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Characterisation of cholera toxin by liquid chromatography - Electrospray mass spectrometry

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Author: Baar, B.L.M. van · Hulst, A.G. · Wils, E.R.J.
Type:article
Date:1999
Institution: TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA, Rijswijk, Netherlands
Source:Toxicon, 1, 37, 85-108
Identifier: 234876
doi: doi:10.1016/S0041-0101(98)00136-6
Keywords: Cholera toxin · Mercaptoethanol · Protein subunit · Amino acid composition · Amino acid sequence · Article · Controlled study · Liquid chromatography · Mass spectrometry · Molecular weight · Priority journal · Toxin analysis · mino Acid Sequence · Chromatography, High Pressure Liquid · Hydrolysis · Mass Spectrometry · Methylation · Molecular Sequence Data · Molecular Weight · Oxidation-Reduction · Peptide Fragments · Bacteria (microorganisms) · Vibrio cholerae

Abstract

Cholera toxin, one of the toxins that may be generated by various strains of the bacterium Vibrio cholerae, can be considered as a substance possibly used in biological warfare. The possibilities of characterising the toxin by liquid chromatography electrospray mass spectrometry (LC-ES-MS) were investigated. The toxin can be detected by flow-injection (FIA) ES-MS of a dialysed solution and observation of the charge envelope signals of its A-unit and B-chain protein; sufficient information for identification by the molecular mass of either protein could be obtained for quantities in the order of 10fmol. Confirmatory analysis was carried out by 2-mercaptoethanol reduction and FIA-ES-MS detection of the product proteins or by tryptic digest LC-ES-MS with ion chromatogram detection of most of the tryptic fragments of the A-unit and B-chain from the singly, doubly or triply charged ion signals. The confirmatory tryptic digest LC-ES-MS analysis could be achieved with quantities as low as 1pmol. Possible biovariations in the toxin can mostly be determined by sequencing, where the amino acid composition of tryptic fragments of the A1-chain, T5 and T15, and of the B-chain, T1, T4 and T5, cover all known biovariations. Partial sequencing of cholera toxin, originating from a classical strain, O1/569B, was achieved by LC-ES-MS/MS of most tryptic fragments larger than three amino acid residues. Copyright (C) 1998 Elsevier Science Ltd.