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ĎuráčováMass SpectrometryTop-down proteomicsNot availableclostridium perfringensPrm; clostridium perfringens; protein toxin; epsilon toxinPrmepsilon toxinprotein toxinhttp://www.ebi.ac.uk/pride/archive/projects/PXD012528Set of 11 different C. perfringens strains were first used to characterize the spectrum of produced toxins. The strains were cultivated anaerobically in two different cultivation media (Schaedler anaerobe broth and BBL Fluid thioglycolate medium), culture filtrates were collected and bacteria were lysed in ultrasonic homogenizer. Samples (culture filtrates as well as the lysates from both media) were solubilized by 1M urea, reduced by tris(2-carboxyethyl)phosphine, alkylated by iodoacetamide and digested by sequencing grade trypsin. The peptides were then desalted using phase extraction cartridges (Empore SPE Cartridges, C18, Merck), according to the manufacturer’s protocol. Due to the lack of commercially available protein toxin standards necessary for the targeted method development, recombinant toxins were prepared. The alpha, beta, beta2, epsilon, iota a, and iota b proteins of C. perfringens were expressed in E. coli and isolated through a polyhistidine tag in sufficient quantity and purity for MS analysis (digestion as above). Peptide mixtures were then analyzed by nano-LC coupled on-line with Q-Exactive mass spectrometer.PrideNot available6x(13)Ciodoacetamide derivatized residuemonohydroxylated residueDatabase search was performed by Proteome Discoverer software v. 1.4.1.14 (Thermo Scientific) and Mascot search algorithm. Data were searched against a database constructed by merging the sets of C. perfringens proteomes (C. perfringens strain 13; ATCC 13124; SM101; ATCC 3626; JGS1495; str. F4969; str. JGS1721; str. JGS1987; NCTC 8239; and WAL-14572). The merged database contained 32,700 sequences (downloaded on May 18th 2016). Search parameters were set as follows: tryptic specificity with maximum of 2 missed cleavages, Cys carbamidomethylation as fixed modification and methionine oxidation as variable modification, mass tolerance of 10 ppm for precursors and 20 mmu for product ions. False discovery rate (FDR) was set to 0.01 (high confidence) and 0.05 (medium confidence). Peptides for targeted assays were selected using Skyline software v. 4.1 (downloaded from https://skyline.ms/project/home/begin.view). The spectral library was created from data-dependent acquisition MS analyses of the recombinant proteins. Skyline parameters for peptide settings were set as follows: trypsin with no missed cleavages; peptide length from 5 to 25 amino acids; methionine-containing peptides were excluded; Cys carbamidomethylation was allowed; isotope label type was set up as heavy (13C6 15N2 lysine and 13C6 15N4 arginine); transitions were set from 2+ and 3+ precursor ions. For each targeted peptide, the Skyline result was manually inspected. For each toxin, two or three unique peptides were selected. Peptides and their labeled forms were included in one common inclusion list.ProteomicsZuzana KrocovaQ ExactiveDepartment of Molecular Pathology, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech RepublicPARTIALClostridium Perfringensmiloslava.duracova@seznam.cz30909561 Duracova M, Klimentova J, Myslivcova Fucikova A, Zidkova L, Sheshko V, Rehulkova H, Dresler J, Krocova Z. Targeted Mass Spectrometry Analysis of Clostridium perfringens Toxins. Toxins (Basel). 