Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #295092

Title: Top-down proteomic identification of Shiga toxin 2 variants from Shiga toxin-producing Escherichia coli (STEC) using MALDI-TOF-TOF-MS/MS-PSD

Author
item Fagerquist, Clifton - Keith
item Zaragoza, William
item Sultan, Omar
item Woo, Nathan
item Quiñones, Beatriz
item Swimley, Michelle
item Cooley, Michael - Mike
item Mandrell, Robert

Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: 6/6/2013
Publication Date: 8/1/2013
Citation: Fagerquist, C.K., Zaragoza, W.J., Sultan, O., Woo, N., Quinones, B., Swimley, M.S., Cooley, M.B., Mandrell, R.E. 2013. Top-down proteomic identification of Shiga toxin 2 variants from Shiga toxin-producing Escherichia coli (STEC) using MALDI-TOF-TOF-MS/MS-PSD. ASMS20139833.3125VER.1.pdf.

Interpretive Summary:

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) are increasingly linked to severe outbreaks of foodborne illness throughout the world, e.g. Germany and France in 2011. STEC infections can result in bloody diarrhea, hemolytic uremic syndrome, kidney failure and death. New analytical techniques are needed for rapid characterization of these pathogens and their virulence factors. Sequence-specific variants of Shiga toxin 2 (Stx2: a, b, c, d, e, f, g) have been linked to differences in STEC virulence. We have recently developed a technique that combines antibiotic-induced over-expression of Stx2 with matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry post-source decay (MALDI-TOF-TOF-MS/MS-PSD) and top-down proteomic analysis. We have applied top-down proteomic analysis and DNA gene sequencing to identify Stx2 variants in 63 STEC strains. Sequence-specific variants (a-g) of Stx2 were identified by top-down in many of the STEC strains analyzed. Serotypes included: O157:H7, O104:H4 (2011 outbreak strain), O157:H-, O91:H21, O139:NM, O177:H25, O111:H8, O113:H4 and O104:H21. Variants were identified from the mass-to-charge (m/z) of the singly charged B-subunit (7.8 kDa) and the A2 fragment (5.3 kDa) of the A-subunit in MS mode as well as from sequence-specific fragment ions generated by MS/MS-PSD. Most of the sequence-specific differences across Stx2 variants reside in the B-subunit and A2. The mature B-subunit (signal peptide removed) has one intramolecular disulfide bond, and the B-subunit was analyzed both with the disulfide bond-intact and disulfide bond-reduced. A more significant top-down identification was obtained for the reduced B-subunit as a result of the increased fragmentation efficiency of the polypeptide backbone due to its linearized structure. MS/MS-PSD of the disulfide bond-intact B-subunit (with its large looped structure) resulted in a much more complex spectrum that displayed fragment ion triplets due to cleavage of the polypeptide backbone between the two cysteines involved in an intramolecular disulfide bond and symmetric and asymmetric cleavage of its disulfide bond. The fragment ion triplets provided confirmatory evidence of its disulfide bond and its looped secondary structure. Top-down proteomic results were confirmed by DNA sequencing of the full Stx2 operon and compared to results from a toxicity assay utilizing a Vero-d2EGFP cell line.