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

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Author: Baar, B.L.M. van · Hulst, A.G. · Jong, A.L. de · Wils, E.R.J.
Institution: Prins Maurits Laboratorium TNO
Source:Journal of Chromatography A, 1, 1035, 97-114
Identifier: 237719
doi: doi:10.1016/j.chroma.2004.02.047
Keywords: Amino acid sequencing · Botulinum toxins · Mass spectrometry · Peptide mapping · Toxins · Genes · Ionization · Laser applications · Liquid chromatography · Mass spectrometry · Proteins · Botulinum toxins · Gene sequences · Bacteria · Botulinum toxin · Botulinum toxin c · Botulinum toxin d · Botulinum toxin E · Botulinum toxin f · Hemagglutinin · Neurotoxin · Trypsin · Unclassified drug · Amino acid sequence · Analytic method · Article · Clostridium botulinum · Controlled study · Electrospray mass spectrometry · Gene sequence · Hemagglutination · Herring · Liquid chromatography · Matrix assisted laser desorption ionization time of flight mass spectrometry · Nonhuman · Priority journal · Tandem mass spectrometry · Amino Acid Sequence · Botulinum Toxins · Molecular Sequence Data · Peptide Mapping · Spectrometry, Mass, Electrospray Ionization · Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization · Bacteria (microorganisms) · Clostridium · Clostridium botulinum · Clupea harengus · Clupeidae


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 33kDa 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. © 2004 Elsevier B.V. All rights reserved. Chemicals / CAS: hemagglutinin, 37333-12-3; neurotoxin, 39386-17-9; trypsin, 9002-07-7; Botulinum Toxins