Ecotoxico-Proteomics for Aquatic Environmental Monitoring: First in Situ Application of a New Proteomics-Based Multibiomarker Assay Using Caged Amphipods.

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 Na+K+/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.

Multiplexed assay for protein quantitation in the invertebrate Gammarus fossarum by liquid chromatography coupled to tandem mass spectrometry.

A highly multiplexed liquid chromatography mass spectrometry-selected reaction monitoring (SRM)-based assay for determination of 40 potential protein biomarkers from Gammarus fossarum, an ecotoxicological relevant species, was described. The assay relies on 71 stable isotope-labeled reported peptide standards for the quantitation of proteins of interest in relation to essential physiological functions such as reproductive cycle, defense mechanism, and enzymes involved in homeostasis process and in energy. A direct linear relationship between the spiked peptide concentration and the area under the peak was clearly demonstrated in biological extracts. Precision and accuracy were determined to be between 1.1 and 21% and between 79 and 120%, respectively, depending on the selected protein in a few samples after optimization of digestion conditions. The validity of the assay was documented for several biomarkers linked with reproduction and the molting process was performed with the assessment of protein levels throughout contrasted physiological process (sex, reproductive status). This assay is easy to use, robust, sensitive, and has high-throughput capabilities. The proposed strategy may be extended to any non-model organisms relevant in environmental science. Graphical abstract ᅟ.