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Toxins 2015, 7(12), 5236-5253; doi:10.3390/toxins7124875

Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum

Western Regional Research Center, United States Department of Agriculture, Albany, CA 94710, USA
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Academic Editor: Florian Lang
Received: 29 September 2015 / Revised: 28 October 2015 / Accepted: 9 November 2015 / Published: 2 December 2015
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Abstract

Shiga-like toxins (verotoxins) are responsible for the virulence associated with a variety of foodborne bacterial pathogens. Direct detection of toxins requires a specific and sensitive technique. In this study, we describe a mass spectrometry-based method of analyzing the tryptic decapeptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. The 15N-labeled protein was prepared by growing the expressing bacteria in minimal medium supplemented with 15NH4Cl. Trypsin digestion of the 15N-labeled protein yields a set of 15N-labeled peptides for use as internal standards to identify and quantify Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx) and Shiga-like toxins (Stx1 and Stx2) in the low attomole range (per injection) in complex media, including human serum. Furthermore, Stx1a could be detected and distinguished from the newly identified Stx1e in complex media. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within 5 h. View Full-Text
Keywords: Keywords: mass spectrometry; Shiga toxins; verotoxins; multiple reaction monitoring method (MRM); STEC; serum; shigatoxigenic and enterohemorrhagic strains of Escherichia coli; EHEC; Enterobacter Keywords: mass spectrometry; Shiga toxins; verotoxins; multiple reaction monitoring method (MRM); STEC; serum; shigatoxigenic and enterohemorrhagic strains of Escherichia coli; EHEC; Enterobacter
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Silva, C.J.; Erickson-Beltran, M.L.; Skinner, C.B.; Patfield, S.A.; He, X. Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum. Toxins 2015, 7, 5236-5253.

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