Analysis of Vero Toxins 1 and 2 by High-performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry During the past 10 years, disease outbreaks of hemorrhagic colitis (HC) and hemolyticÈuremic syndrome (HUS) in humans have been increasing worldwide owing to the ingestion of beef and dairy products contaminated with Vero toxinproducing strains of Escherichia coli (VTEC), in particular serotype O157 : H7.1 In 1996, VTEC caused an extraordinarily huge-scale outbreak in Japan, involving more than 9000 patients ; 11 cases were accompanied by HUS and resulted in death.2 E. coli O157 : H7 and several other serotypes produce two cytotoxins to Vero cells, of which Vero toxin 1 (VT1, Shiga-like toxin 1) has been reported to be immunologically, physicochemically and biologically identical to Shiga toxin, whereas Vero toxin 2 (VT2, Shiga-like toxin 2) is immunologically distinct from VT1 and shares 56% amino acid identity with VT1.3 Vero toxins are subunit toxins comprising an A subunit, which inhibits intracellular protein synthesis via a speciÐc RNA N-glycosidase activity, and Ðve non-covalently associated receptor-binding B subunits, which facilitate the entry of the A subunit into susceptible cells.3
Current methods for the determination of Vero toxins produced by VTEC are classiÐed into two groups : one that Figure 1. Microcapillary HPLC/ESI-MS analysis of VT1 : (a) UV chromatogram monitored at 254 nm and selected ion chromatogram reconstructed from total ion scanning ; (b, c) ESI mass spectra of peaks 1 and 2 respectively ; (d, e) deconvoluted spectra of peaks 1 and 2 respectively. The values in (d) and (e) are the average molecular mass and standard deviation of a mass spectral plot for each multiply charged ion, calculated by using the BIOMASS deconvolution program installed in the Finnigan TSQ-7000 instrument.