2019 11(3) 10.3390/toxins11030177BiomedicalUniversity of DefenceCzech RepublicTargeted proteomics recently proved to be a technique for the detection and absolute quantification of proteins not easily accessible to classical bottom-up approaches. Due to this, it has been considered as a high fidelity tool to detect potential warfare agents in wide spread kinds of biological and environmental matrices. <i>Clostridium perfringens</i> toxins are considered to be potential biological weapons, especially the epsilon toxin which belongs to a group of the most powerful bacterial toxins. Here, the development of a target mass spectrometry method for the detection of <i>C. perfringens</i> protein toxins (alpha, beta, beta2, epsilon, iota) is described. A high-resolution mass spectrometer with a quadrupole-Orbitrap system operating in target acquisition mode (parallel reaction monitoring) was utilized. Because of the lack of commercial protein toxin standards recombinant toxins were prepared within <i>Escherichia coli</i>. The analysis was performed using proteotypic peptides as the target compounds together with their isotopically labeled synthetic analogues as internal standards. Calibration curves were calculated for each peptide in concentrations ranging from 0.635 to 1101 fmol/μL. Limits of detection and quantification were determined for each peptide in blank matrices.Targeted Mass Spectrometry Analysis of <i>Clostridium perfringens</i> Toxins.Duracova Miloslava M, Klimentova Jana J, Myslivcova Fucikova Alena A, Zidkova Lenka L, Sheshko Valeria V, Rehulkova Helena H, Dresler Jiri J, Krocova Zuzana Ztoxin, toxins., Bacillus perfringens, Clostridium welchii, Clostridium plagarum, Bacterium welchii, 'Clostridium plagarum'Atr25D, Forms, AI747449, IPP2A2, ion, Lysine Hydrochloride, Recombinant DNA, L-arginine, DL-Arginine Acetate, Aminosaeure, Str, Amino acid, A4, Monohydrate, H105E3, 5730420M11Rik, peptide, Polypeptides, L-Isomer Methionine, Lysine Acetate, Methionine, Drug Tolerance, peptido, ClvPrd, str, 2, Software Engineering, Arg, Computer Program, WMS, L Lysine, C79691, SET, DNA Recombinant, (S)-2-amino-5-guanidinopentanoic acid, F, amino acids, peptides, DmelCG14026, TAF-I, MUB3_18, K, M, Open, phosphatase 2A inhibitor I2PP2A, (S)-2-Amino-5-guanidinovaleric acid, R, Computer Programs and Programming, epsilon-diaminocaproic acid, Arginine, Monohydrate DL-Arginine Acetate, MUB3.18, ecotype, Lccp, DmelCG4299, set, IGAAD, Recombinant DNA Proteins, LYS, DmelCG10574, ppm, Lysin, GPHYSD2, Racemethionine, drug tolerance, SGS, ratio, L-Arginin, phapii, millidalton, DNA Proteins, PRO, immune system tolerance, Aminokarbonsaeure, 2-Amino-4-(methylthio)butyric acid, Arginine Hydrochloride, StF-IT-1, arginine, Thermo Scientific, CYS, Source Softwares, alpha-amino carboxylic acids, Software Tools, Programs, ACMICD, Program, Computer Applications, Ionen, Algorithm, Computer Applications Software, Computer Applications Softwares, lysine, Immunologic Tolerance, Methionin, cultivar, methionine, Softwares, Hmet, and GLY protein 2, TKV, Tkv, L-Arginine, parent ion, Amino Acid, Acid, and GLY protein 1, Software Applications, HLA-DR-associated protein II, Amino acids, DI-2, Recombinant Proteins, Source Software, I-2Dm, proportionality, mmu, rate, MASCOT, CG4299, MASS, alpha, inhibitor of granzyme A-activated DNase, beta Trypsin, I-2PP1, Atkv, Applications, DMDA, TAF-IBETA, Self