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.