Shiga toxins, also known as verotoxins, are a major virulence factor associated with Shiga toxin producing Escherichia coli (STEC). STEC are the E. coli responsible for many of the serious foodborne outbreaks of disease. We have developed a sensitive and specific mass spectrometry-based method of detecting Shiga toxins. We describe a multiple reaction monitoring-based method of detecting and quantitating tryptic decapeptides derived from the non-toxic B subunits of Shiga toxins. We generated an artificial gene encoding a single protein that, upon digestion with trypsin, yields a set of relevant peptides. When this protein is labeled with 15N, these peptides can be used as internal standards to identify and quantify Shiga toxins. This approach can be used to quantify and distinguish among the known type 1 and type 2 Shiga toxins in the low attomole range in complex media. Human serum contains a protein human amyloid protein P (HuSAP) which binds to type 2 but not type 1 Shiga toxins. HuSAP confounds the detection of the most potent (type 2) Shiga toxins in human serum. Our approach can be used to readily detect both type 1 and type 2 Shiga toxins in human serum, where other methods would fail. Intact Shiga toxins are digested with trypsin prior to analysis, so the need to analyze intact toxins is eliminated by this approach. This analysis is rapid and can be accomplished within five hours.

Detecting and distinguishing among type 1 and type 2 Shiga toxins in human serum

253rd National Meeting (2017)

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