Tolerance, Tolerance, DNA Recombinant Proteins, precursor ion, TAF-Ibeta, Acids, Polypeptide, DNA, i2pp2a, Liquimeth, Proteomes, Computer Software Applications, tkv1, common., 2-amino-4-(methylsulfanyl)butanoic acid, AW549739, False, Aminocarbonsaeure, L Isomer, FBN, high weight, Computer, alpha-amino acid, precursor, TYPE, PHAPII, L-(+)-arginine, DAGA4, template-activating factor I, (2S)-2-amino-5-(carbamimidamido)pentanoic acid, ECTOL1, heavy, L-Methionine, Recombinant Protein, F15E12.6, Lysine, toxins, MAM, SCG3, Biosynthetic, Pedameth, Application, Open Source Softwares, F15E12_6, proportion, Software Application, iones, ipp2a2, beta-Trypsin, Open Source Software, tkva, 2pp2a, ions, Biosynthetic Proteins, Dtfr, CG10574, Computer Software Application, OCTD, mKIAA0989, 2PP2A, 2-amino-4-(methylthio)butanoic acid, Tools, 10^[-6], taf-ibeta, kv, dSET, dSet, L-Arg, dtfr, peptidos, TKV1, metionina, Applications Software, Open Source, data, STK-A, Computer Software, milli unified atomic mass unit, DL-Methionine, Proteins, Brk25D, igaad, alpha-amino acids, L-Isomer, DL Arginine Acetate, Peptide, Tool, LGMD2C, strain, polypeptide, Software Tool, Immune Tolerance, I-2PP2A, L-Isomer Arginine, Acetate, Dm I-2, Protein, I2PP2A, l(2)25Da, Hydrochloride, Bpa, Brk25D2, MFS1, Brk25D1, L Arginine, CG14026, Software, L-Lysine, Library, Data Base, ATCMPG1, MET, Met, WMS2, ATCMPG2, DMDA1, mDa, (2S)-2-amino-5-guanidinopentanoic acid, Engineering, Tripcellim, toxin, Amino, Recombinant, Computer Programs, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, Applications Softwares, Ion, XAP104, SSKS, SCARMD2, alpha-amino-gamma-methylmercaptobutyric acid, quotient, Peptid, l(2)04415, AA407739, 6-diaminohexanoic acid, Enisyl, Immunological Tolerance, Biosynthetic Proteinp27K, DmEpsilonTry, BamF, l(3)DTS7, artificial sequence, single-organism developmental process, AP-2, BamC, determination, AI036344, b2, alpha-Spe, DELTA AND GAMMA, bacterium E3, beta2tub, protein, betaTub2, betaTub1, alpha-Spc, CG31654, peptide, Polypeptides, protein polypeptide chains, l(3)04276, B1t, DSmurf, peptido, ClvPrd, IOTA, alpha-adaptin, Analysis, Enterococcus coli, DmelCG10422, protein aggregate, Toxins, SP88, alpha-spec, Mass Spectrum Analysis, B2t, kus, Escherichia/Shigella coli, CG4260, beta1tub, peptides, Analyses, CG9277, CG6798, T, beta-particle, E coli, proteins, alpha-spectrin, E. coli, Eschericia coli, Enteroaggregative Escherichia coli, 1t, reaction, e, NEUROD, e-, beta1Tub, beta(2)Tub, Smurf, CG18681, Clostridium welchii, CG4943, D-smurf, close to, 2t, BETA 56D, wide/broad, Hbb2, ham, Lack, Enteroaggregative E. coli, Spectrum Analysis, ada, Spectroscopy, alphaSpec, D-Ada, Prosbeta, l(1)G0108, Diffusely Adherent E. coli, cg4260, 3329, TUBB2C, FLOWERING TIME CONTROL PROTEIN FCA ALPHA, dAP-2a, spectrin, l(2)06694, MENE (2L)-A, bHLHa3, beta-tub, Diffusely Adherent Escherichia coli, nAcRbeta, Beta <eudicots>, Enteroinvasive E. coli, DmelCG4943, NeuroD1, Spectrometry, FCAALL.331, fmol, alpha, AP-2alpha, beta56D, DmelCG9277, DmelCG4260, beta1-tub, beta(-), DTB2, organ system, Hbbt2, DTB4, beta-Tub, wide, SYNTHETIC CONSTRUCT sequences, nAcr96Ac, DmelCG6798, 'Clostridium plagarum', i6, DL4180C, artificial, DTS7, Dts7, Polypeptide, beta-Tub56D, betaTub, Specbeta, Gruppe, beta1-Tubulin, beta 2, alpha-Ada, anon-EST:fe2E2, criteria, PROS27, l(3)alpha-Spec, Clostridium plagarum, Beta-cell E-box transcriptional activator 