Characterization of tetanus toxin, neat and in culture supernatant, by electrospray mass spectrometry

A method was developed for the liquid chromatographic-mass spectrometric (LC-MS) identification of extremely neurotoxic toxins. The method combines sample treatment in a safety containment and analysis of detoxified material in a common laboratory facility. The method was applied to the characterization of neat tetanus toxin and subsequent identification of the toxin in cell lysate supernatants and culture supernatants from different Clostridium tetani bacteria strains. Characterization of the neat toxin was accomplished by (1) accurate mass measurement of enzyme digest fragments of the toxin and (2) tandem mass spectrometric (MS/MS) amino acid sequencing of selected peptides. Accurate mass measurement proved no longer feasible for the analysis of supernatants, due to the overwhelming presence of peptides from proteins other than toxin. Even when high-molecular-weight proteins were filtered from the lysates and treated, the retained protein fraction yielded too many peptides. However, MS/MS could successfully be applied when the findings from the characterization of neat toxin were employed. Thus, LC-MS/MS of selected precursor ions from trypsin digest fragments yielded specific sequence data for identification of the toxin. This procedure provided reliable identification of the toxin at levels above 1 microg/ml and within a day. Investigations with the method developed will be extended to the botulinum neurotoxins.