title: Top-down proteomic identification of Shiga toxin 2 subtypes from Shiga toxin-producing Escherichia coli by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry.
abstract: We have analyzed 26 Shiga toxin-producing Escherichia coli (STEC) strains for Shiga toxin 2 (Stx2) production using matrix-assisted laser desorption ionization (MALDI)-tandem time of flight (TOF-TOF) tandem mass spectrometry (MS/MS) and top-down proteomic analysis. STEC strains were induced to overexpress Stx2 by overnight culturing on solid agar supplemented with either ciprofloxacin or mitomycin C. Harvested cells were lysed by bead beating, and unfractionated bacterial cell lysates were ionized by MALDI. The A2 fragment of the A subunit and the mature B subunit of Stx2 were analyzed by MS/MS. Sequence-specific fragment ions were used to identify amino acid subtypes of Stx2 using top-down proteomic analysis using software developed in-house at the U.S. Department of Agriculture (USDA). Stx2 subtypes (a, c, d, f, and g) were identified on the basis of the mass of the A2 fragment and the B subunit as well as from their sequence-specific fragment ions by MS/MS (postsource decay). Top-down proteomic identification was in agreement with DNA sequencing of the full Stx2 operon (stx2) for all strains. Top-down results were also compared to a bioassay using a Vero-d2EGFP cell line. Our results suggest that top-down proteomic identification is a rapid, highly specific technique for distinguishing Stx2 subtypes.
authors.affiliation: Produce Safety Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, California, USA.
authors.forename: Clifton K
authors.initials: CK
authors.lastname: Fagerquist
authors.forename: William J
authors.initials: WJ
authors.lastname: Zaragoza
authors.forename: Omar
authors.initials: O
authors.lastname: Sultan
authors.forename: Nathan
authors.initials: N
authors.lastname: Woo
authors.forename: Beatriz
authors.initials: B
authors.lastname: Quiñones
authors.forename: Michael B
authors.initials: MB
authors.lastname: Cooley
authors.forename: Robert E
authors.initials: RE
authors.lastname: Mandrell
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D040901:Proteomics / Q000379:methods*; D022641:Shiga Toxin 2 / Q000096:biosynthesis / Q000737:chemistry*; D054323:Shiga-Toxigenic Escherichia coli / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D022641:Shiga Toxin 2