Characterization of botulinum progenitor toxins by mass spectrometry

Abstract

Botulinum toxin analysis has renewed importance. This study included the use of nanochromatography-nanoelectrospray-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.

FIG. 1.

Representative base peak chromatogram of a trypsin digestion of botulinum serotype C1 progenitor toxin. The following nLC conditions were used: Aquasil C18 column, 10 cm by 75 μm; flow rate of 500 nl/min; injection volume of 2.5 μl; gradient of linear segments of 0 to 20% B in 40 min, 20 to 60% B in 40 min, and 60 to 100% B in 20 min; for solvent A, 2% acetonitrile in 0.1% (vol/vol) formic acid; for solvent B, 80% acetonitrile in 0.1% (vol/vol) formic acid.

FIG. 2.

Product ion spectrum of standard Glu-fibrinogen peptide (50 ng/ml) demonstrating that all ions necessary for identification were present for this peptide. The amino acid sequence and detected product ions are depicted in this figure also. The nLC conditions were the same as those described in the legend to Fig. 1.