Shiga-toxin-producing Escherichia coli (STEC) are a burden on agriculture and a threat to public health. Rapid methods are needed to identify STEC strains and characterize the Shiga toxin (Stx) they produce. We analyzed three STEC strains for Stx expression, using antibiotic induction, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry, and top-down proteomic analysis. E. coli O157:H- strain 493/89 is a clinical isolate linked to an outbreak of hemolytic uremic syndrome (HUS) in Germany in the late 1980s. E. coli O145:H28 strains RM12367-C1 and RM14496-C1 were isolated from an agricultural region in California. The stx operon of the two environmental strains were determined by whole genome sequencing (WGS). STEC strain 493/89 expressed Shiga toxin 2a (Stx2a) as identified by tandem mass spectrometry (MS/MS) of its B-subunit that allowed identification of the type and subtype of the toxin. RM12367-C1 also expressed Stx2a as identified by its B-subunit. RM14496-C1 expressed Shiga toxin 1a (Stx1a) as identified from its B-subunit. The B-subunits of Stx1 and Stx2 both have an intramolecular disulfide bond. MS/MS was obtained on both the disulfide-bond-intact and disulfide-bond-reduced B-subunit, with the latter being used for top-down proteomic identification. Top-down proteomic analysis was consistent with WGS.
Nano-LC FTICR Tandem Mass Spectrometry for Top-Down Proteomics: Routine Baseline Unit Mass Resolution of Whole Cell Lysate Proteins up to 72 kDa