2, Peptidomics, beta[[2]], DmelCG18681, dSmurf1, epsilon, alpha-Spect, e(-), Gene, TUBB2, broad, Spectrum Analyses, electron, guidelines, protein-containing complex, body system, l(2)SH2 0460, fs(3)neo61, D.m.BETA-85D, beta-tub85D, CG10422, method, alpha-ada, polypeptide chain, B-spec, betaspec, method used in an experiment, Escherchia coli, klo, Gene Products, Mass, system, alpha-Sp, Enteroinvasive Escherichia coli, toxins, synthetic genetic interaction (sensu inequality), CG1977, Bacterium welchii, Mass Spectroscopy, MODY6, prosbeta2, DmelCG9359, anatomical systems, betatub(56D), DTS-7, Acr96Ac, beta2-tub, Elektron, beta[[1]] tubulin, dre3, beta1t, synthetic genetic interaction defined by inequality, Alpha-Adaptin, CG3329, anon-EST:fe1B3, d-smurf, Neurod, grupos, SPEC8, beta2t, peptidos, Prosbeta 2, CG5870, KINDS, beta(2)Tu, l(3)dre3, BETA2, Alkalescens-Dispar Group, beta-tubulin56D, Spec-beta, anon-EST:fe1G6, Dmbeta1, Dmbeta2, Bacterium coli, alpha-Spectrin, grupo, 3A9, protein complex, DmelCG1977, Proteins, beta[[2]]-tubulin, Tubulin, artificial gene, EAggEC, synthetic DNA, Try, i168, Dsmurf, Peptide, Bacterial Toxin, group, near to, development, BETA 85D, dSmurf, polypeptide, beta-Tub85D, Bacillus perfringens, BHF-1, native protein, natural protein, Bacillus coli, Smurf ubiquitin ligase, Protein, chemical analysis, synthetic, MENE(2L)-A, betaSpec, connected anatomical system, alphaSp, b-Spec, Mass Spectrum Analyses, nAcRs, anon-EST:fe1E6, Bacterial, Mass Spectrum, DmelCG5870, l(1)G0074, l(1)G0198, D-alphaAda, mating_type_alpha, negatron, AcrF, beta[[1]]-tubulin, beta2_dm, Beta-2, Rest, FCP-B, b spectrin, toxin, SBD, beta1, beta2, nAChR, synthetic constructs, beta, Protein Gene Products, plan specification, Tub, Dbeta2, Gene Proteins, AP2, Bacterium coli commune, DmelCG3329, l(3)62Bd, beta-spec, approaches, Bam-C, vicinity of, BAM, alpha mating type (yeast), Bam, BETA, Calibrations, l(2)SH0460, Toxin, alfa-Spec, Beta2, Peptid, sbd, CG9359, assay, ms(3)KK[D], groupe, Peptide., Spec, beta85Dtoxin, toxins., Bacillus perfringens, Clostridium welchii, Clostridium plagarum, Bacterium welchii, 'Clostridium plagarum'IPP2A2, p27K, BamF, l(3)DTS7, single-organism developmental process, AP-2, BamC, determination, AI036344, poly-histidine, b2, alpha-Spe, DELTA AND GAMMA, beta2tub, Basodexan, protein, betaTub2, betaTub1, fluid, alpha-Spc, CG31654, poly(L-histidine), Tp53, 5730420M11Rik, peptide, Polypeptides, Background, protein polypeptide chains, l(3)04276, B1t, peptido, ClvPrd, bbl, Cultural, B1, Urea, IOTA, alpha-adaptin, DmelCG10422, protein aggregate, present in fewer numbers in organism, alpha-spec, SET, ethnicity, B2t, Eubacteria, kus, peptides, CG4260, beta1tub, CG9277, TAF-I, E927b, phosphatase 2A inhibitor I2PP2A, hypoplasia, CG6798, T, beta-particle, proteins, alpha-spectrin, DmelCG4299, set, IGAAD, 1t, e, decreased, NEUROD, Bacteria <bacteria>, e-, DmelCG10574, beta1Tub, beta(2)Tub, Prokaryotae, D1, Strains, Procaryotae, phapii, 2t, BETA 56D, Hbb2, (D)-isomer, StF-IT-1, ham, ur, ada, carbamide, alphaSpec, Cultural Background, D-Ada, Prosbeta, l(1)G0108, Cultures, cg4260, 3329, TUBB2C, prokaryotes, FLOWERING TIME CONTROL PROTEIN FCA ALPHA, bfy, dAP-2a, spectrin, l(2)06694, MENE (2L)-A, bHLHa3, beta-tub, nAcRbeta, HLA-DR-associated protein II, Beta <eudicots>, DI-2, NeuroD1, I-2Dm, FCAALL.331, CG4299, alpha, inhibitor of granzyme A-activated DNase, AP-2alpha, beta56D, DmelCG9277, study