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

Authors :
Christopher J. Silva
Melissa L. Erickson-Beltran
Craig B. Skinner
Stephanie A. Patfield
Xiaohua He
Source :
Toxins, Vol 7, Iss 12, Pp 5236-5253 (2015)
Publication Year :
2015
Publisher :
MDPI AG, 2015.

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.

Details

Language :
English
ISSN :
20726651 and 21726612
Volume :
7
Issue :
12
Database :
Directory of Open Access Journals
Journal :
Toxins
Publication Type :
Academic Journal
Accession number :
edsdoj.5713fedb3f4d3bbb21726612a0d58d
Document Type :
article
Full Text :
https://doi.org/10.3390/toxins7124875