Characterization of Tetanus Toxin, Neat and in Culture Supernatant, by Electrospray Mass Spectrometry

Baar, Ben L. M. van; Hulst, A.G.; Roberts, Beverley; Wils, E.R.J.

doi:10.1006/abio.2001.5496

Cited by 19 publications

(17 citation statements)

References 30 publications

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“…Using nLC-nESI in our study represented a refinement of the capillary LC and micro-ESI techniques employed by van Baar et al (28)(29)(30). Because sample requirements are lower for nLC-nESI, total protein quantities used in this study were approximately 40% lower than quantities used by van Baar et al (29,30).…”

Section: Discussionmentioning

confidence: 89%

“…In recent reports, van Baar et al (29,30) addressed some of these issues when they characterized neurotoxin samples from serotypes A (strain 62A), B (strain Okra), C (003-9), D (CB-16), E (no designation; equivalent to NCTC 11219 by analysis), and F (Langeland) that also contained some progenitor toxin proteins. They analyzed these samples with matrix-assisted laser desorption ionization (MALDI)-MS and capillary LC-ESI-MS methods originally developed for tetanus toxin analysis (28). They showed that accurate strain assignments were possible when genetic sequences were available.…”

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confidence: 99%

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Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Hines

Lebeda

Hale

et al. 2005

Appl Environ Microbiol

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Botulinum toxin analysis has renewed importance. This study included the use of nanochromatographynanoelectrospray-mass spectrometry/mass spectrometry to characterize the protein composition of botulinum progenitor toxins and to assign botulinum progenitor toxins to their proper serotype and strain by using currently available sequence information. Clostridium botulinum progenitor toxins from strains Hall, Okra, Stockholm, MDPH, Alaska, and Langeland and 89 representing serotypes A through G, respectively, were reduced, alkylated, digested with trypsin, and identified by matching the processed product ion spectra of the tryptic peptides to proteins in accessible databases. All proteins known to be present in progenitor toxins from each serotype were identified. Additional proteins, including flagellins, ORF-X1, and neurotoxin binding protein, not previously reported to be associated with progenitor toxins, were present also in samples from several serotypes. Protein identification was used to assign toxins to a serotype and strain. Serotype assignments were accurate, and strain assignments were best when either sufficient nucleotide or amino acid sequence data were available. Minor difficulties were encountered using neurotoxin-associated protein identification for assigning serotype and strain. This study found that combined nanoscale chromatographic and mass spectrometric techniques can characterize C. botulinum progenitor toxin protein composition and that serotype/strain assignments based upon these proteins can provide accurate serotype and, in most instances, strain assignments using currently available information. Assignment accuracy will continue to improve as more nucleotide/amino acid sequence information becomes available for different botulinum strains.

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Section: Discussionmentioning

confidence: 89%

mentioning

confidence: 99%

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Hines

Lebeda

Hale

et al. 2005

Appl Environ Microbiol

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“…The sample treatment procedure previously developed with tetanus toxin [7] was used, employing a common autopipette with disposable tips. This procedure allowed handling of any intact toxin inside adequate containment and subsequent mass spectrometry outside that containment.…”

Section: Sample Preparation For Mass Spectrometric Analysismentioning

confidence: 99%

Characterisation of botulinum toxins type C, D, E, and F by matrix-assisted laser desorption ionisation and electrospray mass spectrometry

