Dynamic Multiple Reaction Monitoring of Amphipod Gammarus Fossarum Caeca Expands Molecular Information for Understanding the Impact of Contaminants

Mass spectrometry in multiple reaction monitoring (MRM) mode is a powerful technique that can provide highly selective, multiplexed, and reproducible quantification of peptides derived from proteins. Ideal for the application of molecular biomarkers in biomonitoring surveys, MRM tools have been recently developed to quantify sets of pre-selected biomarkers in freshwater sentinel species. Still limited to the validation and application phase of biomarkers, dynamic MRM (dMRM) acquisition mode has increased the multiplexing capacity of mass spectrometers, expanding opportunities to explore proteome modulations in sentinel species. This study evaluated the feasibility to propose dMRM tools for investigating sentinel species proteomes at the organ level and demonstrated its potential for screening contaminant effects and discovering new protein biomarkers. As a proof of concept, a dMRM assay was developed to comprehensively capture the functional proteome of the caeca of Gammarus fossarum, a freshwater crustacean, commonly used as a sentinel species in environmental biomonitoring. The assay was then used to assess the effects of sub-lethal concentrations of cadmium, silver, and zinc on gammarid caeca. Results showed dose-response and specific metal effects on caecal proteomes, with a slight effect of zinc compared to the two non-essential metals. Functional analyses indicated that cadmium affected proteins involved in carbohydrate metabolism, digestive and immune processes, while silver affected proteins related to oxidative stress response, chaperonin complexes and fatty acid metabolism. Based on these metal-specific signatures, several proteins modulated in a dose-dependent manner were proposed as candidate biomarkers for tracking the level of these metals in freshwater ecosystems. Overall, this study highlights the potential of dMRM to decipher the specific modulations of proteome expression induced by contaminant exposure and pinpoints specific response signatures, offering new perspectives for the de novo identification and development of biomarkers in sentinel species.