatively determined (non-saccharide small molecules: 1.77-3.75%, monosaccharides: 0.93-20.93%, macromolecules: 2.63-5.76% and sucrose: 49.20-65.94%). This study may provide a useful way to comprehensively evaluate the quality of SHL.

  17. Chaperonin GroEL a Brucella immunodominant antigen identified using Nanobody and MALDI-TOF-MS technologies.

    PubMed

    Abbady, A Q; Al-Daoude, A; Al-Mariri, A; Zarkawi, M; Muyldermans, S

    2012-05-15

    The deployment of today's antibodies that are able to distinguish Brucella from the closely similar pathogens, such as Yersinia, is still considered a great challenge since both pathogens share identical LPS (lipopolysaccharide) O-ring epitopes. In addition, because of the great impact of Brucella on health and economy in many countries including Syria, much effort is going to the development of next generation vaccines, mainly on the identification of new immunogenic proteins of this pathogen. In this context, Brucella-specific nanobodies (Nbs), camel genetic engineered heavy-chain antibody fragments, could be of great value. Previously, a large Nb library was constructed from a camel immunized with heat-killed Brucella. Phage display panning of this 'immune' library with Brucella total lysate resulted in a remarkable fast enrichment for a Nb referred to as NbBruc02. In the present work, we investigated the main characteristics of this Nb that can efficiently distinguish under well-defined conditions the Brucella from other bacteria including Yersinia. NbBruc02 showed a strong and specific interaction with its antigen within the crude lysate as tested by a surface plasmon resonance (SPR) biosensor and it was also able to pull down its cognate antigen from such lysate by immuno-capturing. Using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), NbBruc02 specific antigen was identified as chaperonin GroEL, also known as heat shock protein of 60 kDa (HSP-60), which represents a Brucella immunodominant antigen responsible of maintaining proteins folding during stress conditions. Interestingly, the antigen recognition by NbBruc02 was found to be affected by the state of GroEL folding. Thus, the Nb technology applied in the field of infectious diseases, e.g. brucellosis, yields two outcomes: (1) it generates specific binders that can be used for diagnosis, and perhaps treatment, and (2) it identifies the immunogenic candidate

  18. Development of a Rapid and Accurate Identification Method for Citrobacter Species Isolated from Pork Products Using a Matrix-Assisted Laser-Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

    PubMed

    Kwak, Hye-Lim; Han, Sun-Kyung; Park, Sunghoon; Park, Si Hong; Shim, Jae-Yong; Oh, Mihwa; Ricke, Steven C; Kim, Hae-Yeong

    2015-09-01

    Previous detection methods for Citrobacter are considered time consuming and laborious. In this study, we have developed a rapid and accurate detection method for Citrobacter species in pork products, using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A total of 35 Citrobacter strains were isolated from 30 pork products and identified by both MALDI-TOF MS and 16S rRNA gene sequencing approaches. All isolates were identified to the species level by the MALDI-TOF MS, while 16S rRNA gene sequencing results could not discriminate them clearly. These results confirmed that MALDI-TOF MS is a more accurate and rapid detection method for the identification of Citrobacter species.

  19. 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

  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.

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

    PubMed

    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.

  2. 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.

  3. Evaluation of the Bruker Biotyper and VITEK MS MALDI-TOF MS systems for the identification of unusual and/or difficult-to-identify microorganisms isolated from clinical specimens.

    PubMed

    McElvania TeKippe, E; Burnham, C-A D

    2014-12-01

    The purpose of this investigation was to evaluate the analytical performance characteristics of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of unusual organisms. We evaluated the accuracy of two MALDI-TOF MS systems, bioMérieux VITEK MS (database v2.0) and Bruker Biotyper (software version 3.0), for the identification of the most difficult and/or unusual microorganisms isolated from clinical specimens. Our study included 174 bacterial isolates recovered from clinical cultures at Barnes-Jewish Hospital, St. Louis, MO, from 2009 to 2013, representing 50 genera and 52 species. MS identifications were compared to the identification reported by the reference laboratory. Discrepancies were resolved using molecular methods, including 16S rRNA gene sequencing and additional molecular methods. When performed, molecular methods were considered the gold standard. Of the 168 isolates resolved to the genus level, VITEK MS identified 145 (86.3 %), and of the 114 isolates resolved to the species level, 97 (85.1 %) were correctly identified. Bruker Biotyper identified 155 (92.3 %) of 168 isolates to the genus level and 97 (85.1 %) of 114 isolates to the species level. VITEK MS and Bruker Biotyper provided no identification for 17 (10.1 %) and 12 (7.1 %) organisms, respectively, and misidentified six (3.6 %) and one (0.6 %) isolate, respectively. Six isolates (3.6 %) were not resolvable to the genus level and were excluded from data analysis due to the lack of a gold standard for comparison. There was no significant difference in the number of organisms identified to the genus level, species level, unidentified, or misidentified by the two MALDI-TOF MS systems (p = 0.11, 1.0, 0.44, and 0.12, respectively).

  4. 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

  5. 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.

  6. Selected Bioinformatic Tools and MS (MALDI-TOF, PMF) Techniques Used in the Strategy for the Identification of Oat Proteins After 2-DE.

    PubMed

    Szerszunowicz, Iwona; Nałęcz, Dorota; Dziuba, Marta

    2017-01-01

    Computer analysis of protein maps obtained from the separation of proteins with two-dimensional polyacrylamide gel electrophoresis (2-DE), in combination with mass spectrometry (MS) analysis and selected bioinformatic tools is used in the strategy for the identification of oat proteins. In proteomic research the most often used MS technique is the combination of ion sources: matrix-assisted laser desorption/ionization (MALDI) and the analyzer of the time of flight (TOF), i.e., MALDI-TOF MS.This chapter describes the possibilities of the use of selected bioinformatic tools (UniProtKB database, ProtParam, Compute pI/MW programs) for initial identification of separated oat proteins (especially prolamin fractions) with the 2-DE technique. Also the procedure of preparation of samples obtained from cut out protein spots for analysis with the MALDI-TOF MS and peptide mass fingerprinting (PMF) technique is presented.Among oat prolamins separated with the 2-DE technique (see Chapter 17 ), 13 protein spots are considered to be the most characteristic (range of MW 27.0-34.6 kDa, pI 5.7-7.6) for this fraction of proteins. Among them there are four protein spots (MW 27.0-28.0 kDa) and two spots (MW 31.4-32.1 kDa) which can correspond to avenins (Accession numbers (AC) in UniProtKB: L0L5I0, I4EP88, I4EP64, L0L4I8 and F2Q9W5, L0L6J0, respectively).

  7. Identification and characterization of stressed degradation products of prulifloxacin using LC-ESI-MS/Q-TOF, MSn experiments: development of a validated specific stability-indicating LC-MS method.

    PubMed

    Raju, B; Ramesh, M; Srinivas, R; Raju, S Satyanarayana; Venkateswarlu, Y

    2011-11-01

    A rapid, specific and novel gradient LC-MS method has been developed and validated for the identification and characterization of stressed degradation products (DPs) of prulifloxacin (PF) using liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS). PF was subjected to hydrolytic (acidic, alkaline and neutral), oxidation, photolytic and thermal stress, as per ICH guidelines Q1A (R2). The drug showed extensive degradation in hydrolytic and oxidative, while it was stable to thermal and photolytic stress conditions. In total, 13 DPs were formed and the chromatographic separation of drug and its DPs was achieved on a C-18 column (4.6 × 250 mm, 5 μm) using gradient elution method. All the DPs have been identified and characterized using MS(n) experiments and accurate mass measurements. The LC-MS method was validated with respect to specificity, linearity, accuracy, precision and robustness.

  8. Degradation kinetics study of cabozantinib by a novel stability-indicating LC method and identification of its major degradation products by LC/TOF-MS and LC-MS/MS.

    PubMed

    Wu, Chunyong; Xu, Xue; Feng, Chao; Shi, Yuanyuan; Liu, Wenyuan; Zhu, Xiaoyun; Zhang, Junying

    2014-09-01

    The chemical stability of cabozantinib (CBZ) was investigated using a novel stability-indicating LC method. Forced degradation of CBZ was carried out under acidic, basic, thermal, oxidative and photolytic stress conditions. Hydrolysis and oxidation were the primary pathways for this compound and three major unknown degradation products were characterized by LC/TOF-MS and LC-MS/MS. The major oxidative degradation product was isolated by preparative LC and identified by UV, HRMS and NMR techniques to be N-{4-[(N-oxide-6,7-dimethoxyquinolin-4-yl)oxy]phenyl}-N'-(4-fluorophenyl)-cyclopropane-1,1-dicarboxamide. The developed method was validated as per ICH guidelines and then successfully applied to investigate the degradation kinetics of CBZ. Degradation of CBZ followed first-order kinetics under all experimental conditions. A V-shaped pH-rate profile over the pH range 2-10 was observed with maximum stability at pH 6. The effect of temperature on the rate of CBZ degradation was characterized using the Arrhenius equation. The activation energy for hydrolysis was 57.31kJmol(-1) in alkaline solution.

  9. Metabolomics using GC-TOF-MS followed by subsequent GC-FID and HILIC-MS/MS analysis revealed significantly altered fatty acid and phospholipid species profiles in plasma of smokers.

    PubMed

    Müller, Daniel C; Degen, Christian; Scherer, Gerhard; Jahreis, Gerhard; Niessner, Reinhard; Scherer, Max

    2014-09-01

    Mass spectrometry is an ideal tool for investigations of the metabolome in human plasma. To investigate the impact of smoking on the human metabolome, we performed an untargeted metabolic fingerprinting using GC-TOF-MS with EDTA-plasma samples from 25 smokers and 25 non-smokers. The observed elevated levels in the monounsaturated fatty acids (MUFAs) in smokers were verified by a targeted analysis using GC-FID, which revealed also significantly alterations in saturated and polyunsaturated fatty acids in smokers (p<0.05, Mann-Whitney U test). Since the main fraction of fatty acids in plasma is esterified to phospholipids, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species composition in the plasma samples of the same subjects. The profiles of 39 PC and 40 PE species were analyzed with a newly developed and validated HILIC-ESI-MS/MS method. We were able to baseline separate the two lipid classes (PC from PE) by maintaining co-elution of individual lipid species of each class. The method shows a linear range from 0.5μM to 2000μM and an inter- and intraday coefficient of variation (CV)<20% across all analytes. Application of the validated method to the plasma samples of smokers and non-smokers, derived from a diet-controlled smoking study, revealed significantly elevated levels of PC and PE species containing MUFAs in smokers. In summary, we could demonstrate that there is a significantly altered total fatty acid profile, with increased MUFAs, in the plasma of smokers compared to non-smokers. Results obtained with the new HILIC-MS/MS method indicate that the altered fatty acid profile is also reflected in the PC and PE profile of smokers.

  10. Undisturbed and disturbed above canopy ponderosa pine emissions: PTR-TOF-MS measurements and MEGAN 2.1 model results

    SciTech Connect

    Kaser, L.; Karl, T.; Guenther, A.; Graus, M.; Schnitzhofer, R.; Turnipseed, A.; Fischer, L.; Harley, P.; Madronich, M.; Gochis, D.; Keutsch, F. N.; Hansel, A.

    2013-01-01

    We present the first eddy covariance flux measurements of volatile organic compounds (VOCs) using a proton-transfer-reaction time-of-flight mass-spectrometer (PTR-TOFMS) above a ponderosa pine forest in Colorado, USA. The high mass resolution of the PTR-TOF-MS enabled the identification of chemical sum formulas. During a 30 day measurement period in August and September 2010, 649 different ion mass peaks were detected in the ambient air mass spectrum (including primary ions and mass calibration ompounds). Eddy covariance with the vertical wind speed was calculated for all ion mass peaks. On a typical day, 17 ion mass peaks including protonated parent compounds, their fragments and isotopes as well as VOC-H+-water clusters showed a significant flux with daytime average emissions above a reliable flux threshold of 0.1mgcompoundm-2 h-1. These ion mass peaks could be assigned to seven compound classes. The main flux contributions during daytime (10:00-18:00 LT) are attributed to the sum of 2-methyl-3-buten-2-ol (MBO) and isoprene (50 %), methanol (12%), the sum of acetic acid and glycolaldehyde (10%) and the sum of monoterpenes (10 %). The total MBO+isoprene flux was composed of 10% isoprene and 90% MBO. There was good agreement between the light and temperature dependency of the sum of MBO and isoprene observed for this work and those of earlier studies. The above canopy flux measurements of the sum of MBO and isoprene and the sum of 20 monoterpenes were compared to emissions calculated using the Model of Emissions of Gases and Aerosols from Nature (MEGAN 2.1). The best agreement between MEGAN 2.1 and measurements was reached using emission factors determined from site specific leaf cuvette measurements. While the modelled and measured MBO+isoprene fluxes agree well the emissions of the sum of monoterpenes is underestimated by MEGAN 2.1. This is expected as some factors impacting monoterpene emissions, such as physical damage of needles and branches due to storms, are

  11. Rapid characterization of outer-membrane proteins in Neisseria lactamica by SELDI-TOF-MS (surface-enhanced laser desorption ionization-time-of-flight MS) for use in a meningococcal vaccine.

    PubMed

    Mukhopadhyay, Tarit Kumar; Halliwell, Denise; O'Dwyer, Cliona; Shamlou, Parviz Ayazi; Levy, Myriam Susana; Allison, Nigel; Gorringe, Andrew; Reddin, Karen M

    2005-04-01

    Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal Neisseria species, particularly with Neisseria lactamica. We have reported previously that immunization with N. lactamica outer-membrane vesicles containing the major OMPs (outer-membrane proteins) protected mice against lethal challenge with meningococci of diverse serogroups and serotypes and has the potential to form the basis of a vaccine against meningococcal diseases [Oliver, Reddin, Bracegirdle et al. (2002) Infect. Immun. 70, 3621-3626]. In the present study, we have shown that biomass production and the profile of outer-membrane vesicle proteins may be affected by fermentation conditions and, in particular, media composition. Ciphergen SELDI-TOF Protein Chips were used as a rapid and sensitive new method in comparison with conventional SDS/PAGE. SELDI-TOF-MS (surface-enhanced laser-desorption ionization-time-of-flight MS) reproducibly identified three major OMPs (NspA, RmpM and PorB) and detected the changes in the protein profile when the growth medium was altered. The findings of this work indicate that SELDI-TOF-MS is a useful tool for the rapid optimization of OMP production in industrial fermentation processes and can be adapted as a Process Analytical Technology.

  12. Structure elucidation of degradation products of the antibiotic amoxicillin with ion trap MS(n) and accurate mass determination by ESI TOF.

    PubMed

    Nägele, Edgar; Moritz, Ralf

    2005-10-01

    Today, it is necessary to identify relevant compounds appearing in discovery and development of new drug substances in the pharmaceutical industry. For that purpose, the measurement of accurate molecular mass and empirical formula calculation is very important for structure elucidation in addition to other available analytical methods. In this work, the identification and confirmation of degradation products in a finished dosage form of the antibiotic drug amoxicillin obtained under stress conditions will be demonstrated. Structure elucidation is performed utilizing liquid chromatography (LC) ion trap MS/MS and MS3 together with accurate mass measurement of the molecular ions and of the collision induced dissociation (CID) fragments by liquid chromatography electro spray ionization time-of-flight mass spectrometry (LC/ESI-TOF).

  13. Identification of Oxygenated Fatty Acid as a Side Chain of Lipo-Alkaloids in Aconitum carmichaelii by UHPLC-Q-TOF-MS and a Database.

    PubMed

    Liang, Ying; Wu, Jian-Lin; Leung, Elaine Lai-Han; Zhou, Hua; Liu, Zhongqiu; Yan, Guanyu; Liu, Ying; Liu, Liang; Li, Na

    2016-03-31

    Lipo-alkaloid is a kind of C19-norditerpenoid alkaloid usually found in Aconitum species. Structurally, they contain an aconitane skeleton and one or two fatty acid moieties of 3-25 carbon chains with 1-6 unsaturated degrees. Analysis of the lipo-alkaloids in roots of Aconitum carmichaelii resulted in the isolation of six known pure lipo-alkaloids (A1-A6) and a lipo-alkaloid mixture (A7). The mixture shared the same aconitane skeleton of 14-benzoylmesaconine, but their side chains were determined to be 9-hydroxy-octadecadienoic acid, 13-hydroxy-octadecadienoic acid and 10-hydroxy-octadecadienoic acid, respectively, by MS/MS analysis after alkaline hydrolysis. To our knowledge, this is the first time of the reporting of the oxygenated fatty acids as the side chains in naturally-occurring lipo-alkaloids. In order to identify more lipo-alkaloids, a compound database was established based on various combinations between the aconitane skeleton and the fatty acid chain, and then, the identification of lipo-alkaloids was conducted using the database, UHPLC-Q-TOF-MS and MS/MS. Finally, 148 lipo-alkaloids were identified from A. carmichaelii after intensive MS/MS analysis, including 93 potential new compounds and 38 compounds with oxygenated fatty acid moieties.

  14. Comparison of MALDI-TOF MS and VITEK 2 system for laboratory diagnosis of Granulicatella and Abiotrophia species causing invasive infections.

    PubMed

    Ratcliffe, Paul; Fang, Hong; Thidholm, Ellinor; Boräng, Stina; Westling, Katarina; Özenci, Volkan

    2013-11-01

    Granulicatella and Abiotrophia spp. were known as nutritionally variant streptococci (NVS). Such strains have caused major diagnostic difficulties due to fastidious culturing and unspecific colony morphology. The present study is aimed at comparing the performance of laboratory available diagnostic methods for NVS isolates and determining the antimicrobial susceptibility of these isolates. Fourteen clinical invasive isolates, consisting of 10 Granulicatella adiacens, 1 Granulicatella elegans, and 3 Abiotrophia defectiva were in parallel analyzed by 2 matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, i.e., Bruker MS and Vitek MS, as well as Vitek 2 for the species determination. 16S rRNA gene sequencing was applied as a reference method. The Vitek MS gave correct identification for all 14 isolates. The Bruker MS could correctly identify 8/10 G. adiacens, 0/1 G. elegans, and 3/3 A. defectiva isolates at the first analysis occasion, and all 14 isolates became identifiable after repeated tests. The Vitek 2 system could identify 6/10 G. adiacens, 1/1 G. elegans, and 2/3 A. defectiva isolates at the species level. Antimicrobial susceptibilities of 11 antibiotics were determined by Etest. Resistance against ciprofloxacin, ceftriaxone, rifampicin, and tetracycline were observed in 4, 10, 4, and 1 isolates, respectively. In conclusion, MALDI-TOF MS is a useful tool for the rapid diagnosis of NVS. Phenotypic testing by Vitek 2 is only partially effective for the accurate identification of such strains. The emergence of resistant NVS isolates indicates the necessity of monitoring antimicrobial susceptibilities of such uncommon pathogens.

  15. Product ion distributions for the reactions of NO(+) with some physiologically significant aldehydes obtained using a SRI-TOF-MS instrument.

    PubMed

    Mochalski, Paweł; Unterkofler, Karl; Španěl, Patrik; Smith, David; Amann, Anton

    2014-04-15

    Product ion distributions for the reactions of NO(+) with 22 aldehydes involved in human physiology have been determined under the prevailing conditions of a selective reagent ionization time of flight mass spectrometry (SRI-TOF-MS) at an E/N in the flow/drift tube reactor of 130 Td. The chosen aldehydes were fourteen alkanals (the C2-C11 n-alkanals, 2-methyl propanal, 2-methyl butanal, 3-methyl butanal, and 2-ethyl hexanal), six alkenals (2-propenal, 2-methyl 2-propenal, 2-butenal, 3-methyl 2-butenal, 2-methyl 2-butenal, and 2-undecenal), benzaldehyde, and furfural. The product ion fragmentations patterns were determined for both dry air and humid air (3.5% absolute humidity) used as the matrix buffer/carrier gas in the drift tube of the SRI-TOF-MS instrument. Hydride ion transfer was seen to be a common ionization mechanism in all these aldehydes, thus generating (M-H)(+) ions. Small fractions of the adduct ion, NO(+)M, were also seen for some of the unsaturated alkenals, in particular 2-undecenal, and heterocyclic furfural for which the major reactive channel was non-dissociative charge transfer generating the M(+) parent ion. Almost all of the reactions resulted in partial fragmentation of the aldehyde molecules generating hydrocarbon ions; specifically, the alkanal reactions resulted in multiple product ions, whereas, the alkenals reactions produced only two or three product ions, dissociation of the nascent excited product ion occurring preferentially at the 2-position. The findings of this study are of particular importance for data interpretation in studies of aldehydes reactions employing SRI-TOF-MS in the NO(+) mode.

  16. Product ion distributions for the reactions of NO+ with some physiologically significant aldehydes obtained using a SRI-TOF-MS instrument

    PubMed Central

    Mochalski, Paweł; Unterkofler, Karl; Španěl, Patrik; Smith, David; Amann, Anton

    2014-01-01

    Product ion distributions for the reactions of NO+ with 22 aldehydes involved in human physiology have been determined under the prevailing conditions of a selective reagent ionization time of flight mass spectrometry (SRI-TOF-MS) at an E/N in the flow/drift tube reactor of 130 Td. The chosen aldehydes were fourteen alkanals (the C2–C11 n-alkanals, 2-methyl propanal, 2-methyl butanal, 3-methyl butanal, and 2-ethyl hexanal), six alkenals (2-propenal, 2-methyl 2-propenal, 2-butenal, 3-methyl 2-butenal, 2-methyl 2-butenal, and 2-undecenal), benzaldehyde, and furfural. The product ion fragmentations patterns were determined for both dry air and humid air (3.5% absolute humidity) used as the matrix buffer/carrier gas in the drift tube of the SRI-TOF-MS instrument. Hydride ion transfer was seen to be a common ionization mechanism in all these aldehydes, thus generating (M−H)+ ions. Small fractions of the adduct ion, NO+M, were also seen for some of the unsaturated alkenals, in particular 2-undecenal, and heterocyclic furfural for which the major reactive channel was non-dissociative charge transfer generating the M+ parent ion. Almost all of the reactions resulted in partial fragmentation of the aldehyde molecules generating hydrocarbon ions; specifically, the alkanal reactions resulted in multiple product ions, whereas, the alkenals reactions produced only two or three product ions, dissociation of the nascent excited product ion occurring preferentially at the 2-position. The findings of this study are of particular importance for data interpretation in studies of aldehydes reactions employing SRI-TOF-MS in the NO+ mode. PMID:25844049

  17. Chemical derivatization combined with capillary LC or MALDI-TOF MS for trace determination of lipoic acid in cosmetics and integrated protein expression profiling in human keratinocytes.

    PubMed

    Tsai, Chia-Ju; Lin, Ying-Chi; Chen, Yen-Ling; Feng, Chia-Hsien

    2014-12-01

    Lipoic acid (LA) is an essential cofactor in mitochondrial enzymes and an ideal antioxidant in prokaryotic and eukaryotic cells. Capillary liquid chromatography coupled with ultraviolet detection (CapLC-UV) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are two environmentally friendly methods for determining LA. In this study, a pre-column microwave-assisted derivatization with 4-bromomethyl-6,7-dimethoxycoumarin enhanced the UV absorbance of LA and was monitored at 345 nm by CapLC-UV. Gradient separation was performed using a reversed-phase C18 column with a mobile phase consisting of acetonitrile-0.1% formic acid solution. The ionization of LA was increased, and the LA derivative was detected by MALDI-TOF MS at m/z 683 with an α-cyano-4-hydroxycinnamic acid matrix. The linear response ranged from 0.1 to 40 μM with a correlation coefficient of 0.999. The CapLC-UV and MALDI-TOF MS had detection limits of 5 and 4 fmol, respectively. These methods effectively detected LA in dietary supplements and cosmetics. Cellular proteomes of a human keratinocyte cell line (HaCaT) irradiated with UV radiation were also compared with and without LA treatment. The cellular proteomes were identified by nanoultra performance LC with LTQ Orbitrap system after trypsin digestion. Protein identification was performed by simultaneous peptide sequencing and MASCOT search. The analysis revealed changes in several proteins, including CDC42, TPI1, HNRPA2B1, PRDX1, PTGES3 and MYL6.

  18. 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

  19. 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

  20. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and Bayesian phylogenetic analysis to characterize Candida clinical isolates.

    PubMed

    Angeletti, Silvia; Lo Presti, Alessandra; Cella, Eleonora; Dicuonzo, Giordano; Crea, Francesca; Palazzotti, Bernardetta; Dedej, Etleva; Ciccozzi, Massimo; De Florio, Lucia

    2015-12-01

    Clinical Candida isolates from two different hospitals in Rome were identified and clustered by MALDI-TOF MS system and their origin and evolution estimated by Bayesian phylogenetic analysis. The different species of Candida were correctly identified and clustered separately, confirming the ability of these techniques to discriminate between different Candida species. Focusing MALDI-TOF analysis on a single Candida species, Candida albicans and Candida parapsilosis strains clustered differently for hospital setting as well as for period of isolation than Candida glabrata and Candida tropicalis isolates. The evolutionary rates of C. albicans and C. parapsilosis (1.93×10(-2) and 1.17×10(-2)substitutions/site/year, respectively) were in agreement with a higher rate of mutation of these species, even in a narrow period, than what was observed in C. glabrata and C. tropicalis strains (6.99×10(-4) and 7.52×10(-3)substitutions/site/year, respectively). C. albicans resulted as the species with the highest between and within clades genetic distance values in agreement with the temporal-related clustering found by MALDI-TOF and the high evolutionary rate 1.93×10(-2)substitutions/site/year.

