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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #360413
Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Typing/subtyping Shiga toxin from pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis

Author
item Fagerquist, Clifton - Keith
item Zaragoza, William
item Carter, Michelle

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/7/2019
Publication Date: 6/6/2019
Citation: Fagerquist, C.K., Zaragoza, W.J., Carter, M.Q. 2019. Typing/subtyping Shiga toxin from pathogenic Escherichia coli using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis [abstract]. 67th ASMS Conference on Mass Spectrometry and Allied Topics, June 2-6, 2019, Atlanta, GA.

Interpretive Summary:

Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) constitute a continuing threat to public health and agriculture. Rapid and sensitive methods are needed to detect and identify the various types and subtypes of Shiga toxin (Stx) that are generated by these pathogenic bacteria. Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) has the capability to detect and identify types and subtypes of Stx from STEC strains. We have analyzed two non-motile STEC strains for Stx production using antibiotic induction, MALDI-TOF-TOF-MS/MS with post-source decay (PSD) and top-down proteomic analysis. The two STEC strains were isolated from an agricultural region in California. One STEC strain produced Shiga toxin 1a (Stx1a) and the other produced Shiga toxin 2a (Stx2a). Stx types/subtypes were identified by MS/MS-PSD of the disulfide bond-reduced B-subunit of the AB5 holotoxin and top-down proteomic analysis. The most prominent fragment ions result from polypeptide backbone cleavage on the C-terminal side of aspartic acid (D) residues (i.e. aspartic acid effect) with less abundant fragment ions from cleavage on the C-terminal side of glutamic acid (E) residues. On the basis of the B-subunit alone, Stx was typed and subtyped from these strains. The disulfide bond-intact B-subunits were also analyzed by MS/MS-PSD which confirmed the presence of an intramolecular disulfide bond and its lasso-loop secondary structure. The intramolecular disulfide bond was detected due to polypeptide backbone cleavage between the two cysteine residues of the disulfide bond and symmetric and asymmetric cleavage of the disulfide bond (± 33 Da) resulting in characteristic fragment ion triplets which are absent from MS/MS of the disulfide bond-reduced B-subunit. Top-down proteomic analysis was consistent with whole genome sequencing for these STEC strains.