Assessing the relevance of a multiplexed methodology for proteomic biomarker measurement in the invertebrate species Gammarus fossarum: A physiological and ecotoxicological study

Abstract Recently, a protein sequence database was built specifically for the sentinel non-model species Gammarus fossarum using a proteogenomics approach. A quantitative multiplexed targeted proteomics assay (using Selected Reaction Monitoring mass spectrometry) was then developed for a fast and simultaneous quantification of dozens of biomarker peptides specific of this freshwater sentinel crustacean species. In order to assess the relevance of this breakthrough methodology in ecotoxicology, the response patterns of a panel of 26 peptides reporting for 20 proteins from the Gammarus fossarum proteome with putative key functional roles (homeostasis, osmoregulation, nutrition, reproduction, molting,…) were recorded through male and female reproductive cycles and after exposure to environmental concentrations of cadmium and lead in laboratory-controlled conditions. Based on these results, we validated the implication of annotated vtg-like peptides in the oogenesis process, and the implication of Na + /K + ATPase proteins in the molt cycle of organisms. Upon metal (cadmium and lead) contamination, peptides belonging to proteins annotated as involved in antioxidant and detoxification functions, immunity and molting were significantly down-regulated. Overall, this multiplex assay allowed gaining relevant insights upon disruption of different main functions in the sentinel species Gammarus fossarum . This breakthrough methodology in ecotoxicology offers a valid and high throughput alternative to currently used protocols, paving the way for future practical applications of proteogenomics-derived protein biomarkers in chemical risk assessment and environmental monitoring.