  1. Characterization of four new photodegradation products of hydroxychloroquine through LC-PDA, ESI-MSn and LC-MS-TOF studies.

    PubMed

    Saini, Balraj; Bansal, Gulshan

    2013-10-01

    ICH recommended forced degradation on HCQ was carried out under the conditions of hydrolysis, oxidation, dry heat and photolysis. Six degradation products (I-VI) were formed under photolytic conditions in alkaline medium. The products were characterized through +ESI-MS(n), LC-MS-TOF and LC-PDA studies. The product III was identified as N-de-ethylated HCQ which is a known impurity. It was also found to form in trace amounts under acidic and alkaline hydrolytic conditions. The products I, II, IV and V characterized as N-dehydroxyethyl-7-dechloro-7-hydroxy HCQ, dechlorinated HCQ, N-dealkylated HCQ and HCQ N-oxide, respectively were identified as new degradation products of HCQ. The product VI was not characterized due to its trace levels and insufficient mass spectral data. The most probable mechanisms of degradation of HCQ to these products were outlined and discussed.

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

    PubMed

    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.

  3. 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.

  4. Development of a stability-indicating HPLC method of etifoxine with characterization of degradation products by LC-MS/TOF, 1H and 13C NMR.

    PubMed

    Djabrouhou, Nadia; Guermouche, Moulay-Hassane

    2014-11-01

    This paper describes a new LC-MS/TOF method for the degradation products determination when Etifoxine (ETI) is submitted to different stress conditions. Chromatography is performed by using Kromasil C18 column (250mm×4.6mm, 5μm particle size). The selected mobile phase consists of formate buffer 0.02M, pH 3 and methanol (70/30, v/v). ETI is submitted to oxidative, acidic, basic, hydrolytic, thermal and UV light degradations. Detection is made at 254nm by photodiode array detector and mass spectrometry. A number of degradation products (DPs) called DPA, DPB, DPC and DPD are found depending on the stress; DPA with heat, DPA and DPB in acidic media or under UV-light; DPA, DPB and DPC under basic stress; DPA, DPB, DPC and DPD with oxidation. LC-MS/TOF is used to characterize the four DPs of ETI resulting from different stress conditions. (1)H and (13)C NMR are used to confirm the DP structures. The ETI fragmentation pathway is proposed. The method is validated with reference to International Conference on Harmonization guidelines and ETI are selectively determined in presence of its DPs, demonstrating its stability-indicating nature. Finally, for the validation step, specificity, linearity, accuracy and precision are determined for ETI and its DPs.

  5. Effects of berberine and pomegranate seed oil on plasma phospholipid metabolites associated with risks of type 2 diabetes mellitus by U-HPLC/Q-TOF-MS.

    PubMed

    Wu, Xia; Li, Yan; Wang, Qiu; Li, Weimin; Feng, Yifan

    2015-12-15

    A rapid and reliable ultra-performance liquid chromatography coupled with electrospray ionization/quadrupole-time-of-flight mass spectrometry (U-HPLC/Q-TOF-MS) has been firstly used to analyze the changes of plasma phospholipids, in type 2 diabetes mellitus (T2DM) mice after administration of berberine and pomegranate seed oil (PSO). The separation of plasma phospholipids was carried out on an Acquity U-HPLC BEH C18 column (2.1mm×50mm, 1.7μm, Waters) by linear gradient elution using a mobile phase consisting of 10mM ammonium formate in water and acetonitrile: isopropanol (1:1, v/v) mixed solution added by 0.25% water and 10mM ammonium formate. The method demonstrated a good precision and reproducibility. Linear regression analysis showed a good linearity. And potential biomarkers were discovered based on their mass spectra and chemometrics methods. The results demonstrated that the proposed U-HPLC/Q-TOF-MS method was successfully applied to analyze the dynamic changes of phospholipids components in plasma of T2DM mice after drug treatment and could provide a useful data base for meriting further study in humans and investigating pharmacological actions of drugs.

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

    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

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

    PubMed

    Horká, Marie; Salplachta, Jiří; Karásek, Pavel; Kubesová, Anna; Horký, Jaroslav; Matoušková, Hana; Slais, Karel; Roth, Michal

    2013-07-16

    This study was undertaken to investigate feasibility of a combination of capillary isoelectric focusing (CIEF) in a tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and reliable identification of bacteria taken from plant-tissue-containing samples. Eight strains representing different species of the genus Dickeya were selected on the basis of close proximity of their isoelectric points: D. chrysanthemi, D. chrysanthemi bv. parthenii, D. chrysanthemi bv. chrysanthemi, D. dadantii, D. paradisiaca, D. solani, D. diffenbachiae, and D. dianthicola. Because the Dickeya species (spp.) cannot be easily discriminated from each other when CIEF is performed in a cylindrical FS capillary (commonly used in CIEF) even if a narrow pH gradient is used, a tapered FS capillary was employed instead, which enabled satisfactory discrimination of the examined bacteria due to enhanced separation efficiency of CIEF in the tapered FS capillary. CIEF in the tapered FS capillary was also successfully used for the detection and characterization of Dickeya spp. in a plant-tissue-containing sample. Then an off-line combination of CIEF with MALDI-TOF MS was employed for rapid and reliable identification of Dickeya spp. in the plant-tissue-containing sample. It was found that the presence of plant tissue did not affect the results, making the proposed procedure very promising with respect to the fast and reliable detection and identification of bacteria in plant-tissue-containing samples.

  8. Antioxidant activity of leaf extracts from different Hibiscus sabdariffa accessions and simultaneous determination five major antioxidant compounds by LC-Q-TOF-MS.

    PubMed

    Wang, Jin; Cao, Xianshuang; Jiang, Hao; Qi, Yadong; Chin, Kit L; Yue, Yongde

    2014-12-17

    Hibiscus sabdariffa has gained attention for its antioxidant activity. There are many accessions of H. sabdariffa in the world. However, information on the quantification of antioxidant compounds in different accessions is rather limited. In this paper, a liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) method for simultaneous determination of five antioxidant compounds (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and isoquercitrin) in H. sabdariffa leaves was developed. The method was validated for linearity, sensitivity, precision, repeatability and accuracy. The validated method has been successfully applied for determination of the five analytes in eight accessions of H. sabdariffa. The eight accessions of H. sabdariffa were evaluated for their antioxidant activities by DPPH free radical scavenging assay. The investigated accessions of H. sabdariffa were rich in rutin and exhibited strong antioxidant activity. The two accessions showing the highest antioxidant activities were from Cuba (No. 2) and Taiwan (No. 5). The results indicated that H. sabdariffa leaves could be considered as a potential antioxidant source for the food industry. The developed LC-Q-TOF-MS method is helpful for quality control of H. sabdariffa.

  9. Systematic identification and quantification of tetracyclic monoterpenoid oxindole alkaloids in Uncaria rhynchophylla and their fragmentations in Q-TOF-MS spectra.

    PubMed

    Xie, Shuanglu; Shi, Yuanyuan; Wang, Yixiang; Wu, Chunyong; Liu, Wenyuan; Feng, Feng; Xie, Ning

    2013-01-01

    Uncaria rhynchophylla (UR) is a species of Uncaria that is distributed mainly in China and Japan. In this study, the chemical constituents, including alkaloids, flavonoids, and quinic acids, in UR have been systematically identified and quantified by a developed method of high-performance liquid chromatography coupled with diode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF-MS). Tetracyclic monoterpenoid oxindole alkaloids (TMOAs) are characteristic compounds in this herb, and their fragmentations in Q-TOF-MS have been investigated. Diagnostic fragmentation ions (DFIs) were first delineated for isorhynchophylline-type (7S, C20-ethyl) and corynoxeine-type (7R, C20-vinyl) TMOAs, and these were used for identification of these alkaloids in UR. In this study, a total of 29 compounds, comprising 18 alkaloids, six flavonoids, and five quinic acids, were identified. Among them, there are four novel TMOAs, named as 22-O-β-glucopyranosyl isorhynchophyllic acid (10), 22-O-β-glucopyranosyl rhynchophyllic acid (11), 9-hydroxy isocorynoxeine (16), and 9-hydroxy corynoxeine (20), which have not been reported previously. Furthermore, eight marker compounds, namely chlorogenic acid (3), catechin (8), epicatechin (9), isocorynoxeine (24), rhynchophylline (25), isorhynchophylline (27), vincoside lactam (28), and corynoxeine (29), have been simultaneously quantified. The developed method has been validated and successfully applied to analyze three samples of UR from Jiangxi Province. The contents of the marker compounds have been detected and compared.

  10. 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.

  11. Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins

    PubMed Central

    Wu, An-Guo; Kam-Wai Wong, Vincent; Zeng, Wu; Liu, Liang; Yuen-Kwan Law, Betty

    2015-01-01

    With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-flight mass spectrometry {UHPLC-(Q)TOF-MS}. By exhibiting more potent autophagic effect in cells, the active fraction enhances the clearance of mutant huntingtin, and reduces protein level and aggregation of α-synuclein in a higher extent when compared with onjisaponin B. Here, we have reported for the first time the new application of cell-based CMC and UHPLC-(Q)TOF-MS analysis in identifying new autophagy inducers with neuroprotective effects from Chinese medicinal herb. This result has provided novel insights into the possible pharmacological actions of the active components present in the newly identified active fraction of RP, which may help to improve the efficacy of the traditional way of prescribing RP, and also provide new standard for the quality control of decoction of RP or its medicinal products in the future. PMID:26598009

  12. Detection of the metabolites of human plasma and follicular fluid in IVF-ET with microextraction and LC-TOF-MS.

    PubMed

    Wang, Gang; Yao, Shun; Liang, Xin; Zuo, Tao; Zhu, Minghui

    2015-01-01

    The metabolites from human plasma and follicular fluid in IVF-ET are related with the metabolic activity of follicular cells and further with oocyte quality. With liquid chromatography-time-of-flight-mass spectrometry (LC-TOF-MS) and organic extraction from plasma and follicular fluid, a fast and efficient method was established for detecting the metabolites of human plasma and follicular fluid. Certain metabolites were closely related with human physiological activities, such as hormone, amino acid, estrogen and fatty acid, and estrogen fatty acyllipid lipid were successfully detected in human plasma and follicular fluid. The metabolites in follicular fluid and plasma on the day of oocyte retrieval were found to be similar. But those in the latter and basal plasma were partially different. Methyl (3α ,5β ,7α ,12α)-3,7,12-trihydroxycholan-24-oate, androst-4-ene-3,17-dione and tryptophan were identified and might be potential biomarkers on oocyte quality. It demonstrates that LC-TOF-MS combined with organic extraction could be an efficient tool to investigate the metabolites of human plasma and follicular fluid. Based on this study, it is expected to provide the basis for the further steps to explore possible influential factors on oocyte quality.

  13. Evaluation and discrimination of cortex Magnoliae officinalis produced in Zhejiang Province (Wen-Hou-Po) by UPLC-DAD-TOF-MS fingerprint.

    PubMed

    Wang, Lin; Yuana, Ke; Yu, Wei-Wu; Wang, Jing

    2010-10-01

    An ultra performance liquid chromatography tandem TOF mass spectrometric (UPLC-DAD-TOF-MS) fingerprinting method was developed for the quality control and source discrimination of Cortex magnoliae officinalis produced in Zhejiang Province (Wen-Hou-Po). Twelve samples of Wen-Hou-Po collected from two species in five areas in Zhejiang Province of China were used to establish the fingerprint. Data were evaluated statistically using similarity analysis, hierarchical cluster analysis (HCA) and discriminant analysis (DA) in order to establish a similarity standard of fingerprint for quality control of Wen-Hou-Po, then to classify the Wen-Hou-Po samples and to identify key categorizing parameters. The similarity indexes were all above 0.95 between the reference chromatogram and that of each sample. By comparing the UV and MS data with those of the authentic standards and literature, nine main peaks in the fingerprints were identified. The result of hierarchical cluster analysis showed that the samples from two species in five areas could be divided into two distinct groups (the same as the groups of the samples divided by their species) based on their compositional fingerprints. A rapid and convenient discriminant function was then established to discriminate the species of unknown Wen-Hou-Po, and the cross validation result was 100%. In this study, the methods established are reliable, and could be used to evaluate the quality and to identify the species of Wen-Hou-Po in the future.

  14. Application of hydrostatic CCC-TLC-HPLC-ESI-TOF-MS for the bioguided fractionation of anticholinesterase alkaloids from Argemone mexicana L. roots.

    PubMed

    Kukula-Koch, Wirginia; Mroczek, Tomasz

    2015-03-01

    A rapid hydrostatic counter-current chromatography-thin-layer chromatography-electrospray-ionization time-of-flight mass spectrometry (CCC-TLC-ESI-TOF-MS) technique was established for use in seeking potent anti-Alzheimer's drugs among the acethylcholinesterase inhibitors in Argemone mexicana L. underground parts, with no need to isolate components in pure form. The dichloromethane extract from the roots of Mexican prickly poppy that was most rich in secondary metabolites was subjected to hydrostatic-CCC-based fractionation in descending mode, using a biphasic system composed of petroleum ether-ethyl acetate-methanol-water at the ratio of 1.5:3:2.1:2 (v/v). The obtained fractions were analyzed in a TLC-based AChE-inhibition "Fast Blue B" test. All active components in the fractions, including berberine, protopine, chelerithrine, sanguinarine, coptisine, palmatine, magnoflorine, and galanthamine, were identified in a direct TLC-HPLC-ESI-TOF-MS assay with high accuracy. This is the first time galanthamine has been reported in the extract of Mexican prickly poppy and the first time it has been identified in any member of the Papaveraceae family, in the significant quantity of 0.77%.

  15. Identification of NF-κB inhibitors in Qishenyiqi dropping pills for myocardial infarction treatment based on bioactivity-integrated UPLC-Q/TOF MS.

    PubMed

    Han, Yanqi; Zhou, Mengge; Xing, Lu; Jiang, Min; Bai, Gang; Luo, Guoan

    2015-10-01

    Qishenyiqi dropping pills (QSYQ) are a type of standardized cardiovascular multiherb medicine for the treatment of myocardial infarction (MI). Knowledge concerning the systemic identification of nuclear factor-kappa B (NF-κB) inhibitors of QSYQ is generally lacking. Therefore, it is necessary to establish an effective method to screen the bioactive components of NF-κB inhibition. In the present study, a rat model of coronary artery ligation was used to assess the cardioprotective effects of QSYQ. The electrocardiograms, histopathology of heart tissues and serum biochemical indicators, such as brain natriuretic peptide, cardiac troponin I and inflammatory cytokines, were measured. Subsequently, ultra-performance liquid chromatography quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF MS) combined with the NF-κB luciferase reporter assay system was applied to screen the potential anti-inflammatory compounds in QSYQ. The results revealed that the administration of QSYQ could improve heart function, ameliorate neutrophil infiltration and diminish the levels of inflammatory cytokines in MI rats. Furthermore, 22 compounds were determined to be potential NF-κB inhibitors. In conclusion, NF-κB inactivation and cytokine suppression might be the main mechanisms of QSYQ for MI treatment. The method of UPLC-Q/TOF MS combined with a bioactive human cell functional evaluation system was proved to be a simple and effective strategy for screening bioactive compounds in traditional Chinese medicine prescriptions.

  16. Mechanism evaluation of the interactions between flavonoids and bovine serum albumin based on multi-spectroscopy, molecular docking and Q-TOF HR-MS analyses.

    PubMed

    Fu, Ling; Sun, Yiqun; Ding, Lina; Wang, Yangyang; Gao, Zhen; Wu, Zhen; Wang, Shaomin; Li, Wen; Bi, Yuefeng

    2016-07-15

    The mechanism of interactions between a flavonoid glycoside (linarin) and 6 flavonoids with various hydroxyl and methoxyl substituents (luteolin, apigenin, acacetin, tricin, 5,3',4'-trihydroxy-6,7-dimethoxyflavone, and 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone) and bovine serum albumin (BSA) were investigated by multi-spectroscopy, molecular docking, and quadrupole (Q)-time of flight (TOF) high resolution (HR) mass spectrometry (MS). Fluorescence spectra and molecular docking predicted that each of the flavonoids had only one probable binding site inside the hydrophobic cleft of BSA. The binding constants appeared to correlate positively with the number of hydroxyl groups, and negatively with the number of methoxyl groups. In addition, hydroxyls on ring B bound more easily with BSA than those on ring A. The change in conformation of BSA after binding suggested that the quenching mechanism was static quenching combined with nonradiative energy transfer. The results of Q-TOF HR-MS were consistent with fluorescence quenching and molecular docking.

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

    PubMed

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

    2008-12-01

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

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

    PubMed Central

    2014-01-01

    Background Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks. Results The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay. Conclusions The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved. PMID:24943244

  19. Combination of UHPLC/Q-TOF-MS, NMR spectroscopy, and ECD calculation for screening and identification of reactive metabolites of gentiopicroside in humans.

    PubMed

    Han, Han; Xiong, Ai-Zhen; He, Chun-Yong; Liu, Qing; Yang, Li; Wang, Zheng-Tao

    2014-02-01

    The metabolic investigation of natural products is a great challenge because of unpredictable metabolic pathways, little knowledge on metabolic effects, and lack of recommended analytical methodology. Herein, a combined strategy based on ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, and electronic circular dichroism (ECD) calculation was developed and employed for the human metabolism study of gentiopicroside (GPS), a naturally hepato-protective iridoid glycoside. The whole metabolic study consisted of three major procedures. First, an improved UHPLC/Q-TOF-MS method was used to separate and detect a total of 15 GPS metabolites that were obtained from urine samples (0 to 72 h) of 12 healthy male participants after a single 50-mg oral dose of GPS. Second, a developed "MS-NMR-MS" method was applied to accurately identify molecular structures of the observed metabolites. Finally, given that the associated stereochemistry may be a crucial factor of the metabolic activation, the absolute configuration of the reactive metabolites was revealed through chemical calculations. Based on the combined use, a pair of diastereoisomers (G05 and G06) were experimentally addressed as the bioreactive metabolites of GPS, and the stereochemical determination was completed. Whereas several novel metabolic transformations, occurring via oxidation, N-heterocyclization and glucuronidation after deglycosylation, were also observed. The results indicated that GPS has to undergo in vivo metabolism-based activation to generate reactive molecules capable of processing its hepato-protective activity.

  20. SALDI-TOF-MS analyses of small molecules (citric acid, dexasone, vitamins E and A) using TiO2 nanocrystals as substrates.

    PubMed

    Popović, Iva A; Nešić, Maja; Vranješ, Mila; Šaponjić, Zoran; Petković, Marijana

    2016-10-01

    Surface-assisted laser desorption/ionisation time-of-flight mass spectrometry (SALDI-TOF-MS) might be the method of choice for the analysis of low mass molecules (less than m/z 500). Titanium dioxide (TiO2) nanocrystals as a substrate for SALDI-TOF-MS improve the reproducibility of the signal intensities and prevent the fragmentation of some molecules upon laser irradiation, as we have previously shown. In addition, variously shaped and sized TiO2 nanocrystals/substrates for SALDI-MS could be used for quantification of small molecules, which are otherwise difficult to detect with the assistance of organic matrices. TiO2-assisted LDI-MS spectra could be acquired with excellent reproducibility and repeatability and with low detection limit. In the current study, we analysed the spectra of dexasone, citric acid, vitamin E and vitamin A acquired with TiO2 nanocrystals of various shapes and dimensions, i.e. the colloidal TiO2 nanoparticles (TiO2 NPs), TiO2 prolate nanospheroids (TiO2 PNSs) and TiO2 nanotubes (TiO2 NTs). Various shapes and dimensions of substrates were used since these factors determine desorption and ionisation processes. The homogeneity on the target plate was compared based on signal-to-noise values of peaks of interest of analysed molecules as well as the within-day and day-to-day repeatability. In summary, the obtained results show that the applicability of individual TiO2 nanocrystals depends on the analyte. Signals which are acquired with the assistance of TiO2 PNSs have the highest sensitivity and reproducibility (the smallest standard deviation), even compared with those in the LDI mode. This implies that TiO2 PNSs could also be suitable for quantitative analyses of small molecules.

  1. Chip-based nLC-TOF-MS is a highly stable technology for large-scale high-throughput analyses.

    PubMed

    Ruhaak, L Renee; Taylor, Sandra L; Miyamoto, Suzanne; Kelly, Karen; Leiserowitz, Gary S; Gandara, David; Lebrilla, Carlito B; Kim, Kyoungmi

    2013-05-01

    Many studies focused on the discovery of novel biomarkers for the diagnosis and treatment of disease states are facilitated by mass spectrometry-based technology. HPLC coupled to mass spectrometry is widely used; miniaturization of this technique using nano-liquid chromatography (LC)-mass spectrometry (MS) usually results in better sensitivity, but is associated with limited repeatability. The recent introduction of chip-based technology has significantly improved the stability of nano-LC-MS, but no substantial studies to verify this have been performed. To evaluate the temporal repeatability of chip-based nano-LC-MS analyses, N-glycans released from a serum sample were repeatedly analyzed using nLC-PGC-chip-TOF-MS on three non-consecutive days. With an average inter-day coefficient of variation of 4 %, determined on log10-transformed integrals, the repeatability of the system is very high. Overall, chip-based nano-LC-MS appears to be a highly stable technology, which is suitable for the profiling of large numbers of clinical samples for biomarker discovery.

  2. An untargeted metabolomics-driven approach based on LC-TOF/MS and LC-MS/MS for the screening of xenobiotics and metabolites of Zhi-Zi-Da-Huang decoction in rat plasma.

    PubMed

    Wu, Huan; Li, Xixi; Yan, Xuemei; An, Li; Luo, Kaiwen; Shao, Mingjing; Jiang, Yue; Xie, Rui; Feng, Fang

    2015-11-10

    Zhi-Zi-Da-Huang decoction (ZZDHD), a typical traditional Chinese medicine prescription, is widely used in clinical practice for the treatment of alcoholic liver disease. However, due to lack of holistic metabolic research, the active ingredients of ZZDHD have not been fully elucidated. It entails a huge obstacle for the quality evaluation, pharmacokinetic studies and clinical-safe medication administration of ZZDHD. In this work, an untargeted metabolomics-driven approach was proposed to rapidly screen and characterize xenobiotics and related metabolites in vivo conducted by LC-TOF/MS and LC-QqQ/MS. The tR-m/z pairs which were present in the ZZDHD-dosed group and absent in the control group could be clearly displayed by XCMS Online platform combined with supervised orthogonal partial least squares discriminant analysis. Among them, a total of 61 ZZDHD-related xenobiotics and metabolites including 34 prototype components and 27 metabolites were rapidly identified or tentatively characterized in rat plasma. The results indicated that iridoid glycosides and monoterpenoids from Gardenia jasminoides Ellis, flavonoid glycosides from Citrus aurantium L., as well as anthraquinones from Rheum palmatum L. were the main absorbed chemical components of ZZDHD. Hydrolysis, glucuronidation and sulfation were the main metabolic pathways of ZZDHD in vivo. The present study provided a solid basis for further revealing the relationship between the xenobiotic metabolome and pharmacological activity of ZZDHD. In addition, the application of untargeted metabolomics-driven approach offers a fresh insight for rapid screening and identifying xenobiotics and metabolites of ZZDHD and other multiherb prescription.

  3. LCMS/MS and TOF-SIMS identification of the color bodies on the surface of a polymer.

    PubMed

    Moore, Colin; McKeown, Pat

    2005-03-01

    The source of discoloration on a polymer surface can often be identified by washing the surface of the discolored polymer to collect the color bodies, then analyzing the washings using liquid chromatography-mass spectrometry (LCMS), with an in-line ultraviolet (UV) detector set at the optimum wavelength for the particular color bodies. A reference sample having no discoloration is also analyzed in the same way. In this paper, results from this methodology are compared with direct time of flight-secondary ion mass spectrometry (TOF-SIMS) analysis of a discolored polymer. The benefits and shortcomings of each methodology are discussed.

  4. Establishment and application of an analytical in-house database (IHDB) for rapid discrimination of Bacillus subtilis group (BSG) using whole-cell MALDI-TOF MS technology.

    PubMed

    Huang, Chien-Hsun; Huang, Lina; Chang, Mu-Tzu; Chen, Kuo-Lung

    2016-10-01

    Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.

  5. 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

  6. Thin-Layer Matrix Sublimation with Vapor-Sorption Induced Co-Crystallization for Sensitive and Reproducible SAMDI-TOF MS Analysis of Protein Biosensors

    NASA Astrophysics Data System (ADS)

    Roth, Michael J.; Kim, Jaekuk; Maresh, Erica M.; Plymire, Daniel A.; Corbett, John R.; Zhang, Junmei; Patrie, Steven M.

