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

Toxins

Christopher J. Silva¹  Melissa L. Erickson-Beltran²  Craig B. Skinner²  Stephanie A. Patfield²  Xiaohua He² 

[1] Western Regional Research Center, United States Department of Agriculture, Albany, CA 94710, USA

关键词: Keywords: mass spectrometry;    Shiga toxins;    verotoxins;    multiple reaction monitoring method (MRM);    STEC;    serum;    shigatoxigenic and enterohemorrhagic strains of Escherichia coli;    EHEC;    Enterobacter;   

DOI  :  10.3390/toxins7124875

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.