protocol, beta Trypsin, DmelCG4260, beta1-tub, beta(-), I-2PP1, DTB2, Hbbt2, DTB4, beta-Tub, H2NC(O)NH2, nAcr96Ac, TAF-IBETA, DmelCG6798, Acetamide, i6, DL4180C, p44, bhy, TAF-Ibeta, DTS7, Dts7, Polypeptide, beta-Tub56D, i2pp2a, Strains and Sprains, 1728, Prokaryota, betaTub, Specbeta, beta1-Tubulin, beta 2, alpha-Ada, Beliefs, anon-EST:fe2E2, criteria, PROS27, l(3)alpha-Spec, Beta-cell E-box transcriptional activator 2, beta[[2]], p53, alpha-Spect, e(-), Gene, Carbamide, TUBB2, eubacteria, electron, guidelines, protein-containing complex, l(2)SH2 0460, PHAPII, fs(3)neo61, D.m.BETA-85D, beta-tub85D, Karbamid, CG10422, method, alpha-ada, template-activating factor I, reduced, polypeptide chain, B-spec, subnumerary, betaspec, method used in an experiment, Harnstoff, klo, body fluid, Gene Products, alpha-Sp, tiny, toxins, Carmol, CG1977, Sprains, MODY6, Backgrounds, prosbeta2, DmelCG9359, betatub(56D), DTS-7, Acr96Ac, beta2-tub, Elektron, ipp2a2, beta[[1]] tubulin, beta-Trypsin, dre3, beta1t, 2pp2a, Cultural Relativisms, decreased number, Alpha-Adaptin, Monera, CG10574, CG3329, anon-EST:fe1B3, 2PP2A, Neurod, taf-ibeta, SPEC8, dSET, dSet, Customs, beta2t, peptidos, Prosbeta 2, fungi, culture, CG5870, 2-iodo-, TCEP, PTHB1, beta(2)Tu, poly-(alpha-L-histidine), l(3)dre3, small, BETA2, n., beta-tubulin56D, Spec-beta, anon-EST:fe1G6, bacteria, Dmbeta1, Dmbeta2, adequate, alpha-Spectrin, 3A9, protein complex, DmelCG1977, Sprain, beta[[2]]-tubulin, Proteins, igaad, Tubulin, Cultural Backgrounds, i168, Peptide, Belief, polypeptide, development, BETA 85D, beta-Tub85D, BHF-1, native protein, natural protein, I-2PP2A, carbonyldiamide, C18, Dm I-2, Protein, chemical analysis, I2PP2A, Strain, MENE(2L)-A, betaSpec, alphaSp, b-Spec, nAcRs, polyhistidine, anon-EST:fe1E6, DmelCG5870, l(1)G0074, 10^[-9], l(1)G0198, D-alphaAda, mating_type_alpha, negatron, AcrF, underdeveloped, uree, beta[[1]]-tubulin, urea, beta2_dm, Beta-2, Tripcellim, FCP-B, b spectrin, toxin, SBD, beta1, beta2, nAChR, UREA, beta, plan specification, Tub, Dbeta2, Protein Gene Products, Trypure, Gene Proteins, dSET/TAF-Ibeta, AP2, 2610030F17Rik, DmelCG3329, l(3)62Bd, beta-spec, prokaryote, Bam-C, BAM, alpha mating type (yeast), Bam, BETA, l(2)SH0460, Relativisms, alfa-Spec, Peptid, Beta2, sbd, CG9359, assay, ms(3)KK[D], Relativism, AA407739, Cultural Relativism, Spec, beta85D, UltrasonicIPP2A2, human being, Clostridium plagarum, Procedures, Biocatalysts, eubacteria, protein, protein-containing complex, PHAPII, 5730420M11Rik, protein polypeptide chains, Techniques, method, polypeptide chain, template-activating factor I, Method, method used in an experiment, Studies, Impacts, Strain., mammals, Environmental Impacts, toxins, protein aggregate, Bacterium welchii, Sprains, Technique, study, SET, Eubacteria, enzymes, TAF-I, phosphatase 2A inhibitor I2PP2A, ipp2a2, 2pp2a, proteins, present in organism, procedures, man, Monera, CG10574, spore cell, DmelCG4299, Study, Environmental Impact, set, IGAAD, Enzyme, g, 2PP2A, Bacteria <bacteria>, Methodological Studies, DmelCG10574, taf-ibeta, Prokaryotae, dSET, dSet, toxic potential, fungi, Procaryotae, Strains, Clostridium welchii, KINDS, phapii, Spore, bacteria, protein complex, Mammal, Sprain, igaad, StF-IT-1, enzyme activity, Procedure, Impact, Environmental, Bacillus perfringens, native protein, natural protein, I-2PP2A, Protein, Dm I-2, I2PP2A, margin of safety, prokaryotes, techniques, HLA-DR-associated