    2012-10-01

    Coupling immunoassays on self-assembled monolayers (SAMs) to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) provides improved assay selectivity compared with traditional photometric detection techniques. We show that thin-layer-transfer (TLT) of α-cyano-4-hydroxycinnaminic acid (CHCA) MALDI matrix via vacuum sublimation followed by organic solvent-based vapor-sorption induced co-crystallization (VIC) results in unique matrix/analyte co-crystallization tendencies that optimizes assay reproducibility and sensitivity. Unique matrix crystal morphologies resulted from VIC solvent vapors, indicating nucleation and crystal growth characteristics depend upon VIC parameters. We observed that CHCA microcrystals generated by methanol VIC resulted in >10× better sensitivity, increased analyte charging, and improved precision compared with dried droplet measurements. The uniformity of matrix/analyte co-crystallization across planar immunoassays directed at intact proteins yielded low spectral variation for single shot replicates (18.5 % relative standard deviation, RSD) and signal averaged spectra (<10 % RSD). We envision that TLT and VIC for MALDI-TOF will enable high-throughput, reproducible array-based immunoassays for protein molecular diagnostic assays in diverse biochemical and clinical applications.

  7. Classification of 'Chemlali' accessions according to the geographical area using chemometric methods of phenolic profiles analysed by HPLC-ESI-TOF-MS.

    PubMed

    Taamalli, Amani; Arráez Román, David; Zarrouk, Mokhtar; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

    2012-05-01

    The present work describes a classification method of Tunisian 'Chemlali' olive oils based on their phenolic composition and geographical area. For this purpose, the data obtained by HPLC-ESI-TOF-MS from 13 samples of extra virgin olive oils, obtained from different production area throughout the country, were used for this study focusing in 23 phenolics compounds detected. The quantitative results showed a significant variability among the analysed oil samples. Factor analysis method using principal component was applied to the data in order to reduce the number of factors which explain the variability of the selected compounds. The data matrix constructed was subjected to a canonical discriminant analysis (CDA) in order to classify the oil samples. These results showed that 100% of cross-validated original group cases were correctly classified, which proves the usefulness of the selected variables.

  8. Physical characterization of succinylated type I collagen by Raman spectra and MALDI-TOF/MS and in vitro evaluation for biomedical applications

    NASA Astrophysics Data System (ADS)

    Kumar, Ramadhar; Sripriya, R.; Balaji, S.; Senthil Kumar, M.; Sehgal, P. K.

    2011-05-01

    In this study, we report on physical and in vitro biological characterization of succinylated collagen (SC). SC was prepared by succinylation of type I bovine tendon collagen. SC swells and dissolves in physiological pH buffers (pH 7.4) Biocompatibility of SC to collagen for fibroblasts was comparable but L6 myoblasts showed pronounced proliferation and differentiation with SC. Using the MALDI-TOF/MS technique, SC was found with increased molecular mass by 16,359 Da per molecule which corresponds to about 54 succinyl groups covalently linked to the collagen strand. Raman spectroscopy revealed the retention of triple helical structure conformation in the presence of linked succinyl groups. New peaks near 1737, 1675 and 1420 cm -1 and decreased intensities near 2440 and 488 cm -1 provides the most convenient marker bands for succinylation of collagen. The intense band regions near 2856-2934, 2724, and 1445 cm -1 also confirms the existence of succinyl groups.

  9. MALDI-TOF MS analysis of soluble PEG based multi-step synthetic reaction mixtures with automated detection of reaction failure.

    PubMed

    Enjalbal, Christine; Ribière, Patrice; Lamaty, Frédéric; Yadav-Bhatnagar, Neerja; Martinez, Jean; Aubagnac, Jean-Louis

    2005-05-01

    Macromolecules of tunable solubility, used to mimic inert insoluble materials while maintaining solution conditions, allowed the performance of efficient supported organic chemistry and facilitated in situ reaction monitoring. To satisfy the high throughput requirements of automated synthetic processes, organic syntheses carried out on bifunctional polyethylene glycol polymers (PEG(3400)-OH) were monitored step-by-step by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A protocol was designed to control the ionization mechanism of such polymers exhibiting high affinity for alkali metal cations. Automated, rapid, and reliable data interpretation was performed by an in-house developed visual basic application relying on the sodiated ion accurate monoisotopic mass measurement. The methodology was illustrated through the monitoring of a six-step synthetic scheme.

  10. Use of TD-GC–TOF-MS to assess volatile composition during post-harvest storage in seven accessions of rocket salad (Eruca sativa)

    PubMed Central

    Bell, Luke; Spadafora, Natasha D.; Müller, Carsten T.; Wagstaff, Carol; Rogers, Hilary J.

    2016-01-01

    An important step in breeding for nutritionally enhanced varieties is determining the effects of the post-harvest supply chain on phytochemicals and the changes in VOCs produced over time. TD-GC–TOF-MS was used and a technique for the extraction of VOCs from the headspace using portable tubes is described. Forty-two compounds were detected; 39 were identified by comparison to NIST libraries. Thirty-five compounds had not been previously reported in Eruca sativa. Seven accessions were assessed for changes in headspace VOCs over 7 days. Relative amounts of VOCs across 3 time points were significantly different – isothiocyanate-containing molecules being abundant on ‘Day 0’. Each accession showed differences in proportions/types of volatiles produced on each day. PCA revealed a separation of VOC profiles according to the day of sampling. Changes in VOC profiles over time could provide a tool for assessment of shelf life. PMID:26471601

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

    PubMed

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

    2010-06-15

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

  12. Use of TD-GC-TOF-MS to assess volatile composition during post-harvest storage in seven accessions of rocket salad (Eruca sativa).

    PubMed

    Bell, Luke; Spadafora, Natasha D; Müller, Carsten T; Wagstaff, Carol; Rogers, Hilary J

    2016-03-01

    An important step in breeding for nutritionally enhanced varieties is determining the effects of the post-harvest supply chain on phytochemicals and the changes in VOCs produced over time. TD-GC-TOF-MS was used and a technique for the extraction of VOCs from the headspace using portable tubes is described. Forty-two compounds were detected; 39 were identified by comparison to NIST libraries. Thirty-five compounds had not been previously reported in Eruca sativa. Seven accessions were assessed for changes in headspace VOCs over 7days. Relative amounts of VOCs across 3 time points were significantly different - isothiocyanate-containing molecules being abundant on 'Day 0'. Each accession showed differences in proportions/types of volatiles produced on each day. PCA revealed a separation of VOC profiles according to the day of sampling. Changes in VOC profiles over time could provide a tool for assessment of shelf life.

  13. Inductively coupled plasma orthogonal acceleration time-of-flight mass spectrometry (ICP-oa-TOF-MS) analysis of heavy metal content in Indocalamus tesselatus samples.

    PubMed

    Qin, Yuyue; Zhang, Zhihong; Li, Lin; Chen, Chunsheng; Shun, Sha; Huang, Yinchuan

    2013-12-01

    Indocalamus tesselatus is one of the most popular packing materials in China. Heavy metals in Chinese I. tesselatus samples have received great interest because they are related to health. A simple and fast method for the determination of Pb, Cd, Mn, Ni, Cr, As, Hg, Cu and Zn, by inductively coupled plasma orthogonal acceleration time-of-flight mass spectrometry (ICP-oa-TOF-MS), following microwave closed vessel digestion of samples, was proposed. The method was validated using standard reference material (GBW 07605-Tea). Samples of I. tesselatus from five different regions of China were analysed using the proposed method. Heavy metals contents from different regions were found at different levels. Their low contents of heavy metals showed that collection areas were not polluted and all collected I. tesselatus samples could be unreservedly used as food packing materials without any health risk.

  14. Polymeric integrated selective enrichment target (ISET) for solid-phase-based sample preparation in MALDI-TOF MS.

    PubMed

    Ekström, Simon; Wallman, Lars; Helldin, Göran; Nilsson, Johan; Marko-Varga, György; Laurell, Thomas

    2007-11-01

    A polymer microfabricated proteomic sample preparation and MALDI MS sample presentation device, the integrated selective enrichment target (ISET), comprising an array of perforated nanovials is reported. Each perforated nanovial can be filled with selective extraction media (microbeads) for purification and concentration of protein/peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The main areas covered are the influence of the molding-process-induced surface roughness and how to address the lack of inherent conductivity in the polyetheretherketone (PEEK) material for optimal MALDI MS readout. Application of the disposable polymeric ISET devices for solid-phase extraction and phosphopeptide capture is also demonstrated.

  15. MALDI-TOF MS Enables the Rapid Identification of the Major Molecular Types within the Cryptococcus neoformans/C. gattii Species Complex

    PubMed Central

    Firacative, Carolina; Trilles, Luciana; Meyer, Wieland

    2012-01-01

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

  16. The renaissance of high-energy CID for structural elucidation of complex lipids: MALDI-TOF/RTOF-MS of alkali cationized triacylglycerols.

    PubMed

    Pittenauer, Ernst; Allmaier, Günter

    2009-06-01

    Triacylglycerols were analyzed as cationized species (Li(+), Na(+), K(+)) by high-energy CID at 20 keV collisions utilizing MALDI-TOF/RTOF mass spectrometry. Precursor ions, based on [M + Li](+)-adduct ions exhibited incomplete fragmentation in the high and low m/z region whereas [M + K](+)-adducts did not show useful fragmentation. Only sodiated precursor ions yielded product ion spectra with structurally diagnostic product ions across the whole m/z range. The high m/z region of the CID spectra is dominated by abundant charge-remote fragmentation of the fatty acid substituents. In favorable cases also positions of double bonds or of hydroxy groups of the fatty acid alkyl chains could be determined. A-type product ions represent the end products of these charge-remote fragmentations. B- and C-type product ions yield the fatty acid composition of individual triacylglycerol species based on loss of either one neutral fatty acid or one sodium carboxylate residue, respectively. Product ions allowing fatty acid substituent positional determination were present in the low m/z range enabling identification of either the sn-1/sn-3 substituents (E-, F-, and G-type ions) or the sn-2 substituent (J-type ion). These findings were demonstrated with synthetic triacylglycerols and plant oils such as cocoa butter, olive oil, and castor bean oil. Typical features of 20 keV CID spectra of sodiated triacylglycerols obtained by MALDI-TOF/RTOF MS were an even distribution of product ions over the entire m/z range and a mass accuracy of +/-0.1 to 0.2 u. One limitation of the application of this technique is mainly the insufficient precursor ion gating after MS1 (gating window at 4 u) of species separated by 2 u.

  17. A proteomic approach for rapid identification of Weissella species isolated from Korean fermented foods on MALDI-TOF MS supplemented with an in-house database.

    PubMed

    Kim, Eiseul; Cho, Youngjae; Lee, Yoonju; Han, Sun-Kyung; Kim, Chang-Gyeom; Choo, Dong-Won; Kim, Young-Rok; Kim, Hae-Yeong

    2017-02-21

    Weissella are obligate heterofermentative lactic acid bacteria belonging to the Leuconostocaceae family. Some Weissella can be found in salted and fermented foods, such as kimchi and jeotgal, and plays an important role in the fermentation process. In the present study, for the first time, a rapid and accurate identification method for Weissella species from kimchi and jeotgal was developed based on MALDI-TOF MS, supplemented with an in-house database. Of the 135 Weissella spectra aligned with the MALDI bioTyper database, 56 isolates (41.5%) yielded no reliable identification results with low log scores (<1.7). After registering the spectra of six Weissella reference strains, all of the isolates were correctly identified, of which 113 (83.7%) and 22 (16.3%) were identified at the species and genus level, respectively. Moreover, a dendrogram generated by protein profiles of the different Weissella species clearly presented distinctive clusters, and PCA analysis separated the spectra of Weissella species into four clusters. In comparing food origins, different Weissella species were identified from two fermented foods. W. soli and W. cibaria were isolated from kimchi, while W. thailandensis and W. halotolerans were isolated from jeotgal. The results of our proteomic approach confirm that the MALDI bioTyper database, with our in-house Weissella database, is sufficient for Weissella identification. The MALDI-TOF MS method provides fast and reliable discrimination between different species in the genus Weissella and, therefore, will be useful for safety control in fish farms or in the production of fermented foods. This method can also be applied to the control of opportunistic pathogenic Weissella in human clinical infections.

  18. GC-TOF/MS-based metabolomic strategy for combined toxicity effects of deoxynivalenol and zearalenone on murine macrophage ANA-1 cells.

    PubMed

    Ji, Jian; Zhu, Pei; Pi, Fuwei; Sun, Chao; Jiang, Hui; Sun, Jiadi; Wang, Xiumei; Zhang, Yinzhi; Sun, Xiulan

    2016-09-15

    The actual health risk from exposure to combined mycotoxins is unknown, and few studies have focused on changes to cellular biological systems (e.g., metabolomics) caused by combined mycotoxic effects. To evaluate the combined mycotoxic effects of deoxynivalenol (DON) and zearalenone (ZEN) on the level of cellular biological systems, gas chromatographic, time-of-flight mass spectroscopy (GC-TOF/MS) of the complete murine macrophage ANA-1 cell metabolome was implemented in this study. Using optimized chromatography and mass spectrometry parameters, the metabolites detected by GC-TOF/MS were identified and processed using multivariate statistical analysis, including principal component analysis (PCA) and orthogonal projection on latent-structures discriminant analysis (OPLS-DA). The metabolite sets were screened for further pathway analysis under rules of t-test (P) value < 0.05, VIP value > 1, and similarity value > 500. The mainly interfered metabolism pathways were categorized into two dominant types: amino acid metabolism and glycometabolism. Four metabolites, palmitic acid, 1-monopalmitin, ribose-5-phosphate and 2-deoxy-D-galactose, occur only under combined "DON + ZEN" treatment, indicating abnormal metabolism in ANA-1 cells. The metabolic state of ANA-1 cells under induction by combined "DON + ZEN" illustrates that DON may inhibit the estrogenic effects of ZEN. Thus, the combined effect of "DON + ZEN" may exacerbate toxicity in the pentose phosphate pathway, while palmitic acid metabolism is likely a new pathway effected by the combination, "DON + ZEN."

  19. Rapid identification of Bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

    PubMed

    Dybwad, Marius; van der Laaken, Anton L; Blatny, Janet Martha; Paauw, Armand

    2013-09-01

    Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-μg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.

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

    PubMed

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

    2014-12-01

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

  1. Discovery of safety biomarkers for realgar in rat urine using UFLC-IT-TOF/MS and 1H NMR based metabolomics.

    PubMed

    Huang, Yin; Tian, Yuan; Li, Geng; Li, Yuanyuan; Yin, Xinjuan; Peng, Can; Xu, Fengguo; Zhang, Zunjian

    2013-05-01

    As an arsenical, realgar (As4S4) is known as a poison and paradoxically as a therapeutic agent. However, a complete understanding of the precise biochemical alterations accompanying the toxicity and therapy effects of realgar is lacking. Using a combined ultrafast liquid chromatography (UFLC) coupled with ion trap time-of-flight mass spectrometry (IT-TOF/MS) and (1)H NMR spectroscopy based metabolomics approach, we were able to delineate significantly altered metabolites in the urine samples of realgar-treated rats. The platform stability of the liquid chromatography LC/MS and NMR techniques was systematically investigated, and the data processing method was carefully optimized. Our results indicate significant perturbations in amino acid metabolism, citric acid cycle, choline metabolism, and porphyrin metabolism. Thirty-six metabolites were proposed as potential safety biomarkers related to disturbances caused by realgar, and glycine and serine are expected to serve as the central contacts in the metabolic pathways related to realgar-induced disturbance. The LC/MS and NMR based metabolomics approach established provided a systematic and holistic view of the biochemical effects of realgar on rats, and might be employed to investigate other drugs or xenobiotics in the future.

  2. Proteomic study of serum using gel chromatography and MALDI-TOF MS reveals diagnostic biomarkers in male patients with liver-cancer

    NASA Astrophysics Data System (ADS)

    Zeng, Xin-Hua; Huang, He-Qing; Chen, Dong-Shi; Jin, Hong-Wei; Huang, Hui-Ying

    2007-03-01

    Human serum has been widely employed clinically for diagnosing various fatal diseases. However, the concentration of most proteins in human serum is too low to be directly measured using normal analytical methods. In order to obtain reliable analytical results from proteomic analysis of human serum, appropriate sample preparation is essential. A combined off-line analytical technique of gel chromatography and matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) has been successfully established to separate proteins for MS analysis. Using these combined techniques, 176 mass signal peaks of proteins/peptides were found in 6 of 18 fractions from normal male serum (NMS) in the presence of buffer consisting of NH4HCO3 and acetonitrile. A simple gel chromatography column packed with Sephadex G-50 removed most signal-suppressing compounds such as salts and high abundance proteins (HAP). The molecular mass to charge (m/z) ratios of differential peptides revealed in serum of male patient with liver-cancer (LCMPS) compared to NMS were 5365, 5644 and 6462, and these peptides can be used as biomarkers to clinically diagnose liver-cancer. The simple and convenient chromatographic method described here is not only superior to recently described HPLC separation for MS analysis, but also reveals many novel and significant serum biomarkers for the clinical diagnosis of various diseases.

  3. Allelic variation of LMW-GS composition in Chinese wheat landraces of the Yangtze-River region detected by MALDI-TOF-MS

    PubMed Central

    Peng, Yanchun; Yu, Zitong; Islam, Shahidul; Zhang, Yujuan; Wang, Xiaolong; Lei, Zhensheng; Yu, Kan; Sun, Dongfa; Ma, Wujun

    2016-01-01

    Low molecular weight glutenin subunits are important components of wheat storage proteins, which play an important role in determining end-use quality of common wheat. A newly established matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) procedure was used to analyze 478 landraces of bread wheat collected from the Yangtze-River region in China. Results indicated that 17 alleles at three loci: Glu-A3, Glu-B3 and Glu-D3 were identified, resulting in 87 different allele combinations. Of the 17 alleles detected at all the Glu-3 loci, five belonged to Glu-A3, seven to Glu-B3 and five to Glu-D3 locus. MALDI-TOF-MS indicated Glu-A3a/c was present in 72.8%, Glu-A3b in 8.4%, Glu-A3d in 8.4%, Glu-A3f in 5.2% and Glu-A3e in 3.6% lines. Seven types of alleles were identified at the Glu-B3 locus: Glu-B3d/i (25.5%), Glu-B3b (21.3%), Glu-B3c (16.9%), Glu-B3h (13.8%), Glu-B3f (8.4%), Glu-B3a (8.2%), and Glu-B3g (5.2%). Five types of Glu-D3 alleles were detected: Glu-D3a (58.4%), Glu-D3c (22.6%), Glu-D3d (15.5%), Glu-D3b (3.3%) and Glu-D3f (0.2%). Four new alleles that showed abnormal MALDI-TOF spectrum patterns were identified at the Glu-A3 and Glu-B3 loci. A detailed study is needed to further characterize these alleles and their potential usage for wheat improvement. PMID:27795690

  4. Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS

    NASA Astrophysics Data System (ADS)

    Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene

    2016-11-01

    High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

  5. Identification of Enterobacteriaceae and detection of carbapenemases from positive blood cultures by combination of MALDI-TOF MS and Carba NP performed after four hour subculture in Mueller Hinton.

    PubMed

    Fernández, Javier; Rodríguez-Lucas, Carlos; Fernández-Suárez, Jonathan; Vazquez, Fernando; Rodicio, M Rosario

    2016-10-01

    A new protocol for Enterobacteriaceae identification and detection of carbapenemase-producing isolates from blood cultures by combining MALDI-TOF MS and the Carba NP test has been evaluated. Bacterial identification was correct in 129 of the 130 isolates tested while the Carba NP detected 28 out of the 29 carbapenemase producers.

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

    PubMed Central

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

    2016-01-01

    Objective: To evaluate the performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) by using 190 Catalase-negative Gram-Positive Cocci (GPC) clinical isolates. Methods: All isolates were identified by conventional phenotypic tests following the proposed scheme by Ruoff and Christensen and MALDI-TOF MS (Bruker Daltonics, BD, Bremen, Germany). Two different extraction methods (direct transfer formic acid method on spot and ethanol formic acid extraction method) and different cut-offs for genus/specie level identification were used. The score cut-offs recommended by the manufacturer (≥ 2.000 for species-level, 1.700 to 1.999 for genus level and <1.700 no reliable identification) and lower cut-off scores (≥1.500 for genus level, ≥ 1.700 for species-level and score <1.500 no reliable identification) were considered for identification. A minimum difference of 10% between the top and next closest score was required for a different genus or species. MALDI-TOF MS identification was considered correct when the result obtained from MS database agreed with the phenotypic identification result. When both methods gave discordant results, the 16S rDNA or sodA genes sequencing was considered as the gold standard identification method. The results obtained by MS concordant with genes sequencing, although discordant with conventional phenotyping, were considered correct. MS results discordant with 16S or sodA identification were considered incorrect. Results: Using the score cut-offs recommended by the manufacturer, 97.37% and 81.05% were correctly identified to genus and species level, respectively. On the other hand, using lower cut-off scores for identification, 97.89% and 94.21% isolates were correctly identified to genus and species level respectively by MALDI-TOF MS and no significant differences between the results obtained with two extraction methods were obtained. Conclusion: The results obtained suggest that MALDI-TOF

  8. Studies on substantially increased proteins in follicular fluid of bovine ovarian follicular cysts using 2-D PAGE and MALDI-TOF MS

    PubMed Central

    Maniwa, Jiro; Izumi, Shunsuke; Isobe, Naoki; Terada, Takato

    2005-01-01

    Background The objective of this study was to identify substantially increased proteins in bovine cystic follicular fluid (FF) in order to clarify the pathology and etiology of bovine ovarian follicular cysts (BOFC). Methods Proteins in normal and cystic FF samples were subjected to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were compared using silver stained gel images with PDQuest image analysis software. Peptides from these increased spots were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and were identified based on the NCBI database by a peptide mass fingerprinting method. Results Comparative proteomic analysis showed 8 increased protein spots present in cystic FF. MS analysis and database searching revealed that the increased proteins in cystic FF were bovine mitochondrial f1-atpase (BMFA), erythroid associated factor (EAF), methionine synthase (MeS), VEGF-receptor, glyceraldehydes 3-phosphate dehydrogenase (GAPDH), heat shock protein 70 (HSP70), β-lactoglobulin (BLG) and succinate dehydrogenase Ip subunit (SD). Conclusion Our results suggest that these proteins are overexpressed in BOFC, and that they may play important roles in the pathogenesis of BOFC. Furthermore, these proteins in the FF could be useful biomarkers for BOFC. PMID:15941490

  9. New P2X3 receptor antagonists. Part 2: Identification and SAR of quinazolinones.

    PubMed

    Szántó, Gábor; Makó, Attila; Vágó, István; Hergert, Tamás; Bata, Imre; Farkas, Bence; Kolok, Sándor; Vastag, Mónika

    2016-08-15

    Numerous potent P2X3 antagonists have been discovered and the therapeutic potential of P2X3 antagonism already comprises proof-of-concept data obtained in clinical trials with the most advanced compound. We have lately reported the discovery and optimization of thia-triaza-tricycle compounds with potent P2X3 antagonistic properties. This Letter describes the SAR of a back-up series containing a 4-oxo-quinazoline central ring. The discovery of the highly potent compounds 51 is presented.

  10. Exploring in vitro, in vivo metabolism of mogroside V and distribution of its metabolites in rats by HPLC-ESI-IT-TOF-MS(n).

    PubMed

    Xu, Feng; Li, Dian-Peng; Huang, Zhen-Cong; Lu, Feng-Lai; Wang, Lei; Huang, Yong-Lin; Wang, Ru-Feng; Liu, Guang-Xue; Shang, Ming-Ying; Cai, Shao-Qing

    2015-11-10

    Mogroside V, a cucurbitane-type saponin, is not only the major bioactive constituent of traditional Chinese medicine Siraitiae Fructus, but also a widely used sweetener. To clarify its biotransformation process and identify its effective forms in vivo, we studied its metabolism in a human intestinal bacteria incubation system, a rat hepatic 9000g supernatant (S9) incubation system, and rats. Meanwhile, the distribution of mogroside V and its metabolites was also reported firstly. Seventy-seven new metabolites, including 52 oxidation products formed by mono- to tetra- hydroxylation/dehydrogenation, were identified with the aid of HPLC in tandem with ESI ion trap (IT) TOF multistage mass spectrometry (HPLC-ESI-IT-TOF-MS(n)). Specifically, 14 metabolites were identified in human intestinal bacteria incubation system, 4 in hepatic S9 incubation system, 58 in faeces, 29 in urine, 14 in plasma, 34 in heart, 33 in liver, 39 in spleen, 39 in lungs, 42 in kidneys, 45 in stomach, and 51 in small intestine. The metabolic pathways of mogroside V were proposed and the identified metabolic reactions were deglycosylation, hydroxylation, dehydrogenation, isomerization, glucosylation, and methylation. Mogroside V and its metabolites were distributed unevenly in the organs of treated rats. Seven bioactive metabolites of mogroside V were identified, among which mogroside IIE was abundant in heart, liver, spleen and lung, suggesting that it may contribute to the bioactivities of mogroside V. Mogroside V was mainly excreted in urine, whereas its metabolites were mainly excreted in faeces. To our knowledge, this is the first report that a plant constituent can be biotransformed into more than 65 metabolites in vivo. These findings will improve understanding of the in vivo metabolism, distribution, and effective forms of mogroside V and congeneric molecules.

