As a proof of principle, a selected reaction monitoring (SRM) mass spectrometry-based methodology was applied to the simultaneous quantification of dozens of protein biomarkers in caged amphipods (Gammarus fossarum). We evaluated the suitability of the methodology to assess complex field contaminations through its application in the framework of a regional river monitoring network. Thanks to the high throughput acquisition of biomarker levels in G. fossarum exposed in four reference and 13 contaminated sites, we analyzed the individual responses of 38 peptides reporting for 25 proteins of interest in 170 organisms. Responses obtained in contaminated sites included inductions of vitellogenin-like proteins in male organisms, inductions of NaK/ATPases, and strong inhibitions of molt-related proteins such as chitinase and JHE-carboxylesterase. Proteins from detoxification and immunity processes were also found modulated in abundance. Summarizing, the results presented here show that the SRM strategy developed for multibiomarker measurement paves a very promising way to define multiple indicators of the health status of sentinel organisms for environmental hazard assessment.

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