Baar¹,

Hulst²,

Jong³

et al. 2004

Journal of Chromatography A

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In a follow-up of the earlier characterisation of botulinum toxins type A and B (BTxA and BTxB) by mass spectrometry (MS), types C, D, E, and F (BTxC, BTxD, BTxE, BTxF) were now investigated. Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent. Biologically active BTxC, BTxD, BTxE, and BTxF are comprised of a protein complex of the respective neurotoxins with non-toxic non-haemagglutinin (NTNH) and, sometimes, specific haemagglutinins (HA). These protein complexes were observed in mass spectrometric identification. The BTxC complex, from Clostridium botulinum strain 003-9, consisted of a 'type C1 and D mosaic' toxin similar to that of type C strain 6813, a non-toxic non-hemagglutinating and a 33 kDa hemagglutinating (HA-33) component similar to those of strain C-Stockholm, and an exoenzyme C3 of which the sequence was in full agreement with the known genetic sequence of strain 003-9. The BTxD complex, from C. botulinum strain CB-16, consisted of a neurotoxin with the observed sequence identical with that of type D strain BVD/-3 and of an NTNH with the observed sequence identical with that of type C strain C-Yoichi. Remarkably, the observed protein sequence of CB-16 NTNH differed by one amino acid from the known gene sequence: L859 instead of F859. The BTxE complex, from a C. botulinum isolated from herring sprats, consisted of the neurotoxin with an observed sequence identical with that from strain NCTC 11219 and an NTNH similar to that from type E strain Mashike (1 amino acid difference with observed sequence). BTxF, from C. botulinum strain Langeland (NCTC 10281), consisted of the neurotoxin and an NTNH; observed sequences from both proteins were in agreement with the gene sequence known from strain Langeland. As with BTxA and BTxB, matrix-assisted laser desorption/ionisation (MALDI) MS provided provisional identification from trypsin digest peptide maps and liquid chromatography-electrospray (tandem) mass spectrometry (LC-ES MS) afforded unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin digestion.

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“…One is based on the highly specific enzymatic activity of some protein toxins. In this case, instead of detection of the toxin (i.e., botulinum or tetanus), the products resulting from hydrolysis can be analyzed (3,19,40). On the other hand, nonenzymatic toxins, like staphylococcal enterotoxins, could be successfully detected after selective enrichment of the target toxin in the sample.…”

Section: Discussionmentioning

confidence: 99%

“…Over the last 10 years, both matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and electrospray ionization MS have played more important roles in both the detection and structural characterization of bacterial protein toxins (36), such as botulinum (3,7,17), diphtheria (33), tetanus (40), cholera (9,36), and Shiga-like (42) toxins, as well as staphylococcal enterotoxins (9,20,26). MS-based identification of bacterial protein toxins relies on (i) proteomic approaches (5,(38)(39)(40), (ii) indirect measurement of target peptides produced as a result of highly specific enzymatic (i.e., endoprotease) activity of the toxin itself (3,7), and (iii) direct determination of the molecular mass of the toxin after chromatographic or affinity separation (8,20,26,42). MS-based proteomic approaches are used mainly in comparative analyses to obtain an overall picture of the secreted virulence factors, including bacterial protein toxins, in biological samples, such as the exoprotein fraction of culture supernatants (5,25,43).…”

mentioning

confidence: 99%

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B

Schlosser

Kačer

Kuzma

et al. 2007

Appl Environ Microbiol

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The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of paratoluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.Protein toxins secreted by bacteria are the most powerful human poisons known (15, 31). Over the last 10 years, both matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and electrospray ionization MS have played more important roles in both the detection and structural characterization of bacterial protein toxins (36), such as botulinum (3,7,17), diphtheria (33), tetanus (40), cholera (9, 36), and Shiga-like (42) toxins, as well as staphylococcal enterotoxins (9,20,26). MS-based identification of bacterial protein toxins relies on (i) proteomic approaches (5, 38-40), (ii) indirect measurement of target peptides produced as a result of highly specific enzymatic (i.e., endoprotease) activity of the toxin itself (3, 7), and (iii) direct determination of the molecular mass of the toxin after chromatographic or affinity separation (8,20,26,42). MS-based proteomic approaches are used mainly in comparative analyses to obtain an overall picture of the secreted virulence factors, including bacterial protein toxins, in biological samples, such as the exoprotein fraction of culture supernatants (5,25,43). One drawback of MS-based proteomic methods for the detection and identification of bacterial protein toxins, however, is the need to perform separation steps before analysis, which limits application of these methods for rapid screening. Also, the dynamic ranges and sensitivities of proteomic techniques are not able to reach the toxin detection limit at the intoxicat...

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Characterization of Tetanus Toxin, Neat and in Culture Supernatant, by Electrospray Mass Spectrometry

Cited by 19 publications

References 30 publications

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Characterisation of botulinum toxins type C, D, E, and F by matrix-assisted laser desorption ionisation and electrospray mass spectrometry

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B

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