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

    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

  12. 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.

  13. Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory.

    PubMed

    Deak, Eszter; Charlton, Carmen L; Bobenchik, April M; Miller, Shelley A; Pollett, Simon; McHardy, Ian H; Wu, Max T; Garner, Omai B

    2015-01-01

    This study compared the diagnostic performance of Bruker's Microflex LT and bioMérieux's Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. A total of 477 isolates were tested on both instruments. Discrepant results were resolved by sequencing. Overall, there was no statistically significant difference between the proportion of isolates correctly identified, miscalled or not called by each instrument. Although both systems were good at identifying yeast (66/69 to species level), the confidence level was high only to genus level for 30% of the isolates on the Bruker. Both systems performed with high accuracy when evaluated solely on Food and Drug Administration-approved organisms for each database. A user-based assessment of the 2 instruments revealed an overall preference for the Vitek MS instrument.

  14. 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

  15. Window type: 2x3 fixed multipaned steel window flanked by 1x3 ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Window type: 2x3 fixed multipaned steel window flanked by 1x3 multipaned steel casements. Concrete sill and spandrel also illustrated. Building 43, facing east - Harbor Hills Housing Project, 26607 Western Avenue, Lomita, Los Angeles County, CA

  16. Post-acquisition data processing for the screening of transformation products of different organic contaminants. Two-year monitoring of river water using LC-ESI-QTOF-MS and GCxGC-EI-TOF-MS.

    PubMed

    López, S Herrera; Ulaszewska, M M; Hernando, M D; Martínez Bueno, M J; Gómez, M J; Fernández-Alba, A R

    2014-11-01

    This study describes a comprehensive strategy for detecting and elucidating the chemical structures of expected and unexpected transformation products (TPs) from chemicals found in river water and effluent wastewater samples, using liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight mass spectrometer (LC-ESI-QTOF-MS), with post-acquisition data processing and an automated search using an in-house database. The efficacy of the mass defect filtering (MDF) approach to screen metabolites from common biotransformation pathways was tested, and it was shown to be sufficiently sensitive and applicable for detecting metabolites in environmental samples. Four omeprazole metabolites and two venlafaxine metabolites were identified in river water samples. This paper reports the analytical results obtained during 2 years of monitoring, carried out at eight sampling points along the Henares River (Spain). Multiresidue monitoring, for targeted analysis, includes a group of 122 chemicals, amongst which are pharmaceuticals, personal care products, pesticides and PAHs. For this purpose, two analytical methods were used based on direct injection with a LC-ESI-QTOF-MS system and stir bar sorptive extraction (SBSE) with bi-dimensional gas chromatography coupled with a time-of-flight spectrometer (GCxGC-EI-TOF-MS).

  17. Rapid identification of betacyanins from Amaranthus tricolor, Gomphrena globosa, and Hylocereus polyrhizus by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS).

    PubMed

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

    2006-09-06

    Natural betacyanins have attracted great attention as food colorants and potential antioxidants. Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) is a new and powerful technique for the identification of low molecular weight compounds. This study is the first to employ MALDI-QIT-TOF MS to rapidly identify, within a few minutes, a great number of betacyanins in crude extracts from Amaranthus tricolor seedlings, Gomphrena globosa flowers, and Hylocereus polyrhizus fruits. The fresh crude extract samples without any purification were directly used for MALDI-QIT-TOF MS analysis with 2,5-dihydroxybenzoic acid as a matrix. The MS2 and MS3 spectrometric data acquired could provide important characteristic information for structural elucidation of the betacyanins. Fourteen free and acylated betacyanins, belonging to amaranthin-type, betanin-type, and gomphrenin-type betacyanins, respectively, were identified. However, the related isomers should be differentiated with the aid of HPLC.

  18. Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses

    PubMed Central

    Vincent, Delphine; Elkins, Aaron; Condina, Mark R.; Ezernieks, Vilnis; Rochfort, Simone

    2016-01-01

    Cow’s milk is an important source of proteins in human nutrition. On average, cow’s milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600–3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels. PMID:27749892

  19. Challenging the problem of clostridial identification with matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS).

    PubMed

    Grosse-Herrenthey, Anke; Maier, Thomas; Gessler, Frank; Schaumann, Reiner; Böhnel, Helge; Kostrzewa, Markus; Krüger, Monika

    2008-10-01

    Diverse techniques were applied to effect the identification and classification of isolated clostridial strains. Nevertheless, the correct identification of clostridial strains remains a laborious, time-consuming task which entails a not inconsiderable degree of expertise. In addition to this, traditional methods based on the metabolic properties of the bacteria require rigorously standardized media and growth conditions to assure the attainment of reproducible results. Although DNA-based methods, like the PCR of a species specific gene, are known to yield precise and reproducible results, their degree of effectivity is circumscribed by the fact that even the incidence of a toxin encoding gene is not necessarily linked to nor consequently indicative of the presence of an infectious disease. Moreover, most of these methods postulate an initial assumption concerning the expected bacterial species involved before the choice of PCR primer for use can be made. Consequently, the scope of these methods is restricted to that of targeted analyses. The 16S rDNA sequencing which is assumed to be the gold standard for bacterial classification having the unequivocal advantage of being capable of determining even uncultivable bacteria is nonetheless a time-consuming and costly technique. In the present study we describe the utilization of matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for whole cell fingerprinting in combination with a dedicated bioinformatic software tool to distinguish between various clostridial species. Total 64 clostridial strains of 31 different species each displayed a mass spectrum unique to the strain involved, to the effect that it was also possible not only to differentiate between the strains examined, but also to establish to which species the individual strains belonged to. Starting with a single colony it was possible to correctly identify a Clostridium species within minutes. It was even possible

  20. Targeted toxicological screening for acidic, neutral and basic substances in postmortem and antemortem whole blood using simple protein precipitation and UPLC-HR-TOF-MS.

    PubMed

    Telving, Rasmus; Hasselstrøm, Jørgen Bo; Andreasen, Mette Findal

    2016-09-01

    A broad targeted screening method based on broadband collision-induced dissociation (bbCID) ultra-performance liquid chromatography high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS) was developed and evaluated for toxicological screening of whole blood samples. The acidic, neutral and basic substances covered by the method were identified in postmortem and antemortem whole blood samples from forensic autopsy cases, clinical forensic cases and driving under the influence of drugs (DUID) cases by a reverse target database search. The screening method covered 467 substances. Validation was performed on spiked whole blood samples and authentic postmortem and antemortem whole blood samples. For most of the basic drugs, the established cut-off limits were very low, ranging from 0.25ng/g to 50ng/g. The established cut-off limits for most neutral and acidic drugs, were in the range from 50ng/g to 500ng/g. Sample preparation was performed using simple protein precipitation of 300μL of whole blood with acetonitrile and methanol. Ten microliters of the reconstituted extract were injected and separated within a 13.5min UPLC gradient reverse-phase run. Positive electrospray ionization (ESI) was used to generate the ions in the m/z range of 50-1000. Fragment ions were generated by bbCID. Identification was based on retention time, accurate mass, fragment ion(s) and isotopic pattern. A very sensitive broad toxicological screening method using positive electrospray ionization UPLC-HR-TOF-MS was achieved in one injection. This method covered basic substances, substances traditionally analyzed in negative ESI (e.g., salicylic acid), small highly polar substances such as beta- and gamma-hydroxybutyric acid (BHB and GHB, respectively) and highly non-polar substances such as amiodarone. The new method was shown to combine high sensitivity with a very broad scope that has not previously been reported in toxicological whole blood screening when using only one injection.

  1. 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

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

    PubMed

    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.

  3. Evaluation of adenine as scaffold for the development of novel P2X3 receptor antagonists.

    PubMed

    Lambertucci, Catia; Sundukova, Mayya; Kachare, Dhuldeo D; Panmand, Deepak S; Dal Ben, Diego; Buccioni, Michela; Marucci, Gabriella; Marchenkova, Anna; Thomas, Ajiroghene; Nistri, Andrea; Cristalli, Gloria; Volpini, Rosaria

    2013-07-01

    Ligands that selectively block P2X3 receptors localized on nociceptive sensory fibres may be useful for the treatment of chronic pain conditions including neuropathic pain, migraine, and inflammatory pain. With the aim at exploring the suitability of adenine moiety as a scaffold for the development of antagonists of this receptor, a series of 9-benzyl-2-aminoadenine derivatives were designed and synthesized. These new compounds were functionally evaluated at rat or human P2X3 receptors expressed in human embryonic kidney (HEK) cells and on native P2X3 receptors from mouse trigeminal ganglion sensory neurons using patch clamp recording under voltage clamp configuration. The new molecules behaved as P2X3 antagonists, as they rapidly and reversibly inhibited (IC50 in the low micromolar range) the membrane currents induced via P2X3 receptor activation by the full agonist α,β-methyleneATP. Introduction of a small lipophilic methyl substituent at the 6-amino group enhanced the activity, in comparison to the corresponding unsubstituted derivative, resulting in the 9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N(6)-methyl-9H-purine-2,6-diamine (24), which appears to be a good antagonist on recombinant and native P2X3 receptors with IC50 = 1.74 ± 0.21 μM.

  4. Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors.

    PubMed

    Jung, Young-Hwan; Kim, Yeo Ok; Lin, Hai; Cho, Joong-Heui; Park, Jin-Hee; Lee, So-Deok; Bae, Jinsu; Kang, Koon Mook; Kim, Yoon-Gyoon; Pae, Ae Nim; Ko, Hyojin; Park, Chul-Seung; Yoon, Myung Ha; Kim, Yong-Chul

    2017-04-06

    Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.

  5. Studying the interactions of a platinum(II) 9-aminoacridine complex with proteins and oligonucleotides by ESI-TOF MS.

    PubMed

    Samper, Katia G; Vicente, Consuelo; Rodríguez, Venancio; Atrian, Sílvia; Cutillas, Natalia; Capdevila, Mercè; Ruiz, José; Palacios, Òscar

    2012-01-07

    The interaction of a novel Pt complex, [Pt(dmba)(N9-9AA)(PPh(3))](+)1 (dmba = N,N-dimethylbenzylamine-κN,κC; 9AA = 9-aminoacridine), which exhibits anti-tumor activity, with certain key proteins has been monitored by ESI-MS. Also, the interaction of 1 with a designed double-stranded oligonucleotide containing the GG motif has been followed by mass spectrometry as well as by fluorimetry. The results obtained show the low interaction of 1 with the considered proteins and the absence of covalent interaction with the oligonucleotides, but the fluorimetric data confirm the π-π interaction of 1 with the double-stranded DNA, which is probably the reason of the previously reported activity of 1 in several tumor cell lines.

  6. Preparation of longitudinal sections of hair samples for the analysis of cocaine by MALDI-MS/MS and TOF-SIMS imaging.

    PubMed

    Flinders, Bryn; Cuypers, Eva; Zeijlemaker, Hans; Tytgat, Jan; Heeren, Ron M A

    2015-10-01

    Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) for the analysis of intact hair is a powerful tool for the detection of drugs of abuse in toxicology and forensic applications. Here we present a quick, easy, and reproducible method of preparing longitudinal sections of single hairs. This method improves the accessibility of chemicals embedded in the hair matrix for molecular imaging with mass spectrometry. The images obtained from a single, sectioned hair sample show molecular distributions in the exposed medulla, cortex, and a portion of the cuticle observed as a narrow layer surrounding the cortex. Using MALDI-MS/MS imaging, the distribution of cocaine was observed throughout five longitudinally sectioned drug-user hair samples. The images showed the distribution of the product ion at m/z 182, derived from the precursor ion of cocaine at m/z 304. MetA-SIMS images of longitudinally sectioned hair samples showed a more detailed distribution of cocaine at m/z 304, benzoylecgonine the major metabolite of cocaine at m/z 290 and other drugs such as methadone which was observed at m/z 310. Chronological information of drug intake can be obtained more sensitively. The chronological detail is in hours rather than months, which is of great interest in clinical as well as forensic applications.

  7. Quantification of rutin in rat's brain by UHPLC/ESI-Q-TOF-MS/MS after intranasal administration of rutin loaded chitosan nanoparticles

    PubMed Central

    Ahmad, Niyaz; Ahmad, Rizwan; Naqvi, Atta Abbas; Alam, Md Aftab; Samim, Mohd; Iqbal, Zeenat; Ahmad, Farhan Jalees

    2016-01-01

    Rutin (RT), an antioxidant drug, has been utilized to treat cerebral ischemia hence a sensitive quantification method for estimation of RT in brain homogenate is necessary to develop. This study aims to prepare RT loaded Chitosan Nanoparticles (RT-CS-NPs) develop and validate ultra-high performance liquid chromatography-electrospray ionization-synapt mass spectrometric method Synapt Mass Spectrometry (Synapt MS) (UHPLC/ESI-QTOF-MS/MS) for quantification of RT in brain homogenate from Wistar rat. The process of chromatographic separation was carried out on Waters ACQUITY UPLC™ with the components of separation in detail as; column: BEH C-18 with dimension as 2.1 mm×100 mm and particle size 1.7 µm, mobile phase: acetonitrile (85 % v/v/v): 2 mM ammonium formate (15 % v/v/v): formic acid (0.1 % v/v/v) and flow rate: 0.25 mL/min. Liquid-liquid extraction method (LLE), in mixture, i.e. ethyl acetate:acetonitrile, was considered to optimize the recovery of analyte from the brain homogenate of Wistar rat. Over a total run time of 5 minutes, the elution time for RT and internal standard (IS), i.e. Tolbutamide, observed was 2.67 and 2.82 min respectively whereas the transition observed for RT and IS was at m/z 611.1023/303.1071 and 271.1263/155.1073, respectively. Results, regarding various processes and parameters studied for RT as summarized, established a linear dynamic range over a concentration range of 1.00 ng/mL - 1000.0 ng/mL with r2; 0.9991±0.0010. Accuracy for intra and inter-assay in terms of % CV revealed a range of 0.45- 2.11 whereas lower limit of detection (LOD) and quantitation (LOQ) observed was 0.09 ng/mL and 0.142 ng/mL, respectively. The analyte stability as well as method specificity and accuracy, i.e. recovery > 86 %, supports the idea for application of current developed method in order to quantify and evaluate the RT-loaded-CS-NPs for RT determination in brain homogenate after intranasal drug delivery. PMID:28096783

  8. 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

  9. Comparative analysis of salt-responsive phosphoproteins in maize leaves using Ti(4+)--IMAC enrichment and ESI-Q-TOF MS.

    PubMed

    Hu, Yufeng; Guo, Shuangxi; Li, Xuexian; Ren, Xueqin

    2013-02-01

    Salinity is one of the most common abiotic stresses encountered by plants. Reversible protein phosphorylation is involved in plant defense processes against salinity stress. Here, we performed global phosphopeptide mapping through enrichment by our synthesized PVA-phosphate-Ti(4+) IMAC coupled with subsequent identification by ESI-Q-TOF MS. A total of 104 peptide sequences containing 139 phosphorylation sites were determined from 70 phosphoproteins of the control leaves. In contrast, 124 phosphopeptides containing 143 phosphorylated sites from 92 phosphoproteins were identified in salt-stressed maize leaves. Compared with the control, 47 proteins were phosphorylated, 25 were dephosphorylated, and 45 overlapped. Among the 72 differential phosphoproteins, 35 were known salt stress response proteins and the rest had not been reported in the literature. To dissect the differential phosphorylation, gene ontology annotations were retrieved for the differential phosphoproteins. The results revealed that cell signaling pathway members such as calmodulin and 14-3-3 proteins were regulated in response to 24-h salt stress. Multiple putative salt-responsive phosphoproteins seem to be involved in the regulation of photosynthesis-related processes. These results may help to understand the salt-inducible phosphorylation processes of maize leaves.

  10. Phospholipid compositions of sera and synovial fluids from dog, human and horse: a comparison by 31P-NMR and MALDI-TOF MS.

    PubMed

    Fuchs, B; Bondzio, A; Wagner, U; Schiller, J

    2009-08-01

    Alterations of the phospholipid (PL) compositions of body fluids are assumed to be indicative of inflammatory diseases, e.g. rheumatoid arthritis (RA). Recently, we have shown that particularly the phosphatidylcholine/lysophosphatidylcholine (PC/LPC) ratio determined in human synovial fluids (SF) and sera represents a reliable measure of the inflammatory state in RA patients. However, it is not yet clear to what extent the PC/LPC ratio is also affected by nutrition habits. In the present study, the PL and the corresponding acyl chain compositions of human body fluids (SF and serum of RA patients as well as serum from healthy volunteers) are compared with those of two other mammalian species (horses and dogs suffering from degenerative joint diseases as well as healthy controls) by high-resolution 31P-nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). The most important result of this study is that the PL compositions of SF and serum of horse and dog are comparable with those of human body fluids. Compared with humans, however, the horse body fluid contains less PCs with highly unsaturated arachidonoyl residues, while that of dogs possesses the highest content of arachidonoyl-containing PC. These species-related differences stem primarily from different nutrition habits (meat vs. plants).

  11. Metabolic profiles of the Flos Abelmoschus manihot extract by intestinal bacteria from the normal and CKD model rats based on UPLC-Q-TOF/MS.

    PubMed

    Du, Le-Yue; Tao, Jin-Hua; Jiang, Shu; Qian, Da-Wei; Guo, Jian-Ming; Duan, Jin-Ao

    2017-02-01

    Flos Abelmoschus manihot is a traditional herbal medicine widely used in clinical practice to tackle chronic kidney disease (CKD) for thousands of years. Nowadays, many studies indicate that gut bacteria are closely related to the progression of CKD and CKD-related complications. In this study, a UPLC-Q-TOF/MS method coupled with the MetaboLynx™ software was established and successfully applied to investigate the metabolites and metabolic profile of Flos A. manihot extract by intestinal bacteria from normal and CKD rats. Eight parent components and eight metabolites were characterized by their protonated ions. Among these compounds, 15 were detected in the two group samples while M16 was only determined in the CKD model samples. Compared with the quercetin-type glycosides, fewer myricetin-type and gossypetin-type metabolites were obtained in the two group samples. These metabolites suggested that deglycosylation and methylation are the major metabolic pathways of Flos A. manihot extract. Few differences of metabolite classes were observed in the two group samples. However, the concentrations of aglycones such as quercetin, myricetin and gossypetin in the normal samples were notably higher than those in the CKD model samples. The results are important in unravelling the pharmacological effects of A. manihot and clarifying its mechanism of action in vivo.

  12. Anti-melanoma activity of Forsythiae Fructus aqueous extract in mice involves regulation of glycerophospholipid metabolisms by UPLC/Q-TOF MS-based metabolomics study

    PubMed Central

    Bao, Jiaolin; Liu, Fang; Zhang, Chao; Wang, Kai; Jia, Xuejing; Wang, Xiaotong; Chen, Meiwan; Li, Peng; Su, Huanxing; Wang, Yitao; Wan, Jian-Bo; He, Chengwei

    2016-01-01

    Metabolomics is a comprehensive assessment of endogenous metabolites of a biological system in a holistic context. In this study, we evaluated the in vivo anti-melanoma activity of aqueous extract of Forsythiae Fructus (FAE) and globally explored the serum metabolome characteristics of B16-F10 melanoma-bearing mice. UPLC/Q-TOF MS combined with pattern recognition approaches were employed to examine the comprehensive metabolic signatures and differentiating metabolites. The results demonstrated that FAE exhibited remarkable antitumor activity against B16-F10 melanoma in C57BL/6 mice and restored the disturbed metabolic profile by tumor insult. We identified 17 metabolites which were correlated with the antitumor effect of FAE. Most of these metabolites are involved in glycerophospholipid metabolisms. Notably, several lysophosphatidylcholines (LysoPCs) significantly decreased in tumor model group, while FAE treatment restored the changes of these phospholipids to about normal condition. Moreover, we found that lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX) were highly expressed in melanoma, and FAE markedly down-regulated their expression. These findings indicated that modulation of glycerophospholipid metabolisms may play a pivotal role in the growth of melanoma and the antitumor activity of FAE. Besides, our results suggested that serum LysoPCs could be potential biomarkers for the diagnosis and prognosis of melanoma and other malignant tumors. PMID:27991567

  13. Prediction of Chinese green tea ranking by metabolite profiling using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS).

    PubMed

    Jing, Jin; Shi, Yuanzhi; Zhang, Qunfeng; Wang, Jie; Ruan, Jianyun

    2017-04-15

    Metabolomics profiling provides comprehensive picture of the chemical composition in teas therefore may be used to assess tea quality objectively and reliably. In the present experiment, water and methanol extracts of green teas from China were analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with the objectives to establish a model for quality prediction and to identify potential marker metabolites. The blindly evaluated sensory score of green teas was predicted with excellent power (R(2)=0.87 and Q(2)=0.82) and accuracy (RMSEP=1.36) by a partial least-squares (PLS) regression model based on water extract. By contrast, methanol extract failed to reasonably predict the sensory scores. The levels in water extract of neotheaflavin, neotheaflavin 3-O-gallate, trigalloyl-β-d-glucopyranose, myricetin 3,3'-digalactoside, catechin-(4α→8)-epigallocatechin and kaempferol were significantly larger whereas those of theogallin and gallocatechin were less in the low (score<87) than in the high score (⩾90) group.

  14. Identification of bioactive peptides in hypoallergenic infant milk formulas by CE-TOF-MS assisted by semiempirical model of electromigration behavior.

    PubMed

    Català-Clariana, Sergio; Benavente, Fernando; Giménez, Estela; Barbosa, José; Sanz-Nebot, Victoria

    2013-07-01

    Biologically active peptides derived from complex bovine milk protein hydrolysates are of particular interest in food science and nutrition because they have been shown to play different physiological roles, providing benefits in human health. In this study, we used CE-TOF-MS for separation and identification of bioactive peptides in three hypoallergenic infant milk formulas. An appropriate sample cleanup using a citrate buffer with DTT and urea followed by SPE with Sep-Pack® C18 and StrataX™ cartridges allowed the detection of a large number of low molecular mass bioactive peptides. This preliminary identification was solely based on the measured experimental monoisotopic molecular mass values (M(exp)). Later, we evaluated the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (m(e) vs. q/M(α), α = 1/2 for the classical polymer model) to describe their migration behavior. The assistance of migration prediction proved to be useful to improve reliability of the identification, avoiding misinterpretations and solving some identity conflicts. After revision, the identity of 24, 30, and 38 bioactive peptides was confirmed in each of the three infant milk formulas. A significant number of these peptides were reported as inhibitors of angiotensin-converting enzyme, however, the presence of sequences with other biological activities such as antihypertensive, antithrombotic, hypocholesterolemic, immunomodulation, cytotoxicity, antioxidant, antimicrobial, antigenic, or opioid was also confirmed.

  15. Phytochemical, antioxidant and antidiabetic evaluation of eight Bauhinia L. species from Egypt using UHPLC-PDA-qTOF-MS and chemometrics.

    PubMed

    Farag, Mohamed A; Sakna, Sarah T; El-Fiky, Nabaweya M; Shabana, Marawan M; Wessjohann, Ludger A

    2015-11-01

    Bauhinia L. (Fabaceae) comprises ca. 300-350 plant species, many of which are traditionally used in folk medicine for their antidiabetic, antioxidant and anti-inflammatory effects. Bauhinia s.l. recently has been subdivided into 9 genera based on phylogenetic data: Bauhinia s.str., Barklya, Brenierea, Gigasiphon, Lysiphyllum, Phanera, Piliostigma, Schnella (American Phanera) and Tylosema. The aerial parts of 8 species corresponding to 5 genera were analyzed: Bauhinia forficata, Bauhinia variegata, B. variegata var. candida, Bauhinia galpinii, Schnella glabra, Piliostigma racemosa, Phanera vahlii and Lysiphyllum hookeri. Leaves and shoots were subjected to metabolite profiling via UHPLC-PDA-qTOF-MS coupled to multivariate data analyzes to identify compound compositional differences. A total of 90 metabolites were identified including polyphenols and fatty acids; flavonoid conjugates accounted for most of the metabolite variation observed. This study provides a comprehensive map of polyphenol composition in Bauhinia and phytochemical species aggregations are consistent with recent Bauhinia genus taxonomic relationship derived from phylogenetic studies. DPPH radical scavenging and α-glucosidase inhibitory assays were also performed to assess selected aspects of the antioxidant and antidiabetic potential for the examined species with respect to metabolite profiles.

  16. Development and Validation of an UPLC-Q-TOF-MS Method for Quantification of Fuziline in Beagle Dog After Intragastric and Intravenous Administration.

    PubMed

    Gong, Xiao-hong; Li, Yan; Li, Yun-xia; Yuan, An; Zhao, Meng-jie; Zhang, Ruo-qi; Zeng, Dai-wen; Peng, Cheng

    2016-03-01

    A specific and sensitive UPLC-Q-TOF-MS method operated in the positive ion mode was developed and validated for the quantification of Fuziline in Beagle dog plasma. Fuziline and Neoline internal standard were separated on an Acquity UPLC BEH C18 column with the total running time of 4 min using gradient elution at the flow rate of 0.25 mL/min. The calibration curves for Fuziline showed good linearity in the concentrations ranging from 2 to 400 ng/mL with correlation coefficients (r) greater than 0.9971. The lower limit of quantification was 0.8 ng/mL. Intra- and interbatch relative standard deviations ranged from 2.11 to 3.11% and 3.12 to 3.81%, respectively. Fuziline was stable under different sample storage and processing conditions. The developed method was successfully applied to the comparative pharmacokinetic study of Fuziline in Beagle dog after intravenous and oral administration. Low absolute bioavailability of Fuziline (1.45 ± 0.76%) suggested a significant metabolism transformation extent in Beagle dog.

