Putative serum protein biomarkers for epsilon toxin exposure in mouse model using LC-MS/MS analysis.

Epsilon toxin (ETX), produced by Clostridium perfringens Type B or type D strains, is a potential biological and toxin warfare (BTW) agent, largely for its very high toxicity. The toxin is implicated in several animal diseases. Using LC-MS/MS analysis, we report here elucidation of putative serum maker proteins for ETX exposure with an objective of the early diagnosis of intoxication. Of 166 consensus proteins (488 peptides), showing ETX-induced alterations, 119 proteins exhibited increase and 47 proteins showed decreased abundance in serum, as revealed by SWATH (DIA) acquisition on LC-MS/MS and label free quantitative analysis of control and test samples. Complement and coagulation cascade, nitrogen metabolism, negative regulation of peptidase activity, and response to ROS were among the biological processes and pathways perturbed by the ETX exposure. Interaction network indicated enzyme inhibitor activity, detoxification of ROS, and steroid binding functions were the major interaction networks for the proteins with increased abundance, while, hemostasis and structural molecule activity were the prominent networks for the down-regulated proteins. Validation studies were carried out by immunoprecipitation, ELISA, and Western blot analysis of selected proteins to demonstrate diagnostic potential of the putative marker proteins of ETX exposure.