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Open AccessArticle

Top-Down Proteomic Identification of Shiga Toxin 1 and 2 from Pathogenic Escherichia coli Using MALDI-TOF-TOF Tandem Mass Spectrometry

Produce Safety & Microbiology, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA
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Microorganisms 2019, 7(11), 488; https://doi.org/10.3390/microorganisms7110488
Received: 8 September 2019 / Revised: 17 October 2019 / Accepted: 23 October 2019 / Published: 25 October 2019
(This article belongs to the Special Issue Shiga Toxin-Producing Escherichia coli)
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. View Full-Text
Keywords: Shiga-toxin-producing Escherichia coli; STEC; MALDI-TOF-TOF; mass spectrometry; tandem mass spectrometry; MS/MS; antibiotic induction; aspartic acid effect; disulfide bond; top-down proteomics Shiga-toxin-producing Escherichia coli; STEC; MALDI-TOF-TOF; mass spectrometry; tandem mass spectrometry; MS/MS; antibiotic induction; aspartic acid effect; disulfide bond; top-down proteomics
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Fagerquist, C.K.; Zaragoza, W.J.; Carter, M.Q. Top-Down Proteomic Identification of Shiga Toxin 1 and 2 from Pathogenic Escherichia coli Using MALDI-TOF-TOF Tandem Mass Spectrometry. Microorganisms 2019, 7, 488.

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