  17. Adduct formation of Thimerosal with human and rat hemoglobin: a study using liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOF-MS).

    PubMed

    Janzen, Rasmus; Schwarzer, Miriam; Sperling, Michael; Vogel, Martin; Schwerdtle, Tanja; Karst, Uwe

    2011-08-01

    Thimerosal (THI) is used as a preservative in many vaccines throughout the world. Ethylmercury (EtHg(+)), released from THI in aqueous media, has a high affinity to thiol functions of proteins. In blood, hemoglobin is a likely target protein because of its high abundance and its several free thiol functions. In comparison to hemoglobin of human origin, hemoglobin of rats exhibits almost twice as many free thiol groups, which might lead to different binding behavior and therefore a limited comparability between the situation in man and in rats, which are frequently used as models for mercury species toxicity investigations. Thus, the adduct formation of EtHg(+) with hemoglobin of humans and rats was compared under simulated physiological conditions by using gradient reversed-phase liquid chromatography (LC) with electrospray time-of-flight mass spectrometry (ESI-TOF-MS) detection. The binding stoichiometry correlated with the number of free thiols in the α- and β-chain of hemoglobin. The use of rats to verify the safety of additives in vaccines like Thimerosal is therefore doubtful and should be reevaluated.

  18. 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.

  19. Permeability Study of Polyphenols Derived from a Phenolic-Enriched Hibiscus sabdariffa Extract by UHPLC-ESI-UHR-Qq-TOF-MS.

    PubMed

    Borrás-Linares, Isabel; Herranz-López, María; Barrajón-Catalán, Enrique; Arráez-Román, David; González-Álvarez, Isabel; Bermejo, Marival; Fernández Gutiérrez, Alberto; Micol, Vicente; Segura-Carretero, Antonio

    2015-08-07

    Previous findings on the capacity of Hibiscus sabdariffa (HS) polyphenols to ameliorate metabolic disturbances justify the necessity of studies oriented to find the potential metabolites responsible for such an effect. The present study examined the intestinal epithelial membrane permeability of polyphenols present in a phenolic-enriched Hibiscus sabdariffa extract (PEHS), free and encapsulated, using the Caco-2 cell line. Additionally, selected polyphenols (quercetin, quercetin-3-glucoside, quercetin-3-glucuronide, and N-feruloyltyramine) were also studied in the same absorption model. The powerful analytical platform used ultra-high-performance liquid chromatography coupled with ultra-high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-ESI-UHR-Qq-TOF-MS), and enabled the characterization of seven new compounds in PEHS. In the permeation study, only a few compounds were able to cross the cell monolayer and the permeability was lower when the extract was in an encapsulated form. Pure compounds showed a moderate absorption in all cases. Nevertheless, these preliminary results may need further research to understand the complete absorption mechanism of Hibiscus polyphenols.

  20. Determination of free and bound phenolic compounds in buckwheat spaghetti by RP-HPLC-ESI-TOF-MS: effect of thermal processing from farm to fork.

    PubMed

    Verardo, Vito; Arraez-Roman, David; Segura-Carretero, Antonio; Marconi, Emanuele; Fernandez-Gutierrez, Alberto; Caboni, Maria Fiorenza

    2011-07-27

    Nowadays there is considerable interest in the consumption of alternative crops as potential recipes for gluten-free products production. Therefore, the use of buckwheat for the production of gluten-free pasta has been investigated in the present study. RP-HPLC-ESI-TOF-MS has been applied for the separation and characterization of free and bound phenolic compounds in buckwheat flour and buckwheat spaghetti. Thus, 32 free and 24 bound phenolic compounds in buckwheat flour and spaghetti have been characterized and quantified. To the authors' knowledge, protochatechuic-4-O-glucoside acid and procyanidin A have been detected in buckwheat for the first time. The results have demonstrated a decrease of total free phenolic compounds from farm to fork (from flour to cooked spaghetti) of about 74.5%, with a range between 55.3 and 100%, for individual compounds. The decrease in bound phenols was 80.9%, with a range between 46.2 and 100%. The spaghetti-making process and the cooking caused losses of 46.1 and 49.4% of total phenolic compounds, respectively. Of the total phenolic compounds present in dried spaghetti, 11.6% were dissolved in water after cooking.

  1. In Vitro Antiproliferative Effect of Arthrocnemum indicum Extracts on Caco-2 Cancer Cells through Cell Cycle Control and Related Phenol LC-TOF-MS Identification

    PubMed Central

    Boulaaba, Mondher; Mkadmini, Khaoula; Tsolmon, Soninkhishig; Han, Junkyu; Smaoui, Abderrazak; Kawada, Kiyokazu; Ksouri, Riadh; Isoda, Hiroko; Abdelly, Chedly

    2013-01-01

    This study aimed to determinate phenolic contents and antioxidant activities of the halophyte Arthrocnemum indicum shoot extracts. Moreover, the anticancer effect of this plant on human colon cancer cells and the likely underlying mechanisms were also investigated, and the major phenols were identified by LC-ESI-TOF-MS. Results showed that shoot extracts had an antiproliferative effect of about 55% as compared to the control and were characterised by substantial total polyphenol content (19 mg GAE/g DW) and high antioxidant activity (IC50 = 40 μg/mL for DPPH test). DAPI staining revealed that these extracts decrease DNA synthesis and reduce the proliferation of Caco-2 cells which were stopped at the G2/M phase. The changes in the cell-cycle-associated proteins (cyclin B1, p38, Erk1/2, Chk1, and Chk2) correlate with the changes in cell cycle distribution. Eight phenolic compounds were also identified. In conclusion, A. indicum showed interesting antioxidant capacities associated with a significant antiproliferative effect explained by a cell cycle blocking at the G2/M phase. Taken together, these data suggest that A. indicum could be a promising candidate species as a source of anticancer molecules. PMID:24348703

  2. Multi-residue analysis method for analysis of pharmaceuticals using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS) in water sample

    NASA Astrophysics Data System (ADS)

    Al-Qaim, Fouad Fadhil; Abdullah, Md Pauzi; Othman, Mohamed Rozali

    2013-11-01

    In this work, a developed method using solid - phase extraction (SPE) followed by liquid chromatography - time of flight mass spectrometry (LC-ESI-TOF/MS) was developed and validated for quantification and confirmation of eleven pharmaceuticals with different therapeutic classes in water samples, Malaysia. These compounds are caffeine (CAF), prazosin (PRZ), enalapril (ENL), carbamazepine (CBZ), nifedipine (NFD), levonorgestrel (LNG), simvastatin (SMV), hydrochlorothiazide (HYD), gliclazide (GLIC), diclofenac-Na (DIC-Na) and mefenamic acid (MEF). LC was performed on a Dionex Ultimate 3000/LC 09115047 (USA) system. Chromatography was performed on a Thermo Scientific C18 (250 mm × 2.1 mm, i.d.: 5μm) column. Several parameters were optimised such as; mobile phase, gradient elution, collision energy and solvent elution for extraction of compounds from water. The recoveries obtained ranged from 30-148 % in river water. Five pharmaceutical compounds were detected in the surface water samples: caffeine, prazosin, enalpril, diclofenac-Na and mefenamic acid. The developed method is precise and accepted recoveries were got. In addition, this method is suitable to identify and quantify trace concentrations of pharmaceuticals in surface water.

  3. 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.

  4. 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.

  5. 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.

  6. Permeability Study of Polyphenols Derived from a Phenolic-Enriched Hibiscus sabdariffa Extract by UHPLC-ESI-UHR-Qq-TOF-MS

    PubMed Central

    Borrás-Linares, Isabel; Herranz-López, María; Barrajón-Catalán, Enrique; Arráez-Román, David; González-Álvarez, Isabel; Bermejo, Marival; Gutiérrez, Alberto Fernández; Micol, Vicente; Segura-Carretero, Antonio

    2015-01-01

    Previous findings on the capacity of Hibiscus sabdariffa (HS) polyphenols to ameliorate metabolic disturbances justify the necessity of studies oriented to find the potential metabolites responsible for such an effect. The present study examined the intestinal epithelial membrane permeability of polyphenols present in a phenolic-enriched Hibiscus sabdariffa extract (PEHS), free and encapsulated, using the Caco-2 cell line. Additionally, selected polyphenols (quercetin, quercetin-3-glucoside, quercetin-3-glucuronide, and N-feruloyltyramine) were also studied in the same absorption model. The powerful analytical platform used ultra-high-performance liquid chromatography coupled with ultra-high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-ESI-UHR-Qq-TOF-MS), and enabled the characterization of seven new compounds in PEHS. In the permeation study, only a few compounds were able to cross the cell monolayer and the permeability was lower when the extract was in an encapsulated form. Pure compounds showed a moderate absorption in all cases. Nevertheless, these preliminary results may need further research to understand the complete absorption mechanism of Hibiscus polyphenols. PMID:26262611

  7. Research on the change of chemical composition in productive process of Re Du Ning injection by HPLC/Q-TOF MS.

    PubMed

    Zhang, Shan; Li, Yan-Jing; Zhang, Chun-Xiao; Huang, Wen-Zhe; Ding, Gang; Wang, Zhen-Zhong; Bi, Yu-An; Xiao, Wei

    2016-02-01

    A high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) was developed for the analysis of chemical composition change in the production process of Re Du Ning injection, a Chinese medicine preparation with a combination of Lonicera japonica Thunb., Gardenia jasminoides Ellis and Artemisia annua L. A total of 90 compounds from raw materials-intermediates-Re Du Ning injection were detected; among them, 55 compounds were identified or tentatively characterized, and the characteristic ions of different types of compounds were described. Based on these studies, the different types of compounds in the various process routes were analyzed. A total of 28 compounds, including seven iridoid glycosides and six monoterpenes from G. jasminoides Ellis, five iridoid glycosides, nine phenolic acids and one unknown compound from L. japonica Thunb., were transferred to Re Du Ning injection, and two unknown compounds were generated in the production process of Re Du Ning injection. The results indicated that the Chinese Medicine Pharmaceutical process control is very important. This method could provide some reference for other Chinese medicine preparations.

  8. Desensitization properties of P2X3 receptors shaping pain signaling

    PubMed Central

    Giniatullin, Rashid; Nistri, Andrea

    2013-01-01

    ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called “high affinity desensitization.” We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called “left flipper”) converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain. PMID:24367291

  9. Identification of keto- and hydroxy-dicarboxylic acids in remote marine aerosols from the western North Pacific: GC and GC/TOF-MS measurements

    NASA Astrophysics Data System (ADS)

    Vani, D.; Kawamura, K.; Tachibana, E.; Boreddy, S. K. R.

    2015-12-01

    Dicarboxylic acids (diacids) are dominant components of organic aerosols in the atmosphere. They contribute significantly to the total aerosol mass and have a serious impacts on global climate changes. However, studies on keto- and hydroxy-diacids in marine aerosols are limited. Compare to diacids, keto- and hydroxy-diacids are more hygroscopic due to the additional polar groups (OH and CO) and, hence, acts as cloud condensation nuclei (CCN). Molecular characterization of these compounds provides insight into organic aerosols sources and transformation pathways. We collected marine aerosols from remote Chichijima Island in the western North Pacific from December 2010 to November 2011 and studied for water-soluble keto- and hydroxy-diacids. Carboxyl groups were derivatized to dibutyl esters with 14% boron trifluoride/n-butanol, whereas hydroxyl groups were derivatized to trimethylsilyl ethers using N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA). After two-step derivatization, samples were injected to GC, GC/MS and GC/TOF-MS. In the GC chromatogram, we detected several new peaks after BSTFA derivatization of dibutyl ester fraction. Based on mass spectral interpretation, we found these peaks as homologues series of hydroxy-diacids and keto-diacids. Some of these hydroxy-diacids have been individually reported in literature in the laboratory photo-oxidation experiments and forest environments samples. But, there are no evidences to prove their sources and formation mechanism in the atmosphere. Here, we report for the first time homologous series of hydroxy-diacids (hC3di-hC6di) and keto-diacid (oxaloacetic acid, enol and keto forms) in remote marine atmosphere. Molecular distributions of hydroxy-diacids generally showed the predominance of malic acid followed by tartronic acid. Both hydroxy- and keto-diacids show significant positive correlation with oxalic acid and SO42-, suggesting that they are generated in the atmosphere and play an important role in the

  10. Identification of four new degradation products of epirubicin through forced degradation, LC-UV, MSn and LC-MS-TOF studies.

    PubMed

    Kaushik, Dheeraj; Saini, Balraj; Bansal, Gulshan

    2015-01-01

    Epirubicin (EPI) was subjected to International Conference on Harmonization recommended forced degradation under the conditions of hydrolysis, oxidation, dry heat and photolysis to characterize its possible impurities and/or degradation products. The drug was found highly unstable to alkaline hydrolysis even at room temperature, unstable to acid hydrolysis at 80°C and to oxidation at room temperature. The hydrolytic and oxidative degradation products were resolved on an Agilent RP8 (150 mm × 4.6 mm; 5 µm) column with isocratic elution using mobile phase composed of ammonium formate (10 mM, pH 3.0), acetonitrile and methanol. The drug degraded to four oxidative products (O-I, O-II, O-III and O-IV) and to one acid hydrolyzed product (A-I). Purity of each peak in liquid chromatography-ultraviolet (LC-UV) chromatogram was ascertained through photodiode array (LC-PDA) analysis. The products were characterized through electrospray ionization-mass spectrometry (+ESI-MS(n)) studies on EPI and liquid chromatography-time of flight mass spectrometry (LC-MS-TOF) studies on degraded drug solutions. The products, O-I-O-IV, were characterized as 2-hydroxy-8-desacetylepirubicin-8-hydroperoxide, 4-hydroxy-8-desacetylepirubicin-8-hydroperoxide, 8-desacetylepirubicin-8-hydroperoxide and 8-desacetylepirubicin, respectively, and product A-I was characterized as deglucosaminylepirubicin. While A-I was found to be a pharmacopoeial impurity, all oxidative products were found to be new degradation impurities. The mechanisms and pathways of degradation of EPI were discussed and outlined.

  11. UHPLC-Q-TOF-MS-based metabolomics approach to compare the saponin compositions of Xueshuantong injection and Xuesaitong injection.

    PubMed

    Yao, Changliang; Yang, Wenzhi; Zhang, Jingxian; Qiu, Shi; Chen, Ming; Shi, Xiaojian; Pan, Huiqin; Wu, Wanying; Guo, Dean

    2017-02-01

    Various traditional Chinese medicine preparations developed from Notoginseng total saponins, including Xueshuantong injection and Xuesaitong injection, are extensively used in China to treat cardiocerebrovascular diseases. However, the difference of their saponin compositions remains unknown. An ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry based metabolomics approach was developed to probe the saponin discrimination between Xueshuantong and Xuesaitong and the related factors by large sample analysis. A highly efficient chromatographic separation was achieved on an HSS T3 column within 20 min with the holistic metabolites information recorded in the negative MS(E) mode. A six-step data pretreatment procedure mainly based on Progenesis QI and mass defect filtering was established. Pattern recognition chemometrics was used to discover the potential saponin markers. The saponin composition of Wuzhou Xueshuantong showed distinct discrimination from the other products. Wuzhou Xueshuantong contains more abundant protopanaxatriol-type noto-R1 , Rg1 , Re, and protopanaxadiol-type Rb1 , but less Rd and other low-polarity protopanaxadiol-type ginsenosides. These differences could not directly correlate to the use of different parts of Panax notoginseng, but possibly to the different preparation techniques employed by different manufacturers. These results are beneficial to the establishment of pharmacopoeia standards and the assessment of the efficacy and adverse drug reactions for these homologous products.

  12. UPLC-qTOF-MS/MS-based phenolic profile and their biosynthetic enzyme activity used to discriminate between cashew apple (Anacardium occidentale L.) maturation stages.

    PubMed

    Cunha, Aline G; Brito, Edy S; Moura, Carlos F H; Ribeiro, Paulo R V; Miranda, Maria Raquel A

    2017-04-15

    Cashew immature and ripe peduncles (Anacardium occidentale L.) from orange- and red-colored clones CCP 76 and BRS 189, respectively, were prepared as juice or fibrous fraction and submitted to UPLC-MS analyses, while the soluble fraction was also submitted to enzymatic evaluation. Cinnamoyl glucoside was present in ripe juice samples from both cashew clones, while monogalloyl diglucoside and digalloyl glucoside were present in immature juice samples from both cashew clones. Four compounds were found at immature fiber of both clones, anacardic acids (1, 2, 3) and GA19. The phenolic biosynthetic pathway was evaluated in juice samples and phenylalanine ammonia-lyase activity decreased significantly during the development, although it was much higher in ripe CCP 76. UDP-glycosyltransferases activity differed between clones, however its product cinnamoyl glucoside was a possible chemical marker of ripe juice samples from both clones. Flavonol synthase showed the highest specific activity in both cashew clones and its product, flavonols were identified in cashew apple at immature and ripe stages.

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

    PubMed

    Nomura, Fumio

    2015-06-01

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

  14. Elucidation of riverine and lacustrine dissolved organic matter (DOM) composition using comprehensive GC×GC time-of-flight mass spectrometry (GC×GC-TOF-MS)

    NASA Astrophysics Data System (ADS)

    Ball, G. I.; Goldberg, S. J.; Aluwihare, L. I.

    2012-12-01

    Rivers and streams play a key role in mediating the transfer of organic carbon (both particulate and dissolved) from terrestrial to aquatic settings. Dissolved organic carbon represents the majority of the carbon pool in low alkalinity riverine and lacustrine waters, and its composition plays important roles, including affecting water clarity and stimulating heterotrophic productivity, which influences its rate of reconversion to CO2. Yet, the chemical complexity and heterogeneity of this reservoir have limited structural elucidation to primarily describing common bulk-level characteristics. Seasonal SPE-DOM samples from the Upper Truckee River, Lake Tahoe, and two surrounding lakes, as well as SPE-DOM isolated from two dissimilar California rivers, were first characterized using δ13C, δ15N, 1H-NMR, and then subjected to CuO oxidation followed by TMS derivatization and were analyzed using comprehensive GC×GC time-of-flight mass spectrometry (GC×GC-TOF-MS). Thousands of peaks were identified per sample. Simultaneous, orthogonal separation of components in two dimensions (on the basis of volatility and polarity) allowed for the identification of oxidation mixture components by both their MS spectra and, when MS spectra alone were insufficient for structural assignment and standards were absent, by the observed trajectories of homologues compound series assumed in 2-D retention-time space. Several homologous compound series were observed, including mid-to-long chain fatty acids, keto (ω-1) fatty acids, (α, ω)-dioic acids, and the resolution and identification of closely related isomers, such as the benzene di-, and tricarboxylic acids, were also facilitated by this method. Furthermore, in mixed samples containing two or more end-members, such as in lake DOM samples characterized by mixed terrestrial and algal OM sources, the intensity of the phenolic elution space, which includes the lignin phenols and lignin phenolic dimers, correlates with ancillary

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

    PubMed

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

    2015-01-01

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

  16. An automated GCxGC-TOF-MS protocol for batch-wise extraction and alignment of mass isotopomer matrixes from differential 13C-labelling experiments: a case study for photoautotrophic-mixotrophic grown Chlamydomonas reinhardtii cells.

    PubMed

    Kempa, Stefan; Hummel, Jan; Schwemmer, Thorsten; Pietzke, Matthias; Strehmel, Nadine; Wienkoop, Stefanie; Kopka, Joachim; Weckwerth, Wolfram

    2009-02-01

    Two dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS) is a promising technique to overcome limits of complex metabolome analysis using one dimensional GC-TOF-MS. Especially at the stage of data export and data mining, however, convenient procedures to cope with the complexity of GCxGC-TOF-MS data are still in development. Here, we present a high sample throughput protocol exploiting first and second retention index for spectral library search and subsequent construction of a high dimensional data matrix useful for statistical analysis. The method was applied to the analysis of (13)C-labelling experiments in the unicellular green alga Chlamydomonas reinhardtii. We developed a rapid sampling and extraction procedure for Chlamydomonas reinhardtii laboratory strain (CC503), a cell wall deficient mutant. By testing all published quenching protocols we observed dramatic metabolite leakage rates for certain metabolites. To circumvent metabolite leakage, samples were directly quenched and analyzed without separation of the medium. The growth medium was adapted to this rapid sampling protocol to avoid interference with GCxGC-TOF-MS analysis. To analyse batches of samples a new software tool, MetMax, was implemented which extracts the isotopomer matrix from stable isotope labelling experiments together with the first and second retention index (RI1 and RI2). To exploit RI1 and RI2 for metabolite identification we used the Golm metabolome database (GMD [1] with RI1/RI2-reference spectra and new search algorithms. Using those techniques we analysed the dynamics of (13)CO(2) and (13)C-acetate uptake in Chlamydomonas reinhardtii cells in two different steady states namely photoautotroph and mixotroph growth conditions.

  17. The role of P2X3 receptors in bilateral masseter muscle allodynia in rats

    PubMed Central

    Tariba Knežević, Petra; Vukman, Robert; Antonić, Robert; Kovač, Zoran; Uhač, Ivone; Simonić-Kocijan, Sunčana

    2016-01-01

    Aim To determine the relationship between bilateral allodynia induced by masseter muscle inflammation and P2X3 receptor expression changes in trigeminal ganglia (TRG) and the influence of intramasseteric P2X3 antagonist administration on bilateral masseter allodynia. Methods To induce bilateral allodynia, rats received a unilateral injection of complete Freund’s adjuvant (CFA) into the masseter muscle. Bilateral head withdrawal threshold (HWT) was measured 4 days later. Behavioral measurements were followed by bilateral masseter muscle and TRG dissection. Masseter tissue was evaluated histopathologically and TRG tissue was analyzed for P2X3 receptor mRNA expression by using quantitative real-time polymerase chain reaction (PCR) analysis. To assess the P2X3 receptor involvement in nocifensive behavior, two doses (6 and 60 μg/50 μL) of selective P2X3 antagonist A-317491 were administrated into the inflamed masseter muscle 4 days after the CFA injection. Bilateral HWT was measured at 15-, 30-, 60-, and 120-minute time points after A-317491 administration. Results HWT was bilaterally reduced after the CFA injection (P < 0.001). Intramasseteric inflammation was confirmed ipsilaterally to the CFA injection. Quantitative real-time PCR analysis demonstrated enhanced P2X3 expression in TRG ipsilaterally to CFA administration (P < 0.01). In comparison with controls, the dose of 6 μg of A-317491 significantly increased bilateral HWT at 15-, 30-, and 60-minute time points after the A-317491 administration (P < 0.001), whereas the dose of 60 μg of A-317491 was efficient at all time points ipsilaterally (P = 0.004) and at 15-, 30-, and 60-minute time points contralaterally (P < 0.001). Conclusion Unilateral masseter inflammation can induce bilateral allodynia in rats. The study provided evidence that P2X3 receptors can functionally influence masseter muscle allodynia and suggested that P2X3 receptors expressed in TRG neurons are involved in masseter

  18. Real-time analysis of aromatics in combustion engine exhaust by resonance-enhanced multiphoton ionisation time-of-flight mass spectrometry (REMPI-TOF-MS): a robust tool for chassis dynamometer testing.

    PubMed

    Adam, T W; Clairotte, M; Streibel, T; Elsasser, M; Pommeres, A; Manfredi, U; Carriero, M; Martini, G; Sklorz, M; Krasenbrink, A; Astorga, C; Zimmermann, R

    2012-07-01

    Resonance-enhanced multiphoton ionisation time-of-flight mass spectrometry (REMPI-TOF-MS) is a robust method for real-time analysis of monocyclic and polycyclic aromatic hydrocarbons in complex emissions. A mobile system has been developed which enables direct analysis on site. In this paper, we utilize a multicomponent calibration scheme based on the analytes' photo-ionisation cross-sections relative to a calibrated species. This allows semi-quantification of a great number of components by only calibrating one compound of choice, here toluene. The cross-sections were determined by injecting nebulised solutions of aromatic compounds into the TOF-MS ion source with the help of a HPLC pump. Then, REMPI-TOF-MS was implemented at various chassis dynamometers and test cells and the exhaust of the following vehicles and engines investigated: a compression ignition light-duty (LD) passenger car, a compression ignition LD van, two spark ignition LD passenger cars, 2 two-stroke mopeds, and a two-stroke engine of a string gas trimmer. The quantitative time profiles of benzene are shown. The results indicate that two-stroke engines are a significant source for toxic and cancerogenic compounds. Air pollution and health effects caused by gardening equipment might still be underestimated.

  19. 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.

  20. Screening for illicit and medicinal drugs in whole blood using fully automated SPE and ultra-high-performance liquid chromatography with TOF-MS with data-independent acquisition.

