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 bio-markers 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 pro-teome modulations in sentinel species. This study evaluated the feasibility to propose dMRM tools for investigating senti-nel 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 environmen-tal 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 car-bohydrate 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 eco-systems. 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.

Automated summary

Mass spectrometry in multiple reaction monitoring (MRM) mode enables selective and multiplexed quantification of peptides derived from proteins. This study evaluates the feasibility of using dynamic MRM (dMRM) tools to investigate proteomes in sentinel species and demonstrates their potential for discovering new protein biomarkers. A dMRM assay was developed to capture the functional proteome of a freshwater crustacean and assess the effects of metal exposure. Metal-specific signatures were identified as potential biomarkers for tracking metal levels in freshwater ecosystems. This study highlights the potential of dMRM for identifying and developing biomarkers in sentinel species.