protein II, DI-2, I-2Dm, common, toxin, Methodological, CG4299, inhibitor of granzyme A-activated DNase, Methodological Study, human, plan specification, I-2PP1, dSET/TAF-Ibeta, 2610030F17Rik, TAF-IBETA, prokaryote, 'Clostridium plagarum', Biocatalyst, Environments, Mammalia, TAF-Ibeta, AA407739, i2pp2a, Strains and Sprains, Prokaryota, methodologytrueTargeted Mass Spectrometry Analysis of Clostridium perfringens ToxinsClostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium spread throughout the environment. This bacterium is a common agent in the gastrointestinal tracts of healthy human beings and other mammals. Simultaneously, this agent is one of the most significant producers of toxins among all known bacteria. This expressive toxicity is due to the bacterium’s ability collectively to produce different protein toxins and/or enzymes with diverse modes of action. The present study uses currently developed targeted proteomic methods for the simultaneous detection of selected C. perfringens protein toxins. The method was applied in different kinds of environmental matrices and was used to analyze toxins production in a set of collection strains.2019-03-252019-01-28PXD0125284454464152499175593117388043330432359839062887053271601603293581127583851187947135122511775058555714683536492158910306291593641388580332715910022638886915135588146479692828038365458333985801182590632957272563928672813954513408169139036345301355477142450732644355512744327146945597293751461575464784224220896431744712701390114319323074111481194669489693529372512646907275802402165993665812165952941281283296543128033952321818431832920435315257554787575930797253623114321381849222426191096976226155153111795745586265281736309431796989361604885664678253175136303555432046NCBITaxon:1313456586068860769938942795545351201488779592161294100264203676310761854312157457794144242324113NCBITaxon:17731023910117870974990799131122731701873674510116569156939833466330399258839100058987329940224911343053115655442193287263178723666987246426872754762422311151043055180066125522856213616299767982510216998231174673116761080772446896776744685960039376511822635735784588086363266689NCBITaxon:26970491031298382413681089319975961231975228319662776689605960615729513722117110125673712334351052311009040549986387833578840193733496151982157574878272151908985076480256318418985623919026454637463601134506253145943NCBITaxon:615797784081263380394526411459536123517861672272432307469112386940979541NCBITaxon:29544243277627912924970361251802848124264281504751029938865990346941734751541554036329979662877323962899548657558462823823522862871736231399463601068842043994796858355847954440426071016780781440772NCBITaxon:61918402393128118361003610502318557919080237499070381692259106592260704618326070533454226070788531870299103209285124966842528619212638541094343197192875NCBITaxon:1035928291268063956029598704483006418022235443224308469008575412983964271191364516996328104121697927262327262430085212389936436801097791356223347472697049583318030297603818583342409061147787127441415368163913076749402243264081721281612042546876138370812744239901191274420127442651953491137021316467506028532189517731193501846451450511492412153239564818822970915800257125614801549031384736031423166385962859631015104932275969373332648373153NCBITaxon:96152972210976771121114493760833341911079276283332237561266625555929248479749200360094340733524725151514389923747309095610000-0003-0197-8708