    PubMed

    Pedersen, Anders Just; Dalsgaard, Petur Weihe; Rode, Andrej Jaroslav; Rasmussen, Brian Schou; Müller, Irene Breum; Johansen, Sys Stybe; Linnet, Kristian

    2013-07-01

    A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4-methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of δ-9-tetrahydrocannabinol, which should be handled in a separate method.

  1. Sensomics analysis of key hazelnut odorants (Corylus avellana L. 'Tonda Gentile') using comprehensive two-dimensional gas chromatography in combination with time-of-flight mass spectrometry (GC×GC-TOF-MS).

    PubMed

    Kiefl, Johannes; Pollner, Gwendola; Schieberle, Peter

    2013-06-05

    Comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) has been used a few times to identify and quantitate single aroma-active compounds, but the capability of this technique to monitor a complete set of key odorants evoking the aroma of a given food in one run has not been exploited so far. A fast, multiodorant analysis using GC×GC-TOF-MS in combination with stable isotope dilution assays (SIDA) was developed to quantitate the entire set of aroma compounds, the sensometabolome, of raw and roasted hazelnuts ( Corylus avellana L. 'Tonda Gentile') previously established by GC-olfactometry. The capability of the method to evaluate the aroma contribution of each sensometabolite was evaluated by introducing a new term, the limit of odor activity value (LOAV), indicating whether a given aroma compound can be determined down to an odor activity value (OAV) of 1 (odor activity value = ratio of concentration to odor threshold). The advantage of the new method was proven by comparing the performance parameters with a traditional one-dimensional approach using GC-ion trap mass-spectrometry (GC-IT-MS). The results showed that the detector linearity and sensitivity of GC×GC-TOF-MS was on average higher by a factor of 10 compared to GC-IT-MS, thus enabling the quantitation of the aroma relevant amounts of 22 key odorants of hazelnuts in one run of the 30 aroma-active compounds. Seven novel isotopically labeled internal standards were synthesized to meet the analytical requirements defined by electron impact ionization in TOF-MS, that is, to keep the label. On the basis of the quantitative results obtained, it was possible to closely mimic the aroma of raw and roasted 'Tonda Gentile' hazelnuts by preparing an aroma recombinate containing the key odorants at their natural concentrations occurring in the nuts.

  2. The potential of using laser ablation inductively coupled plasma time of flight mass spectrometry (LA-ICP-TOF-MS) in the forensic analysis of micro debris.

    PubMed

    Scadding, Cameron J; Watling, R John; Thomas, Allen G

    2005-08-15

    The majority of crimes result in the generation of some form of physical evidence, which is available for collection by crime scene investigators or police. However, this debris is often limited in amount as modern criminals become more aware of its potential value to forensic scientists. The requirement to obtain robust evidence from increasingly smaller sized samples has required refinement and modification of old analytical techniques and the development of new ones. This paper describes a new method for the analysis of oxy-acetylene debris, left behind at a crime scene, and the establishment of its co-provenance with single particles of equivalent debris found on the clothing of persons of interest (POI). The ability to rapidly determine and match the elemental distribution patterns of debris collected from crime scenes to those recovered from persons of interest is essential in ensuring successful prosecution. Traditionally, relatively large amounts of sample (up to several milligrams) have been required to obtain a reliable elemental fingerprint of this type of material [R.J. Walting , B.F. Lynch, D. Herring, J. Anal. At. Spectrom. 12 (1997) 195]. However, this quantity of material is unlikely to be recovered from a POI. This paper describes the development and application of laser ablation inductively coupled plasma time of flight mass spectrometry (LA-ICP-TOF-MS), as an analytical protocol, which can be applied more appropriately to the analysis of micro-debris than conventional quadrupole based mass spectrometry. The resulting data, for debris as small as 70mum in diameter, was unambiguously matched between a single spherule recovered from a POI and a spherule recovered from the scene of crime, in an analytical procedure taking less than 5min.

  3. 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

  4. Comprehensive non-targeted analysis of contaminated groundwater of a former ammunition destruction site using 1H-NMR and HPLC-SPE-NMR/TOF-MS.

    PubMed

    Godejohann, Markus; Heintz, Lea; Daolio, Cristina; Berset, Jean-Daniel; Muff, Daniel

    2009-09-15

    The aim of the present study was to explore the capabilities of the combination of 1H NMR (proton nuclear magnetic resonance) mixture analysis and HPLC-SPE-NMR/TOF-MS (high-performance liquid chromatography coupled to solid-phase extraction and nuclear magnetic resonance and time-of-flight mass spectrometry) for the characterization of xenobiotic contaminants in groundwater samples. As an example, solid-phase extracts of two groundwater samples taken from a former ammunition destruction site in Switzerland were investigated. 1H NMR spectra of postcolumn SPE enriched compounds, together with accurate mass measurements, allowed the structural elucidation of unknowns. This untargeted approach allowed us to identify expected residues of explosives such as 2,4,6-trinitrotoluene (2,4,6-TNT), Hexogen (RDX) and Octogen (HMX), degradation products of TNT (1,3,5-trinitrobenzene (1,3,5-TNB), 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT), 3,5-dinitrophenol (3,5-DNP), 3,5-dinitroaniline (3,5-DNA), 2,6-dinitroanthranite, and 2-Hydroxy-4,6-dinitrobenzonitrile), benzoic acid, Bisphenol A (a known endocrine disruptor compound), and some toxicologically relevant additives for propelling charges: Centralite I (1,3-diethyl-1,3-diphenylurea), DPU (N,N-diphenylurethane), N,N-diphenylcarbamate (Acardite II), and N-methyl-N-phenylurethane. To our knowledge, this is the first report of the presence of these additives in environmental samples. Extraction recoveries for Centralite I and DPU have been determined. Contaminants identified by our techniques were quantified based on HPLC-UV (HPLC-ultraviolet detection) and 1H NMR mixture analysis. The concentrations of the contaminants ranged between 0.1 and 48 microg/L assuming 100% recovery for the SPE step.

  5. Comparison of different extraction methods for the determination of statin drugs in wastewater and river water by HPLC/Q-TOF-MS.

    PubMed

    Martín, Julia; Buchberger, Wolfgang; Alonso, Esteban; Himmelsbach, Markus; Aparicio, Irene

    2011-07-15

    Three preconcentration techniques including solid phase extraction (SPE), dispersive liquid-liquid microextraction (DLLME) and stir-bar sorptive extraction (SBSE) have been optimized and compared for the analysis of six hypolipidaemic statin drugs (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin) in wastewater and river water samples by high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (HPLC/Q-TOF-MS). Parameters that affect the efficiency of the different extraction methods such as solid phase material, sample pH and elution solvent in the case of SPE; the type and volume of the extracting and dispersive solvent, pH of sample, salt addition and number of extraction steps in the case of DLLME; and the stirring time, pH of sample, sample volume and salt addition for SBSE were evaluated. SPE allowed the best recoveries for most of the analytes. Pravastatin was poorly extracted by DLLME and could not be determined. SBSE was only applicable for lovastatin and simvastatin. However, despite the limitations of having poorer recovery than SPE, DLLME and SBSE offered some advantages because they are simple, require low organic solvent volumes and present low matrix effects. DLLME required less time of analysis, and for SBSE the stir-bar was re-usable. SPE, DLLME and SBSE provided method detection limits in the range of 0.04-11.2 ng L(-1), 0.10-17.0 ng L(-1) for 0.52-2.00 ng L(-1), respectively, in real samples. To investigate and compare their applicability, SPE, DLLME and SBSE procedures were applied to the detection of statin drugs in effluent wastewater and river samples.

  6. Development of a two-step screening ESI-TOF-MS method for rapid determination of significant stress-induced metabolome modifications in plant leaf extracts: the wound response in Arabidopsis thaliana as a case study.

    PubMed

    Grata, Elia; Boccard, Julien; Glauser, Gaetan; Carrupt, Pierre-Alain; Farmer, Edward E; Wolfender, Jean-Luc; Rudaz, Serge

    2007-09-01

    To study the stress-induced effects caused by wounding under a new perspective, a metabolomic strategy based on HPLC-MS has been devised for the model plant Arabidopsis thaliana. To detect induced metabolites and precisely localise these compounds among the numerous constitutive metabolites, HPLC-MS analyses were performed in a two-step strategy. In a first step, rapid direct TOF-MS measurements of the crude leaf extract were performed with a ballistic gradient on a short LC-column. The HPLC-MS data were investigated by multivariate analysis as total mass spectra (TMS). Principal components analysis (PCA) and hierarchical cluster analysis (HCA) on principal coordinates were combined for data treatment. PCA and HCA demonstrated a clear clustering of plant specimens selecting the highest discriminating ions given by the complete data analysis, leading to the specific detection of discrete-induced ions (m/z values). Furthermore, pool constitution with plants of homogeneous behaviour was achieved for confirmatory analysis. In this second step, long high-resolution LC profilings on an UPLC-TOF-MS system were used on pooled samples. This allowed to precisely localise the putative biological marker induced by wounding and by specific extraction of accurate m/z values detected in the screening procedure with the TMS spectra.

  7. Identification of metabolites of oridonin in rats with a single run on UPLC-Triple-TOF-MS/MS system based on multiple mass defect filter data acquisition and multiple data processing techniques.

    PubMed

    Tian, Tingting; Jin, Yiran; Ma, Yinghua; Xie, Weiwei; Xu, Huijun; Zhang, Kerong; Zhang, Lantong; Du, Yingfeng

    2015-12-01

    Oridonin (ORI) is an active natural ent-kaurane diterpenoid ingredient originating from well-known traditional Chinese herb medicine and is expected to be pursued as a new anticancer agent. In the present study, a novel and efficient approach was developed for in vivo screening and identification of ORI metabolites using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). This analytical strategy was as follows: an effective on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS), was developed to trace all of potential metabolites of ORI. The MMDF and DBS method could trigger an information dependent acquisition scan, which could give the information of low-level metabolites masked by background noise and endogenous components in complex matrix. Moreover, the sensitive and specific multiple data-mining techniques including extracted ion chromatography, mass defect filtering, product ion filtering and neutral loss filtering were employed to identify the metabolites of ORI. Then, structures for the metabolites were successfully assigned based on accurate masses, the mass fragmentation of ORI and metabolic knowledge. Finally, an important parameter Clog P was used to estimate the retention time of isomers. Based on the proposed strategy, 16 phase I and 2 phase II metabolites were detected in rats after oral administration of ORI. The main biotransformation route of ORI was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. This is the first study of ORI metabolism in vivo. This study not only proposed a practical strategy for rapidly screening and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of ORI in vivo. At the same time this methodology can be widely applied for the structural characterization of the metabolites

  8. Screening and identification of the main metabolites of 2-amino-9H-pyrido[2,3-b]indole (AαC) in liver microsomes and rat urine by using UPLC-Q-TOF-MS/MS.

    PubMed

    Hu, Kai; Zhao, Ge; Fu, Yufeng; Wang, Sheng; Yuan, Hang; Xie, Fuwei; Zhang, Shusheng; Liu, Huimin; Liu, Minying

    2017-03-01

    2-Amino-9H-pyrido[2,3-b]indole (AαC), which has been reported to be 40-258ng per cigarette, was regarded as a probable human carcinogen (Group 2B) and harmful composition in Hoffman list. Thus, it is of great significance to develop an effective method for the accurate identification of AαC and its metabolites. In the present study, we have investigated for the first time the in vivo and in vitro metabolites of AαC using ultra performance liquid chromatography combined with diode array detector and time-of-flight mass spectrometry (UPLC-DAD and UPLC-Q-TOF-MS/MS). A comparative study showed that the metabolic patterns of AαC in beagle, mouse, rat and human liver microsomes were of significant difference with these in rat urine. For the metabolism of AαC in liver microsomes, nine metabolites of AαC, including five hydroxy metabolites, two quinone metabolites and two N-dimer metabolites, have been found. However, metabolism of AαC in rats is a phase II process with complex enzyme catalysis, 23 metabolites including C- and N-oxidation, O- and N-glycosylation, O- and N-sulfonation, and N-acetylation were identified in rat urine. In addition, five new N-acetyl-AαC-OH metabolites were identified for the first time, indicating a possible new pathway for the metabolism. This study significantly enriched our knowledge about the metabolism of AαC, and will be useful for a better understanding of its harmfulness and toxicity.

  9. Analysis of the chemical composition of the essential oil of Polygonum minus Huds. using two-dimensional gas chromatography-time-of-flight mass spectrometry (GC-TOF MS).

    PubMed

    Baharum, Syarul Nataqain; Bunawan, Hamidun; Ghani, Ma'aruf Abd; Mustapha, Wan Aida Wan; Noor, Normah Mohd

    2010-10-12

    The essential oil in leaves of Polygonum minus Huds., a local aromatic plant, were identified by a pipeline of gas chromatography (GC) techniques coupled with mass-spectrometry (MS), flame ionization detector (FID) and two dimensional gas chromatography time of flight mass spectrometry (GC x GC-TOF MS). A total of 48 compounds with a good match and high probability values were identified using this technique. Meanwhile, 42 compounds were successfully identified in this study using GC-MS, a significantly larger number than in previous studies. GC-FID was used in determining the retention indices of chemical components in P. minus essential oil. The result also showed the efficiency and reliability were greatly improved when chemometric methods and retention indices were used in identification and quantification of chemical components in plant essential oil.

  10. Characterization of the organic matter in submicron urban aerosols using a Thermo-Desorption Proton-Transfer-Reaction Time-of-Flight Mass Spectrometer (TD-PTR-TOF-MS)

    NASA Astrophysics Data System (ADS)

    Salvador, Christian Mark; Ho, T.-T.; Chou, Charles C.-K.; Chen, M.-J.; Huang, W.-R.; Huang, S.-H.

    2016-09-01

    Organic matter is the most complicated and unresolved major component of atmospheric aerosol particles. Its sources and global budget are still highly uncertain and thereby necessitate further research efforts with state-of-the-art instrument. This study employed a Thermo-Desorption Proton-Transfer-Reaction Time-of-Flight Mass Spectrometer (TD-PTR-TOF-MS) for characterization of ambient organic aerosols. First, five authentic standard substances, which include phthalic acid, levoglucosan, arabitol, cis-pinonic acid and glutaric acid, were utilized to examine the response of the instrument. The results demonstrated the linearity of the TD-PTR-TOF-MS signals against a range of mass loading of specific species on filters. However, it was found that significant fragmentation happened to those challenging compounds, although the proton-transfer-reaction (PTR) was recognized as a soft ionization technique. Consequently, quantitative characterization of aerosols with the TD-PTR-TOF-MS depended on the availability of the fragmentation pattern in mass spectra and the recovery rate with the quantification ion peak(s). The instrument was further deployed to analyze a subset of submicron aerosol samples collected at the TARO (Taipei Aerosol and Radiation Observatory) in Taipei, Taiwan during August 2013. The results were compared with the measurements from a conventional DRI thermo-optical carbon analyzer. The inter-comparison indicated that the TD-PTR-TOF-MS underestimated the mass of total organic matter (TOM) in aerosol samples by 27%. The underestimation was most likely due to the thermo-decomposition during desorption processes and fragmentation in PTR drift tube, where undetectable fragments were formed. Besides, condensation loss of low vapor pressure species in the transfer components was also responsible for the underestimation to a certain degree. Nevertheless, it was showed that the sum of the mass concentrations of the major detected ion peaks correlated strongly

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

    PubMed

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Gnat, Sebastian; Trościańczyk, Aleksandra; Adaszek, Łukasz

    2017-01-01

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

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

    PubMed Central

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Gnat, Sebastian; Trościańczyk, Aleksandra; Adaszek, Łukasz

    2017-01-01

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

  13. The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment.

    PubMed

    Rodrigues, Naiara Miranda Bento; Bronzato, Greiciane França; Santiago, Gabrielli Stefaninni; Botelho, Larissa Alvarenga Batista; Moreira, Beatriz Meurer; Coelho, Irene da Silva; Souza, Miliane Moreira Soares de; Coelho, Shana de Mattos de Oliveira

    Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n=47) and fecal samples (n=94) from cows, and samples from water (n=23) and milk lines (n=19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

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

    PubMed Central

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

    2017-01-01

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

  15. Generators for the elliptic curve y2 = x3-nx II

    NASA Astrophysics Data System (ADS)

    Fujita, Yasutsugu; Terai, Nobuhiro

    2011-09-01

    Let E be an elliptic curve defined by y2 = x3-nx with n a positive integer. In our previous work, we gave several infinite families of n for which certain two points of infinite order can be in a system of generators for the Mordell-Weil group E(Q). In this paper, we extend this result and give those examples of infinite families of n for which the generators for rank three part of E(Q) can be explicitly described.

  16. The 2nd order focusing sector field type TOF mass analyzer with an orthogonal ion acceleration for LC-IMS-MS.

    PubMed

    Poteshin, S S; Zarakovsky, A I

    2017-03-15

    Original orthogonal acceleration (OA) electrostatic sector time of flight (TOF) mass analyzer is proposed those allows the second order focusing of time of flight by initial ions position. Resolving power aberration limit exceeding 80,000 FW (full width mass peak) was shown to be obtainable for mass analyzer with the total length of flight L=133.2cm, the average ion energy 3700V and the ion energy spread of 2.5% on the entrance of sector field.

  17. Gas Chromatography Time-Of-Flight Mass Spectrometry (GC-TOF-MS)-Based Metabolomics for Comparison of Caffeinated and Decaffeinated Coffee and Its Implications for Alzheimer’s Disease

    PubMed Central

    Chang, Kai Lun; Ho, Paul C.

    2014-01-01

    Findings from epidemiology, preclinical and clinical studies indicate that consumption of coffee could have beneficial effects against dementia and Alzheimer’s disease (AD). The benefits appear to come from caffeinated coffee, but not decaffeinated coffee or pure caffeine itself. Therefore, the objective of this study was to use metabolomics approach to delineate the discriminant metabolites between caffeinated and decaffeinated coffee, which could have contributed to the observed therapeutic benefits. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS)-based metabolomics approach was employed to characterize the metabolic differences between caffeinated and decaffeinated coffee. Orthogonal partial least squares discriminant analysis (OPLS-DA) showed distinct separation between the two types of coffee (cumulative Q2 = 0.998). A total of 69 discriminant metabolites were identified based on the OPLS-DA model, with 37 and 32 metabolites detected to be higher in caffeinated and decaffeinated coffee, respectively. These metabolites include several benzoate and cinnamate-derived phenolic compounds, organic acids, sugar, fatty acids, and amino acids. Our study successfully established GC-TOF-MS based metabolomics approach as a highly robust tool in discriminant analysis between caffeinated and decaffeinated coffee samples. Discriminant metabolites identified in this study are biologically relevant and provide valuable insights into therapeutic research of coffee against AD. Our data also hint at possible involvement of gut microbial metabolism to enhance therapeutic potential of coffee components, which represents an interesting area for future research. PMID:25098597

  18. Identification of mycobacterium spp. and nocardia spp. from solid and liquid cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

    PubMed

    Girard, Victoria; Mailler, Sandrine; Welker, Martin; Arsac, Maud; Cellière, Béatrice; Cotte-Pattat, Pierre-Jean; Chatellier, Sonia; Durand, Géraldine; Béni, Anne-Marie; Schrenzel, Jacques; Miller, Elizabeth; Dussoulier, Rahima; Dunne, W Michael; Butler-Wu, Susan; Saubolle, Michael A; Sussland, Den; Bell, Melissa; van Belkum, Alex; Deol, Parampal

    2016-11-01

    Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

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

    PubMed

    Badía, J D; Strömberg, E; Ribes-Greus, A; Karlsson, S

    2011-04-29

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

  20. 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.

  1. Study of baicalin on sympathoexcitation induced by myocardial ischemia via P2X3 receptor in superior cervical ganglia.

    PubMed

    Zhang, Jun; Liu, Shuangmei; Xu, Baohua; Li, Guodong; Li, Guilin; Huang, An; Wu, Bing; Peng, Lichao; Song, Miaomiao; Xie, Qiuyu; Lin, Weijian; Xie, Wei; Wen, Shiyao; Zhang, Zhedong; Xu, Xiaoling; Liang, Shangdong

    2015-05-01

    After the myocardial ischemia, injured myocardial tissues released large quantity of ATP, which activated P2X3 receptor in superior cervical ganglia and made the SCG postganglionic neurons excited. Excitatory of sympathetic postganglionic efferent neurons increased the blood pressure and heart rates, which aggravated the myocardial ischemic injury. Baicalin has anti-inflammatory and anti-oxidant properties. Our study showed that baicalin reduced the incremental concentration of serum CK-MB, cTn-T, epinephrine and ATP, decreased the up-regulated expression levels of P2X3 mRNA and protein in SCG after MI, and then inhibited the sympathetic excitatory activity triggered by MI injury. These results indicated that baicalin acted on P2X3 receptor was involved in the transmission of sympathetic excitation after the myocardial ischemic injury. Baicalin might decrease sympathetic activity via inhibiting P2X3 receptor in rat SCG to protect the myocardium.

  2. Matching unknown empirical formulas to chemical structure using LC/MS TOF accurate mass and database searching: example of unknown pesticides on tomato skins.

    PubMed

    Thurman, E Michael; Ferrer, Imma; Fernández-Alba, Amadeo Rodriguez

    2005-03-04

    Traditionally, the screening of unknown pesticides in food has been accomplished by GC/MS methods using conventional library searching routines. However, many of the new polar and thermally labile pesticides and their degradates are more readily and easily analyzed by LC/MS methods and no searchable libraries currently exist (with the exception of some user libraries, which are limited). Therefore, there is a need for LC/MS approaches to detect unknown non-target pesticides in food. This report develops an identification scheme using a combination of LC/MS time-of-flight (accurate mass) and LC/MS ion trap MS (MS/MS) with searching of empirical formulas generated through accurate mass and a ChemIndex database or Merck Index database. The approach is different than conventional library searching of fragment ions. The concept here consists of four parts. First is the initial detection of a possible unknown pesticide in actual market-place vegetable extracts (tomato skins) using accurate mass and generating empirical formulas. Second is searching either the Merck Index database on CD (10,000 compounds) or the ChemIndex (77,000 compounds) for possible structures. Third is MS/MS of the unknown pesticide in the tomato-skin extract followed by fragment ion identification using chemical drawing software and comparison with accurate-mass ion fragments. Fourth is the verification with authentic standards, if available. Three examples of unknown, non-target pesticides are shown using a tomato-skin extract from an actual market place sample. Limitations of the approach are discussed including the use of A + 2 isotope signatures, extended databases, lack of authentic standards, and natural product unknowns in food extracts.

  3. Capture and identification of the volatile components in crude and processed herbal medicines through on-line purge and trap technique coupled with GC × GC-TOF MS.

    PubMed

    Cao, Gang; Xu, Zhiwei; Wu, Xin; Li, Qingli; Chen, Xiaocheng

    2014-01-01

    This work aimed to investigate the volatile components in crude and processed herbal medicines (HMs). Using Atractylodis Macrocephalae Rhizoma (AMR) as a model HM, the volatile components were captured through on-line purge and trap technique and identified by using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF MS) system. A total of 224 and 171 volatile compounds were identified in crude and processed AMR samples, respectively. After frying with honey-bran, 52 compounds which were found in crude AMR samples disappeared in processed AMR samples, and 15 compounds were newly generated in processed AMR. The established method can be applied in different research areas such as HM and food processing.

  4. NMR spectroscopy and MALDI-TOF MS characterisation of end-functionalised PVP oligomers prepared with different esters as chain transfer agents.

    PubMed

    Ranucci, Elisabetta; Ferruti, Paolo; Annunziata, Rita; Gerges, Irini; Spinelli, Giuseppe

    2006-03-14

    Ester-functionalised poly(1-vinylpyrrolidin-2-one) (PVP) oligomers obtained by radical polymerisation in methyl propionate, diethyl malonate and diethyl 2-methylmalonate were characterised by NMR spectroscopy, and MALDI-TOF mass spectrometry. The chain-transfer constants were determined as 5.54 x 10(-4), 1.22 x 10(-3) and 1.70 x 10(-2), respectively, by measuring the variation of the number-average molecular weight on conversion. These values were compared with those of methyl isobutyrate (1.65 x 10(-3)) and ethyl lactate (1.03 x 10(-2)), which had been previously determined. A clear dependence was found on the reactivity of the mobile hydrogen atoms alpha with the ester group. All of the macromolecules carried a single ester function. Therefore, the re-initiation step by the CTA-derived radicals overwhelmingly prevailed over initiation by the primary radicals.

  5. Lipidomics study of plasma phospholipid metabolism in early type 2 diabetes rats with ancient prescription Huang-Qi-San intervention by UPLC/Q-TOF-MS and correlation coefficient.

    PubMed

    Wu, Xia; Zhu, Jian-Cheng; Zhang, Yu; Li, Wei-Min; Rong, Xiang-Lu; Feng, Yi-Fan

    2016-08-25

    Potential impact of lipid research has been increasingly realized both in disease treatment and prevention. An effective metabolomics approach based on ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) along with multivariate statistic analysis has been applied for investigating the dynamic change of plasma phospholipids compositions in early type 2 diabetic rats after the treatment of an ancient prescription of Chinese Medicine Huang-Qi-San. The exported UPLC/Q-TOF-MS data of plasma samples were subjected to SIMCA-P and processed by bioMark, mixOmics, Rcomdr packages with R software. A clear score plots of plasma sample groups, including normal control group (NC), model group (MC), positive medicine control group (Flu) and Huang-Qi-San group (HQS), were achieved by principal-components analysis (PCA), partial least-squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). Biomarkers were screened out using student T test, principal component regression (PCR), partial least-squares regression (PLS) and important variable method (variable influence on projection, VIP). Structures of metabolites were identified and metabolic pathways were deduced by correlation coefficient. The relationship between compounds was explained by the correlation coefficient diagram, and the metabolic differences between similar compounds were illustrated. Based on KEGG database, the biological significances of identified biomarkers were described. The correlation coefficient was firstly applied to identify the structure and deduce the metabolic pathways of phospholipids metabolites, and the study provided a new methodological cue for further understanding the molecular mechanisms of metabolites in the process of regulating Huang-Qi-San for treating early type 2 diabetes.

  6. Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS.

    PubMed

    Chu, Fong Lam; Sleno, Lekha; Yaylayan, Varoujan A

    2011-11-09

    Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.

  7. SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence

    PubMed Central

    Mazzatti, Dawn J; Pawelec, Graham; Longdin, Robin; Powell, Jonathan R; Forsey, Rosalyn J

    2007-01-01

    Background The adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies. The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown in vitro until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays. Results Discriminant analysis identified several protein or peptide peaks in the region of 14.5–16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis. Conclusion Due to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence. PMID:17550585

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

    PubMed Central

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

    2016-01-01

    Background The technical, biological, and inter-center reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) typing data has not yet been explored. The aim of this study is to compare typing data from multiple centers employing bioinformatics using bacterial strains from two past outbreaks and non-related strains. Material/Methods Participants received twelve extended spectrum betalactamase-producing E. coli isolates and followed the same standard operating procedure (SOP) including a full-protein extraction protocol. All laboratories provided visually read spectra via flexAnalysis (Bruker, Germany). Raw data from each laboratory allowed calculating the technical and biological reproducibility between centers using BioNumerics (Applied Maths NV, Belgium). Results Technical and biological reproducibility ranged between 96.8–99.4% and 47.6–94.4%, respectively. The inter-center reproducibility showed a comparable clustering among identical isolates. Principal component analysis indicated a higher tendency to cluster within the same center. Therefore, we used a discriminant analysis, which completely separated the clusters. Next, we defined a reference center and performed a statistical analysis to identify specific peaks to identify the outbreak clusters. Finally, we used a classifier algorithm and a linear support vector machine on the determined peaks as classifier. A validation showed that within the set of the reference center, the identification of the cluster was 100% correct with a large contrast between the score with the correct cluster and the next best scoring cluster. Conclusions Based on the sufficient technical and biological reproducibility of MALDI-TOF MS based spectra, detection of specific clusters is possible from spectra obtained from different centers. However, we believe that a shared SOP and a bioinformatics approach are required to make the analysis robust and reliable. PMID:27798637

  9. Proton sponge-functionalized silica as high performance adsorbents for solid-phase extraction of trace perfluoroalkyl sulfonates in the environmental water samples and their direct analysis by MALDI-TOF-MS.

    PubMed

    Cao, Dong; Hu, Ming; Han, Chunguang; Yu, Jiyao; Cui, Lin; Liu, Yongxue; Wang, Hailin; Cai, Yaqi; Kang, Yuehui; Zhou, Yiqi

    2012-05-07

    1,8-Bis(dimethylamino)naphthalene (DMAN), a classical 'proton sponge', was functionalized on silica particles as a novel solid-phase extraction (SPE) adsorbent (DMAN@silica) for extracting perfluoroalkyl sulfonates (PFSs). High reproducibility and excellent extraction capability for PFSs were obtained in a wide pH range (3.0~8.5). The adsorbed PFSs on DMAN@silica sorbents could be efficiently eluted by 1,8-bis(tetramethylguanidino)naphthalene (TMGN) solution which is a proton sponge with higher proton affinity than DMAN. The elution could be directly analyzed by MALDI-TOF-MS using TMGN as matrix. Clear mass spectra for the PFSs were obtained due to no matrix ions interference observed. Furthermore, a novel strategy based on the DMAN@silica-SPE enrichment, followed by MALDI-TOF-MS analysis, was proposed and applied for PFSs quantification in environmental water samples. The calibration curves of each of the target analytes showed a wide linear dynamic range of response (0.1-10 ng L(-1) for perfluorooctane sulfonate (PFOS), perfluorohexyl sulfonate (PFHxS) and perfluorobutylsulfonate (PFBS)), which were over 2 orders of magnitude. The detection limits for PFOS, PFHxS, and PFBS were 0.021, 0.016, and 0.013 ng L(-1), respectively (S/N = 3). Recoveries of PFOS, PFHxS, and PFBS are in the ranges of 92-104%, 95-102%, and 98-109% for spiked river water samples. These results indicated that the prepared DMAN@silica adsorbents could efficiently enrich PFSs and that the proposed method is reliable.

  10. Comprehensive characterization of the in vitro and in vivo metabolites of ziyuglycoside I in rat microsome, intestinal flora, excretion specimen and fresh tissues based on LC-Q-TOF/MS.

    PubMed

    Wang, Guangji; Fu, Hanxu; Ye, Wei; Zheng, Xiao; Xiao, Jingcheng; Kang, Dian; Rao, Tai; Shao, Yuhao; Xie, Lin; Liang, Yan

    2016-09-05

    Ziyuglycoside I is one of the major active ingredients in Sanguisorba officinalis, a popular medicinal plant in China. In the present study, the metabolites of ziyuglycoside I in rat liver microsome and intestinal flora were identified and structurally characterized, and the metabolic rules were summed based on the LC-Q-TOF/MS system. Then, the metabolites in rat excreta samples were rapidly screened and identified according to the in vitro metabolic rules. Finally, ziyuglycoside I was incubated with fresh liver/lung/kidney/stomach homogenates to further explore the source of the metabolites and reveal the possible metabolic organs involved. Four metabolites in liver microsome were identified as M0-Glu, M0-CH2OH, M0-Glu+CH3, M0-Glu-Ara+CH3. In intestinal flora incubation system, 6 degradation products including M0-Glu-Ara+O, M0-Ara, M0-Glu-COOH, M0-Glu, M0-Glu-Ara+O and M0-Ara+H2O were tentatively identified by interpretation of their accurate MS(1) and MS(2) data. Fifteen metabolites in rat urine and feces were identified, and most of the metabolites were attributed to the transformation in liver microsome and intestinal flora. Specifically, more than a dozen of new metabolites were identified in rat fresh tissues, and ziyuglycoside II was confirmed as the major metabolite in rats.

  11. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    PubMed Central

    Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-01-01

    Summary P2X receptors are trimeric, non-selective cation channels activated by ATP that play important roles in cardiovascular, neuronal and immune systems. Despite their central function in human physiology and as potential targets of therapeutic agents, there are no structures of human P2X receptors. Mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structure of the pore-forming transmembrane domains remain unclear. We report x-ray crystal structures of human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/desensitized and antagonist-bound closed states. The open state structure harbors an intracellular motif we term the “cytoplasmic cap”, that stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. Competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements underpinning P2X receptor gating and provide a foundation for development of new pharmacologic agents. PMID:27626375

  12. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    NASA Astrophysics Data System (ADS)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-10-01

    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the ‘cytoplasmic cap’, which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

  13. Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice.

    PubMed

    Vandenbeuch, Aurelie; Larson, Eric D; Anderson, Catherine B; Smith, Steven A; Ford, Anthony P; Finger, Thomas E; Kinnamon, Sue C

    2015-03-01

    Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca(2+) transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities.

  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. Stable, synthetic analogs of diadenosine tetraphosphate inhibit rat and human P2X3 receptors and inflammatory pain

    PubMed Central

    Viatchenko-Karpinski, Viacheslav; Novosolova, Natalia; Ishchenko, Yevheniia; Azhar, M Ameruddin; Wright, Michael; Tsintsadze, Vera; Kamal, Ahmed; Burnashev, Nail; Voitenko, Nana; Giniatullin, Rashid; Lozovaya, Natalia

    2016-01-01

    Background A growing body of evidence suggests that ATP-gated P2X3 receptors (P2X3Rs) are implicated in chronic pain. We address the possibility that stable, synthetic analogs of diadenosine tetraphosphate (Ap4A) might induce antinociceptive effects by inhibiting P2X3Rs in peripheral sensory neurons. Results The effects of two stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) are studied firstly in vitro on HEK293 cells expressing recombinant rat P2XRs (P2X2Rs, P2X3Rs, P2X4Rs, and P2X7Rs) and then using native rat brain cells (cultured trigeminal, nodose, or dorsal root ganglion neurons). Thereafter, the action of these stable, synthetic Ap4A analogs on inflammatory pain and thermal hyperalgesia is studied through the measurement of antinociceptive effects in formalin and Hargreaves plantar tests in rats in vivo. In vitro inhibition of rat P2X3Rs (not P2X2Rs, P2X4Rs nor P2X7Rs) is shown to take place mediated by high-affinity desensitization (at low concentrations; IC50 values 100–250 nM) giving way to only weak partial agonism at much higher concentrations (EC50 values ≥ 10 µM). Similar inhibitory activity is observed with human recombinant P2X3Rs. The inhibitory effects of AppNHppA on nodose, dorsal root, and trigeminal neuron whole cell currents suggest that stable, synthetic Ap4A analogs inhibit homomeric P2X3Rs in preference to heteromeric P2X2/3Rs. Both Ap4A analogs mediate clear inhibition of pain responses in both in vivo inflammation models. Conclusions Stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) being weak partial agonist provoke potent high-affinity desensitization-mediated inhibition of homomeric P2X3Rs at low concentrations. Therefore, both analogs demonstrate clear potential as potent analgesic agents for use in the management of chronic pain associated with heightened P2X3R activation. PMID:27030723

  16. The effect of sinomenine in diabetic neuropathic pain mediated by the P2X3 receptor in dorsal root ganglia.

    PubMed

    Rao, Shenqiang; Liu, Shuangmei; Zou, Lifang; Jia, Tianyu; Zhao, Shanhong; Wu, Bing; Yi, Zhihua; Wang, Shouyu; Xue, Yun; Gao, Yun; Xu, Changshui; Li, Guilin; Xu, Hong; Zhang, Chunping; Liang, Shangdong

    2017-01-04

    Type 2 diabetes mellitus (T2DM) accounts for more than 90% of all cases of diabetes mellitus (DM). Diabetic neuropathic pain (DNP) is a common complication of T2DM. Sinomenine is a natural bioactive component extracted from the Sinomenium acutum and has anti-inflammatory effects. The aim of our study was to investigate the effects of sinomenine on DNP mediated by the P2X3 receptor in dorsal root ganglia (DRG). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in T2DM rats were lower than those of control rats. MWT and TWL in T2DM rats treated with sinomenine were higher compared with those in T2DM rats. The expression levels of the P2X3 protein and mRNA in T2DM rat DRG were higher compared with those of the control, while those in T2DM rats treated with sinomenine were significantly lower compared with those of the T2DM rats. Sinomenine significantly inhibited P2X3 agonist ATP-activated currents in HEK293 cells transfected with the P2X3 receptor. Sinomenine decreased the phosphorylation and activation of P38MAPK in T2DM DRG. Therefore, sinomenine treatment may suppress the up-regulated expression and activation of the P2X3 receptor and relieve the hyperalgesia potentiated by the activation of P38MAPK in T2DM rats.

  17. UPLC-Q-TOF/MS-based metabolomic studies on the toxicity mechanisms of traditional Chinese medicine Chuanwu and the detoxification mechanisms of Gancao, Baishao, and Ganjiang.

    PubMed

    Dong, Hui; Yan, Guang-Li; Han, Ying; Sun, Hui; Zhang, Ai-Hua; Li, Xian-Na; Wang, Xi-Jun

    2015-09-01

    Chuanwu (CW), a famous traditional Chinese medicine (TCM) from the mother roots of Aconitum carmichaelii Debx.. (Ranunculaceae), has been used for the treatment of various diseases. Unfortunately, its toxicity is frequently reported because of its narrow therapeutic window. In the present study, a metabolomic method was performed to characterize the phenotypically biochemical perturbations and potential mechanisms of CW-induced toxicity. Meanwhile, the expression level of toxicity biomarkers in the urine were analyzed to evaluate the detoxification by combination with Gancao (Radix Glyeyrrhizae, CG), Baishao (Radix Paeoniae Alba, CS) and Ganjiang (Rhizoma Zingiberis, CJ), which were screened from classical TCM prescriptions. Urinary metabolomics was performed by UPLC-Q-TOF-HDMS, and the mass spectra signals of the detected metabolites were systematically analyzed using pattern recognition methods. As a result, seventeen biomarkers associated with CW toxicity were identified, which were associated with pentose and glucuronate interconversions, alanine, aspartate, and glutamate metabolism, among others. The expression levels of most toxicity biomarkers were effectively modulated towards the normal range by the compatibility drugs. It indicated that the three compatibility drugs could effectively detoxify CW. In summary, our work demonstrated that metabolomics was vitally significant to evaluation of toxicity and finding detoxification methods for TCM.

  18. 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.

  19. A pre-classification strategy based on UPLC-Triple-TOF/MS for metabolic screening and identification of Radix glehniae in rats.

    PubMed

    Wang, Shuang; Qi, Pengcheng; Zhou, Na; Zhao, Minmin; Ding, Weijing; Li, Song; Liu, Minyan; Wang, Qiao; Jin, Shumin

    2016-10-01

    Traditional Chinese Medicines (TCMs) have gained increasing popularity in modern society. However, the profiles of TCMs in vivo are still unclear owing to their complexity and low level in vivo. In this study, UPLC-Triple-TOF techniques were employed for data acquiring, and a novel pre-classification strategy was developed to rapidly and systematically screen and identify the absorbed constituents and metabolites of TCMs in vivo using Radix glehniae as the research object. In this strategy, pre-classification for absorbed constituents was first performed according to the similarity of their structures. Then representative constituents were elected from every class and analyzed separately to screen non-target absorbed constituents and metabolites in biosamples. This pre-classification strategy is basing on target (known) constituents to screen non-target (unknown) constituents from the massive data acquired by mass spectrometry. Finally, the screened candidate compounds were interpreted and identified based on a predicted metabolic pathway, well - studied fragmentation rules, a predicted metabolic pathway, polarity and retention time of the compounds, and some related literature. With this method, a total of 111 absorbed constituents and metabolites of Radix glehniae in rats' urine, plasma, and bile samples were screened and identified or tentatively characterized successfully. This strategy provides an idea for the screening and identification of the metabolites of other TCMs.

  20. Enrichment and Detection of Molecules Secreted by Tumor Cells Using Magnetic Reversed-Phase Particles and LC-MALDI-TOF-MS

    PubMed Central

    Peter, Jochen F.; Otto, Angela M.; Wolf, Bernhard

    2007-01-01

    Tumor cells change their genetic expression pattern as they progress to states of increasing malignancy. Investigations at the DNA and RNA level alone cannot provide all the information resulting after the translation and processing of the corresponding proteins, which is one reason for a poor correlation between mRNA and the respective protein abundance. In diagnostics, differentially expressed peptides or proteins are important markers for the early detection of cancer. Unfortunately, tumor cells secrete peptides and proteins in only very low amounts, making mass spectrometric determination very difficult. In this publication, methods have been developed for the effective enrichment and cleanup of substances secreted by cultivated cancer cells. To obviate peptides from fetal calf serum used in cell culture, a serum surrogate was developed, which maintained growth of the cancer cells. After the binding of substances from cell-culture supernatants to custom-made magnetic reversed-phase particles, the substances were eluted and separated by capillary high-performance liquid chromatography. Fractions were spotted directly on a MALDI target, and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode. This technology was used to detect substances secreted by two mammary carcinoma cell lines differing in their malignancy (MCF-7, MDA-MB231). Unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensitive investigation of peptides secreted by cancer cells in culture and provides a valuable tool for the investigation of cancer cells in different states of malignancy. PMID:18166672

  1. Two-Dimensional Correlation Spectroscopy for Multimodal Analysis of FT-IR, Raman, and MALDI-TOF MS Hyperspectral Images with Hamster Brain Tissue.

    PubMed

    Lasch, Peter; Noda, Isao

    2017-04-11

    Hyperspectral imaging (HSI) techniques are useful for obtaining very detailed structural and compositional information from biomedical, pharmaceutical, or clinical samples, among others. The informative value of these methods can be further increased through the application of different HSI techniques and joint analysis of the data. However, interpretation and understanding of multimodal HSI have been impeded by difficulties in registration of the different HSI data sets and by the lack of integrative analysis concepts. Here, we introduce two-dimensional correlation spectroscopy (2DCOS) as a novel technique for jointly analyzing HSI data which allows one to obtain deeper insights into the chemistry of complex samples by decrypting auto- and heterospectral correlations that may exist between features of the different HSI data. The general workflow of 2DCOS analysis is demonstrated by HSI examples acquired from cryo-sections of hamster brain tissue using Fourier-transform infrared (FT-IR) microspectroscopy, confocal Raman microspectroscopy (CRM), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Multimodal hyperspectral image analysis by 2DCOS opens up new opportunities for spectral band assignments and thus the interpretation of structure-spectra and composition-spectra relationships. We foresee wide application potential for describing complex samples in various fields ranging from biomedicine to industrial applications.

  2. Tracing the pathogen Staphylococcus aureus on laboratory ants using physical preconcentration coupled ZnO nanoparticle assisted MALDI-TOF MS.

    PubMed

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

    2012-01-21

    Ants and humans coexist closely and for the most part happily. We consider ants to be harmless, small beings--we have no problem picking them out of our tea cups or sugar jars, throwing them away and continuing to consume the food. This paper is an eye-opener that these ants are not as harmless as they may seem. In particular, our relationship with those present in bacteria-rich environments (e.g. a microbiological lab) need to be reconsidered. From an analytical point of view we have applied the physical preconcentration coupled ZnO NPs assisted MALDI-MS (PP-MALDI-MS) as a novel and sensitive technique for detecting bacteria on the surface of a species of ant present in our laboratory. The preconcentration methods consist of simple techniques comprising of vortex combined with centrifugation or ultrasonication resulting in increasing sample concentration up to the MALDI-MS detection limit. ZnO NPs were used to further enhance the bacterial signals for culture free rapid analysis using MALDI-MS. The importance of a vortex-combined centrifugation approach, using a large number of samples (large number of ants) and decreasing the suspension volume and addition of sample to ZnO NPs (3.5g L(-1)) were found to be crucial prerequisites for increasing MALDI-MS detection of bacteria on ants. We were able to identify the pathogenic clinically important Staphylococcus aureus on the surface of the ants. The bacterial identification was validated using ClinPro 2.1.

  3. X-ray standing wave study of the Sr/Si(001)-(2 x 3) surface.

    SciTech Connect

    Goodner, D. M.; Marasco, D. L.; Escuadro, A. A.; Cao, L.; Tinkham, B. P.; Bedzyk, M. J.; Northwestern Univ.

    2003-12-10

    Sub-monolayer surface phases of Sr on Si(0 0 1) have been studied with low-energy electron diffraction (LEED) and X-ray standing waves (XSW). A (3 x 1) phase was observed after depositing 0.6-0.8 ML Sr on room-temperature Si(0 0 1). Annealing at 750-800 {sup o}C caused a portion of the Sr to desorb and resulted in a sharp (2 x 3) LEED pattern. Normal Si(0 0 4) and off-normal Si(0 2 2) and Si(1 1 1) XSW measurements made on the (2 x 3) phase indicate that Sr atoms must sit at either cave or bridge sites. The XSW results also suggest that if a sufficiently low anneal temperature is used, the (2 x 3) phase co-exists with short-range ordered regions of Sr atoms located at valley-bridge sites.

  4. Quantitative analysis of cortisol and 6β-hydroxycortisol in urine by fully automated SPE and ultra-performance LC coupled with electrospray and atmospheric pressure chemical ionization (ESCi)-TOF-MS.

    PubMed

    Lang, Lotte M; Dalsgaard, Petur W; Linnet, Kristian

    2013-01-01

    An ultra-performance LC TOF MS method for quantitative analysis of cortisol and 6β-hydroxycortisol in urine was developed. The method was used for determination of the ratio between 6β-hydroxycortisol and cortisol in urine received from autopsy cases and living persons as a measure of cytochrome P450 3A enzyme activity. Urine samples (0.25 mL) were extracted with an in-house developed fully automated 96-well SPE system. The compounds were quantified using a Waters ACQUITY UPLC system coupled to a Waters SYNAPT G2. The MS sensitivity was optimized by using negative ionization in sensitivity mode (resolution >10 000 full-width at half-maximum), and further optimized by using the enhanced duty cycle around the 410 m/z. ESCi (simultaneous electrospray and atmospheric pressure chemical ionization) mode was used to compensate for the matrix effects of postmortem urine. Finally, the SYNAPT G2 was tested as a quantitative instrument. The developed method has a measurement range from 2.5-300 ng/mL for cortisol to 10-1200 ng/mL for 6β-hydroxycortisol. Mean overall process efficiencies were 29.4 and 23.0% for cortisol and 6β-hydroxycortisol, respectively. In 20 forensic reference cases, the range of the 6β-hydroxycortisol/cortisol ratio was 0.29-14.2 with a median of 3.04.

  5. Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.

    PubMed

    Shcherbatko, Anatoly; Foletti, Davide; Poulsen, Kris; Strop, Pavel; Zhu, Guoyun; Hasa-Moreno, Adela; Melton Witt, Jody; Loo, Carole; Krimm, Stellanie; Pios, Ariel; Yu, Jessica; Brown, Colleen; Lee, John K; Stroud, Robert; Rajpal, Arvind; Shelton, David

    2016-06-03

    Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

  6. Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

    PubMed Central

    Gu, Yanping; Li, Guangwen; Chen, Yong; Huang, Li-Yen Mae

    2016-01-01

    Abstract Sensitization of purinergic P2X3 receptors (P2X3Rs) is a major mechanism contributing to injury-induced exaggerated pain responses. We showed in a previous study that cyclic adenosine monophosphate (cAMP)–dependent guanine nucleotide exchange factor 1 (Epac1) in rat sensory dorsal root ganglia (DRGs) is upregulated after inflammatory injury, and it plays a critical role in P2X3R sensitization by activating protein kinase C epsilon (PKCε) inside the cells. protein kinase C epsilon has been established as the major PKC isoform mediating injury-induced hyperalgesic responses. On the other hand, the role of PKCα in receptor sensitization was seldom considered. Here, we studied the participation of PKCα in Epac signaling in P2X3R-mediated hyperalgesia. The expression of both Epac1 and Epac2 and the level of cAMP in DRGs are greatly enhanced after complete Freund adjuvant (CFA)–induced inflammation. The expression of phosphorylated PKCα is also upregulated. Complete Freund adjuvant (CFA)–induced P2X3R-mediated hyperalgesia is not only blocked by Epac antagonists but also by the classical PKC isoform inhibitors, Go6976, and PKCα-siRNA. These CFA effects are mimicked by the application of the Epac agonist, 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (CPT), in control rats, further confirming the involvement of Epacs. Because the application of Go6976 prior to CPT still reduces CPT-induced hyperalgesia, PKCα is downstream of Epacs to mediate the enhancement of P2X3R responses in DRGs. The pattern of translocation of PKCα inside DRG neurons in response to CPT or CFA stimulation is distinct from that of PKCε. Thus, in contrast to prevalent view, PKCα also plays an essential role in producing complex inflammation-induced receptor-mediated hyperalgesia. PMID:26963850

  7. Overview of VOC emissions and chemistry from PTR-TOF-MS measurements during the SusKat-ABC campaign: high acetaldehyde, isoprene and isocyanic acid in wintertime air of the Kathmandu Valley

    NASA Astrophysics Data System (ADS)

    Sarkar, Chinmoy; Sinha, Vinayak; Kumar, Vinod; Rupakheti, Maheswar; Panday, Arnico; Mahata, Khadak S.; Rupakheti, Dipesh; Kathayat, Bhogendra; Lawrence, Mark G.

    2016-03-01

    The Kathmandu Valley in Nepal suffers from severe wintertime air pollution. Volatile organic compounds (VOCs) are key constituents of air pollution, though their specific role in the valley is poorly understood due to insufficient data. During the SusKat-ABC (Sustainable Atmosphere for the Kathmandu Valley-Atmospheric Brown Clouds) field campaign conducted in Nepal in the winter of 2012-2013, a comprehensive study was carried out to characterise the chemical composition of ambient Kathmandu air, including the determination of speciated VOCs, by deploying a proton transfer reaction time-of-flight mass spectrometer (PTR-TOF-MS) - the first such deployment in South Asia. In the study, 71 ion peaks (for which measured ambient concentrations exceeded the 2σ detection limit) were detected in the PTR-TOF-MS mass scan data, highlighting the chemical complexity of ambient air in the valley. Of the 71 species, 37 were found to have campaign average concentrations greater than 200 ppt and were identified based on their spectral characteristics, ambient diel profiles and correlation with specific emission tracers as a result of the high mass resolution (m / Δm > 4200) and temporal resolution (1 min) of the PTR-TOF-MS. The concentration ranking in the average VOC mixing ratios during our wintertime deployment was acetaldehyde (8.8 ppb) > methanol (7.4 ppb) > acetone + propanal (4.2 ppb) > benzene (2.7 ppb) > toluene (1.5 ppb) > isoprene (1.1 ppb) > acetonitrile (1.1 ppb) > C8-aromatics ( ˜ 1 ppb) > furan ( ˜ 0.5 ppb) > C9-aromatics (0.4 ppb). Distinct diel profiles were observed for the nominal isobaric compounds isoprene (m / z = 69.070) and furan (m / z = 69.033). Comparison with wintertime measurements from several locations elsewhere in the world showed mixing ratios of acetaldehyde ( ˜ 9 ppb), acetonitrile ( ˜ 1 ppb) and isoprene ( ˜ 1 ppb) to be among the highest reported to date. Two "new" ambient compounds, namely formamide (m / z = 46.029) and acetamide (m / z

  8. Overview of VOC emissions and chemistry from PTR-TOF-MS measurements during the SusKat-ABC campaign: high acetaldehyde, isoprene and isocyanic acid in wintertime air of the Kathmandu Valley

    NASA Astrophysics Data System (ADS)

    Sarkar, C.; Sinha, V.; Kumar, V.; Rupakheti, M.; Panday, A.; Mahata, K. S.; Rupakheti, D.; Kathayat, B.; Lawrence, M. G.

    2015-09-01

    The Kathmandu Valley in Nepal suffers from severe wintertime air pollution. Volatile organic compounds (VOCs) are key constituents of air pollution, though their specific role in the Valley is poorly understood due to insufficient data. During the SusKat-ABC (Sustainable Atmosphere for the Kathmandu Valley-Atmospheric Brown Clouds) field campaign conducted in Nepal in the winter of 2012-2013, a comprehensive study was carried out to characterize the chemical composition of ambient Kathmandu air, including the determination of speciated VOCs by deploying a Proton Transfer Reaction Time of Flight Mass Spectrometer (PTR-TOF-MS)-the first such deployment in South Asia. 71 ion peaks (for which measured ambient concentrations exceeded the 2 σ detection limit) were detected in the PTR-TOF-MS mass scan data, highlighting the chemical complexity of ambient air in the Valley. Of the 71 species, 37 were found to have campaign average concentrations greater than 200 ppt and were identified based on their spectral characteristics, ambient diel profiles and correlation with specific emission tracers as a result of the high mass resolution (m/Δm > 4200) and temporal resolution (1 min) of the PTR-TOF-MS. The highest average VOC mixing ratios during the measurement period were (in rank order): acetaldehyde (8.8 ppb), methanol (7.4 ppb), acetone (4.2 ppb), benzene (2.7 ppb), toluene (1.5 ppb), isoprene (1.1 ppb), acetonitrile (1.1 ppb), C8-aromatics (~ 1 ppb), furan (~ 0.5 ppb), and C9-aromatics (0.4 ppb). Distinct diel profiles were observed for the nominal isobaric compounds isoprene (m/z = 69.070) and furan (m/z = 69.033). Comparison with wintertime measurements from several locations elsewhere in the world showed mixing ratios of acetaldehyde (~ 9 ppb), acetonitrile (~ 1 ppb) and isoprene (~ 1 ppb) to be among the highest reported till date. Two "new" ambient compounds namely, formamide (m/z = 46.029) and acetamide (m/z = 60.051), which can photochemically produce isocyanic

  9. Rapid identification of bacteria from bioMérieux BacT/ALERT blood culture bottles by MALDI-TOF MS.

    PubMed

    Haigh, J D; Green, I M; Ball, D; Eydmann, M; Millar, M; Wilks, M

    2013-01-01

    Several studies have reported poor results when trying to identify microorganisms directly from the bioMérieux BacT/ALERT blood culture system using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry. The aim of this study is to evaluate two new methods, Sepsityper and an enrichment method for direct identification of microorganisms from this system. For both methods the samples were processed using the Bruker Microflex LT mass spectrometer (Biotyper) using the Microflex Control software to obtain spectra. The results from direct analysis were compared with those obtained by subculture and subsequent identification. A total of 350 positive blood cultures were processed simultaneously by the two methods. Fifty-three cultures were polymocrobial or failed to grow any organism on subculture, and these results were not included as there was either no subculture result, or for polymicrobial cultures it was known that the Biotyper would not be able to distinguish the constituent organisms correctly. Overall, the results showed that, contrary to previous reports, it is possible to identify bacteria directly from bioMérieux blood culture bottles, as 219/297 (74%) correct identifications were obtained using the Bruker Sepsityper method and 228/297 (77%) were obtained for the enrichment method when there is only one organism was present. Although the enrichment method was simpler, the reagent costs for the Sepsityper method were approximately pound 4.00 per sample compared to pound 0.50. An even simpler and cheaper method, which was less labour-intensive and did not require further reagents, was investigated. Seventy-seven specimens from positive signalled blood cultures were analysed by inoculating prewarmed blood agar plates and analysing any growth after 1-, 2- and 4-h periods of incubation at 37 degrees C, by either direct transfer or alcohol extraction. This method gave the highest number of correct identifications, 66/77 (86

  10. Volatile Organic Compound emissions from soil: using Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-TOF-MS) for the real time observation of microbial processes

    NASA Astrophysics Data System (ADS)

    Veres, P. R.; Behrendt, T.; Klapthor, A.; Meixner, F. X.; Williams, J.

    2014-08-01

    In this study we report on the emissions of volatile organic compounds (VOC) and nitric oxide (NO) from two contrasting soils (equatorial rainforest and arid cotton field) analyzed in a laboratory based dynamic chamber system. The effect of soil moisture and soil temperature on VOC and NO emission was examined in laboratory incubation experiments by measuring as a pre-saturated soil dried out. Our results suggest that real time monitoring of VOC emissions from soil using a proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS) instrument can be used to improve our understanding of the release mechanisms of trace gases (e.g. NO, N2O) that are involved in the nitrogen cycle. Moreover, we report on the release rate of various VOC species, many of which exhibit a temperature dependent response indicative of biological production, namely a temperature amplification factor (Q10) ∼ 2-3. Contrary to the conventional modeling of NO emissions from soils, that the release of NO from the overall community across the range of soil water content can be modeled as an optimum function, we suggest that VOC measurements indicate there exist multiple distinct contributing microbial guilds releasing NO. These microbial guilds could likely be individually identified with the observed VOC profiles. Using a cotton field soil sample from a Sache oasis (Taklimakan desert, Xinijang, P. R. China), we identify five VOC emission groups with varying degrees of NO co-emission. An equatorial rainforest soil (Suriname) was shown to emit a variety of VOC including acetaldehyde, acetone, DMS, formaldehyde, and isoprene that vary strongly and individually as a function of temperature and soil moisture content. PTR-TOF-MS with high time resolution, sensitivity, and molecular specificity is an ideal tool for the real time analysis of VOC and NO emitting processes in soil systems. These experiments can be used as a template for future experiments to more completely and specifically

  11. Overview of VOC emissions and chemistry from PTR-TOF-MS measurements during the SusKat-ABC campaign: high acetaldehyde, ketene, isoprene and isocyanic acid in wintertime air of the Kathmandu Valley

    NASA Astrophysics Data System (ADS)

    Sarkar, C.; Sinha, V.; Kumar, V.; Rupakheti, M.; Panday, A. K.; Mahata, K.; Rupakheti, D.; Kathayat, B.; Lawrence, M. G.

    2015-12-01

    During SusKat-ABC (Sustainable Atmosphere for the Kathmandu Valley-Atmospheric Brown Clouds) field campaign conducted in the winter of 2012-2013, a comprehensive study was carried out to characterize the chemical composition of ambient Kathmandu air for speciated VOCs by deploying a Proton Transfer Reaction Time of Flight Mass Spectrometer (PTR-TOF-MS), the first time to be deployed in South Asia. Due to its high mass resolution (m/Δm > 4200) and temporal resolution (1 minute), 71 ion peaks were detected in the PTR-TOF-MS mass scan data, highlighting the chemical complexity of ambient air in the Valley. Of the 71, 38 species were found to have campaign average concentrations > 200 ppt and were identified based on their spectral characteristics, ambient diel profiles and correlation with specific emission tracers. Distinct diel profiles were observed for the nominal isobaric compounds isoprene (m/z=69.070) and furan (m/z=69.033). Comparison with several sites elsewhere in the world showed mixing ratios of acetaldehyde (~ 9 ppb), acetonitrile (~1 ppb) and isoprene (~ 1 ppb) to be among the highest measured anywhere in the world. Two "new" ambient compounds namely, methanamide (m/z = 46.029) and acetamide (m/z=60.051) which can photochemically produce isocyanic acid in the atmosphere, are reported in this study alongwith nitromethane (a tracer for diesel exhaust) and ketene (a very reactive compound). Two distinct periods were identified during the campaign based on high daytime biogenic emissions of isoprene even in winter and biomass fired brick kiln emissions of acetonitrile, benzene and isocyanic acid. Biomass burning and biomass fired brick kiln emissions were found to be the dominant source for compounds such as propyne, propene, benzene and propanenitrile which correlated strongly with biomass burning tracer acetonitrile (r2 > 0.7). The calculated total VOC OH reactivity was dominated by acetaldehyde (20.1%), ketene (ethenone) (17.1%), isoprene (16.8 %) and

  12. Postsynaptic P2X3-containing receptors in gustatory nerve fibres mediate responses to all taste qualities in mice

    PubMed Central

    Vandenbeuch, Aurelie; Larson, Eric D; Anderson, Catherine B; Smith, Steven A; Ford, Anthony P; Finger, Thomas E; Kinnamon, Sue C

    2015-01-01

    Abstract Taste buds release ATP to activate ionotropic purinoceptors composed of P2X2 and P2X3 subunits, present on the taste nerves. Mice with genetic deletion of P2X2 and P2X3 receptors (double knockout mice) lack responses to all taste stimuli presumably due to the absence of ATP-gated receptors on the afferent nerves. Recent experiments on the double knockout mice showed, however, that their taste buds fail to release ATP, suggesting the possibility of pleiotropic deficits in these global knockouts. To test further the role of postsynaptic P2X receptors in afferent signalling, we used AF-353, a selective antagonist of P2X3-containing receptors to inhibit the receptors acutely during taste nerve recording and behaviour. The specificity of AF-353 for P2X3-containing receptors was tested by recording Ca2+ transients to exogenously applied ATP in fura-2 loaded isolated geniculate ganglion neurons from wild-type and P2X3 knockout mice. ATP responses were completely inhibited by 10 μm or 100 μm AF-353, but neither concentration blocked responses in P2X3 single knockout mice wherein the ganglion cells express only P2X2-containing receptors. Furthermore, AF-353 had no effect on taste-evoked ATP release from taste buds. In wild-type mice, i.p. injection of AF-353 or simple application of the drug directly to the tongue, inhibited taste nerve responses to all taste qualities in a dose-dependent fashion. A brief access behavioural assay confirmed the electrophysiological results and showed that preference for a synthetic sweetener, SC-45647, was abolished following i.p. injection of AF-353. These data indicate that activation of P2X3-containing receptors is required for transmission of all taste qualities. Key points Acute inhibition of purinergic receptors with a selective P2X3 antagonist prevents transmission of information from taste buds to sensory nerves. The P2X3 antagonist has no effect on taste-evoked release of ATP, confirming the effect is postsynaptic. The

  13. The Effect of Temperature on Pressurised Hot Water Extraction of Pharmacologically Important Metabolites as Analysed by UPLC-qTOF-MS and PCA

    PubMed Central

    Khoza, B. S.; Chimuka, L.; Mukwevho, E.; Steenkamp, P. A.; Madala, N. E.

    2014-01-01

    Metabolite extraction methods have been shown to be a critical consideration for pharmacometabolomics studies and, as such, optimization and development of new extraction methods are crucial. In the current study, an organic solvent-free method, namely, pressurised hot water extraction (PHWE), was used to extract pharmacologically important metabolites from dried Moringa oleifera leaves. Here, the temperature of the extraction solvent (pure water) was altered while keeping other factors constant using a homemade PHWE system. Samples extracted at different temperatures (50, 100, and 150°C) were assayed for antioxidant activities and the effect of the temperature on the extraction process was evaluated. The samples were further analysed by mass spectrometry to elucidate their metabolite compositions. Principal component analysis (PCA) evaluation of the UPLC-MS data showed distinctive differential metabolite patterns. Here, temperature changes during PHWE were shown to affect the levels of metabolites with known pharmacological activities, such as chlorogenic acids and flavonoids. Our overall findings suggest that, if not well optimised, the extraction temperature could compromise the “pharmacological potency” of the extracts. The use of MS in combination with PCA was furthermore shown to be an excellent approach to evaluate the quality and content of pharmacologically important extracts. PMID:25371697

  14. Development of a direct in-matrix extraction (DIME) protocol for MALDI-TOF-MS detection of glycated phospholipids in heat-treated food samples.

    PubMed

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

    2014-09-01

    In foodstuffs, one of the main factors inducing modifications in phospholipids (PLs) structure is the heat treatment. Among PLs, only phosphatidylethanolamines and phosphatidylserines, due to their free amino group, can be involved in Maillard reaction and can form adducts with reducing sugars, besides other by-products called advanced glycation end-products. To date, glycated lipid products are less characterized in comparison to proteins. The aim of this work was to develop a novel, rapid and sensitive extraction protocol for the detection and characterization of modified PLs (glycated and oxidized) by means of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). At first, to investigate the formation of glycated and/or short chain by-products in different classes of PLs, representative standards were heated with or without sugar (lactose or glucose) and subjected to traditional lipid extraction methods as Bligh and Dyer and to the novel direct in matrix extraction (DIME) using 1,8-bis(dimethylamino)naphthalene as preconcentrating matrix. MALDI-MS analysis in negative ion mode allowed detecting glycation and oxidation products both on fatty acid and glucose moieties. Then, the procedure was successfully applied to different heat-treated and powdered samples (milk powders, pasteurized milk, ultra-high-temperature milk and soy flour) for the detection of modified PLs in complex foods. The currently developed DIME protocol could be a powerful tool for understanding lipid glycation also in biological samples.

  15. 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.

  16. Characterization of Polyphenols from Lycium ruthenicum Fruit by UPLC-Q-TOF/MS(E) and Their Antioxidant Activity in Caco-2 Cells.

    PubMed

    Wu, Tao; Lv, Haiyang; Wang, Fengzhong; Wang, Yi

    2016-03-23

    The fruit of Lycium ruthenicum Murr. (LRF) has long been used in folk medicine. Nevertheless, detailed information related to its polyphenol compositions remains scarce. In this study, we confirmed that the total phenolic and anthocyanin contents of LRF fruit extracts (LRFEs) were 4906.5 ± 60.6 mg of gallic acid equivalents/100 g DW and 787.6 ± 34.1 mg of cyanindin-3-glucoside equivalents/100 g DW, respectively. A characterization of LRFEs was performed by ultrahigh performance liquid chromatography/quadrupole time-of-flight mass spectrometry using an MS(E) data acquisition. A total of 26 polyphenols were tentatively identified, of which 19 represent the first reports of these polyphenols in LRFEs. Furthermore, the cellular antioxidant array showed that LRFEs could protect Caco-2 cells against H2O2-induced oxidative damage based on microscopic fluorometric imaging.

  17. On-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct MALDI-QIT-TOF MS analysis.

    PubMed

    Tang, Jia; Liu, Yingchao; Qi, Dawei; Yao, Guoping; Deng, Chunhui; Zhang, Xiangmin

    2009-11-01

    In this study, an on-plate-selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless-steel plate, then modified with 4-mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI-MS simply by deposition of 2,5-dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on-plate strategy promising for online enrichment of glycopeptides, which could be applied in high-throughput proteome research.

  18. 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.

  19. Lipidomic fingerprint of almonds (Prunus dulcis L. cv Nonpareil) using TiO₂ nanoparticle based matrix solid-phase dispersion and MALDI-TOF/MS and its potential in geographical origin verification.

    PubMed

    Shen, Qing; Dong, Wei; Yang, Mei; Li, Linqiu; Cheung, Hon-Yeung; Zhang, Zhifeng

    2013-08-14

    A matrix solid-phase dispersion (MSPD) procedure with titanium dioxide (TiO2) nanoparticles (NP) as sorbent was developed for the selective extraction of phospholipids from almond samples, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) was employed for analysis. A remarkable increase in the signals of phospholipid accompanied by a decrease in those of triacylglycerols and diacylglycerols was observed in the relevant mass spectra. The proposed method was applied to five batches of almonds originating from four geographical areas, whereas principal component analysis (PCA) was utilized to normalize the relative amounts of the identified phospholipid species. The results indicated that the lipidomic fingerprint of almonds was successfully established by the negative ion mode spectrum, and the ratio of m/z 833.6 to 835.6 as well as m/z 821.6 could be introduced as potential markers for the differentiation of the tested almonds with different geographical origins. The whole method is of great promise for selective separation of phospholipids from nonphospholipids, especially the glycerides, and superior in fast screening and characterization of phospholipids in almond samples.

  20. Structural, MALDI-TOF-MS, magnetic and spectroscopic studies of new dinuclear copper(II), cobalt(II) and zinc(II) complexes containing a biomimicking μ-OH bridge.

    PubMed

    Núñez, Cristina; Bastida, Rufina; Macías, Alejandro; Valencia, Laura; Neuman, Nicolás I; Rizzi, Alberto C; Brondino, Carlos D; González, Pablo J; Capelo, José Luis; Lodeiro, Carlos

    2010-12-28

    The Py(2)N(4)S(2) octadentate coordinating ligand afforded dinuclear cobalt, copper and zinc complexes and the corresponding mixed metal compounds. The overall geometry and bonding modes have been deduced on the basis of elemental analysis data, MALDI-TOF-MS, IR, UV-vis and EPR spectroscopies, single-crystal X-Ray diffraction, conductivity and magnetic susceptibility measurements. In the copper and zinc complexes, a μ-hydroxo bridge links the two metal ions. In both cases, the coordination geometry is distorted octahedral. Magnetic and EPR data reveal weakly antiferromagnetic high spin Co(II) ions, compatible with a dinuclear structure. The magnetic characterization of the dinuclear Cu(II) compound indicates a ferromagnetically coupled dimer with weak antiferromagnetic intermolecular interactions. The intra-dimer ferromagnetic behaviour was unexpected for a Cu(II) dimer with such μ-hydroxo bridging topology. We discuss the influence on the magnetic properties of non-covalent interactions between the bridging moiety and the lattice free water molecules.

  1. Metabolomic analysis using ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS) uncovers the effects of light intensity and temperature under shading treatments on the metabolites in tea.

    PubMed

    Zhang, Qunfeng; Shi, Yuanzhi; Ma, Lifeng; Yi, Xiaoyun; Ruan, Jianyun

    2014-01-01

    To investigate the effect of light intensity and temperature on the biosynthesis and accumulation of quality-related metabolites, field grown tea plants were shaded by Black Net and Nano-insulating Film (with additional 2-4°C cooling effect) with un-shaded plants as a control. Young shoots were subjected to UPLC-Q-TOF MS followed by multivariate statistical analysis. Most flavonoid metabolites (mainly flavan-3-ols, flavonols and their glycosides) decreased significantly in the shading treatments, while the contents of chlorophyll, β-carotene, neoxanthin and free amino acids, caffeine, benzoic acid derivatives and phenylpropanoids increased. Comparison between two shading treatments indicated that the lower temperature under Nano shading decreased flavonols and their glycosides but increased accumulation of flavan-3-ols and proanthocyanidins. The comparison also showed a greater effect of temperature on galloylation of catechins than light intensity. Taken together, there might be competition for substrates between the up- and down-stream branches of the phenylpropanoid/flavonoid pathway, which was influenced by light intensity and temperature.

  2. Analysis of Wheat Prolamins, the Causative Agents of Celiac Sprue, Using Reversed Phase High Performance Liquid Chromatography (RP-HPLC) and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS)

    PubMed Central

    Mejías, Jaime H.; Lu, Xiaoqiao; Osorio, Claudia; Ullman, Jeffrey L.; von Wettstein, Diter; Rustgi, Sachin

    2014-01-01

    Wheat prolamins, commonly known as “gluten”, are a complex mixture of 71–78 proteins, which constitute ~80% of the proteins in the wheat grains and supply 50% of the global dietary protein demand. Prolamins are also responsible for numerous gluten-induced disorders and determine the unique visco-elastic properties of the wheat dough. These properties necessitate the reliable determination of the prolamin composition in wheat grains and their derived products. Therefore, this study examined the impact of HPLC conditions, including column type, column temperature, flow rate, and the gradient of polar and non-polar solvents in the mobile phase, to improve the analytical resolution of prolamins. The following conditions were found optimal for analyses: column temperature 60 °C, flow rate 1.0 mL/min and an elution gradient of 20%–60% of 0.1% trifluoroacetic acid + acetonitrile in 60 min. For further improvement of resolution, gliadin and glutenin extracts were analyzed using MALDI-TOF-MS in combination with HPLC fractionation. Two semi-quantitative methods, densitometry of stained polyacrylamide gels and HPLC, were used to determine relative prolamin quantities and the correspondence between the methods was established. The combinatorial gluten analyses approach developed during the present study was used to analyze prolamin profiles of wheat transformants expressing DEMETER silencing artificial microRNA, and the results are discussed. PMID:24739977

  3. Simultaneous qualitative and quantitative analysis of phenolic acids and flavonoids for the quality control of Apocynum venetum L. leaves by HPLC-DAD-ESI-IT-TOF-MS and HPLC-DAD.

    PubMed

    An, Haijuan; Wang, Hong; Lan, Yuexiang; Hashi, Yuki; Chen, Shizhong

    2013-11-01

    A reliable method based on high performance liquid chromatography-diode array detector-electrospray ionization-ion trap-time of flight-mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) was developed for the identification of phenolic acids and flavonoids in Apocynum venetum L. leaves and its adulterant, Pocynum hendersonii (Hook. f.) Woodson leaves. A total of 21 compounds were identified or tentatively identified, including 4 phenolic acids and 17 flavonoids. 3-O-caffeoylquinic acid (3-CQA) and caffeic acid were detected for the first time in A. venetum leaves; 4-O-caffeoylquinic acid (4-CQA), 3-CQA, caffeic acid, quercetin-3-O-(6"-O-malonyl)-galactoside, quercetin-3-O-(6"-O-malonyl)-glucoside, kaempferol-3-O-(6"-O-malonyl)-glucoside, kaempferol-3-O-(6"-O-acetyl)-glucoside, and kaempferol-3-O-dihexoside were detected for the first time in P. hendersonii leaves. Cluster analysis was employed to analyze 24 batches of A. venetum leaves and 5 batches of P. hendersonii leaves collected from various regions in China. The analysis, which was based on the 21 compounds, indicated that profiles of these compounds were distinct between the two species, and among A. venetum leaf samples from different origins. 18 of these 21 compounds were selected as the markers and simultaneously analyzed by HPLC-DAD for the first time. The quantitative analytical method was validated and subsequently applied to the comprehensive quality evaluation of 24 batches of A. venetum leaves.

  4. HPLC-DAD-q-TOF-MS as a powerful platform for the determination of phenolic and other polar compounds in the edible part of mango and its by-products (peel, seed, and seed husk).

    PubMed

    Gómez-Caravaca, Ana María; López-Cobo, Ana; Verardo, Vito; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

    2016-04-01

    Free and bound phenolic and other polar compounds in mango edible fraction and its by-products (peel, seed, and seed husk) have been determined by HPLC-DAD-ESI-qTOF-MS. This analytical technique has demonstrated to be a valuable platform for the identification and quantification of these compounds in mango. In fact, UV-Vis and mass spectra data allowed the determination of 91 free compounds and 13 bound (cell wall linked) compounds taking into account the four fractions of mango. To our knowledge, this is the first time that mango seed husk has been studied regarding its phenolic compounds. The method proposed showed LODs between 0.006 and 0.85 μg/mL and accuracy ranged from 94.8 and 100.7%. Mango peel presented the highest concentration of free polar compounds followed by seed, pulp, and seed husk. It is also important to highlight that bound phenolic compounds had never been determined in mango pulp, seed, and seed husk before. Furthermore, ellagic acid was the most abundant bound compound in the four mango fractions analyzed. These results show that mango pulp and its by-products are a good source of phenolic and other polar compounds. In particular, mango seed contains a high total concentration of ellagic acid (650 mg/100 g dry weight).

  5. Identification of “Multiple Components-Multiple Targets-Multiple Pathways” Associated with Naoxintong Capsule in the Treatment of Heart Diseases Using UPLC/Q-TOF-MS and Network Pharmacology

    PubMed Central

    Ma, Xianghui; Lv, Bin; Li, Pan; Jiang, Xiaoqing; Zhou, Qian; Wang, Xiaoying; Gao, Xiumei

    2016-01-01

    Naoxintong capsule (NXT) is a commercial medicinal product approved by the China Food and Drug Administration which is used in the treatment of stroke and coronary heart disease. However, the research on the composition and mechanism of NXT is still lacking. Our research aimed to identify the absorbable components, potential targets, and associated pathways of NXT with network pharmacology method. We explored the chemical compositions of NXT based on UPLC/Q-TOF-MS. Then, we used the five principles of drug a