Organ-oriented proteogenomics functional atlas of three aquatic invertebrate sentinel species.

Proteogenomic methodologies have enabled the identification of protein sequences in wild species without annotated genomes, shedding light on molecular mechanisms affected by pollution. However, proteomic resources for sentinel species are limited, and organ-level investigations are necessary to expand our understanding of their molecular biology. This study presents proteomic resources obtained from proteogenomic analyses of key organs (hepatopancreas, gills, hemolymph) from three established aquatic sentinel invertebrate species of interest in ecotoxicological/ecological research and environmental monitoring: Gammarus fossarum, Dreissena polymorpha, and Palaemon serratus. Proteogenomic analyses identified thousands of proteins for each species, with over 90% of them being annotated to putative function. Functional analysis validated the relevance of the proteomic atlases by revealing similarities in functional annotation of catalogues of proteins across analogous organs in the three species, while deep contrasts between functional profiles are delimited across different organs in the same organism. These organ-level proteomic atlases are crucial for future research on these sentinel animals, aiding in the evaluation of aquatic environmental risks and providing a valuable resource for ecotoxicological studies.

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.

Development of a multi-omics extraction method for ecotoxicology: investigation of the reproductive cycle of Gammarus fossarum.

Omics study exemplified by proteomics, lipidomics or metabolomics, provides the opportunity to get insight of the molecular modifications occurring in living organisms in response to contaminants or in different physiological conditions. However, individual omics discloses only a single layer of information leading to a partial image of the biological complexity. Multiplication of samples preparation and processing can generate analytical variations resulting from several extractions and instrumental runs. To get all the -omics information at the proteins, metabolites and lipids level coming from a unique sample, a specific sample preparation must be optimized. In this study, we streamlined a biphasic extraction procedure based on a MTBE/Methanol mixture to provide the simultaneous extraction of polar (proteins, metabolites) and apolar compounds (lipids) for multi-omics analyses from a unique biological sample by a liquid chromatography (LC)/mass spectrometry (MS)/MS-based targeted approach. We applied the methodology for the study of female amphipod Gammarus fossarum during the reproductive cycle. Multivariate data analyses including Partial Least Squares Discriminant Analysis and multiple factor analysis were applied for the integration of the multi-omics data sets and highlighted molecular signatures, specific to the different stages.

Multiple reaction monitoring mass spectrometry for the discovery of environmentally modulated proteins in an aquatic invertebrate sentinel species, Gammarus fossarum.

Multiple reaction monitoring (MRM) mass spectrometry is emerging as a relevant tool for measuring customized molecular markers in freshwater sentinel species. While this technique is typically used for the validation of protein molecular markers preselected from shotgun experiments, recent gains of MRM multiplexing capacity offer new possibilities to conduct large-scale screening of animal proteomes. By combining the strength of active biomonitoring strategies and MRM technologies, this study aims to propose a new strategy for the discovery of candidate proteins that respond to environmental variability. For this purpose, 249 peptides derived from 147 proteins were monitored by MRM in 273 male gammarids caged in 56 environmental sites, representative of the diversity of French water bodies. A methodology is here proposed to identify a set of customized housekeeping peptides (HKPs) used to correct analytical batch effects and allow proper comparison of peptide levels in gammarids. A comparative analysis performed on HKPs-normalized data resulted in the identification of peptides highly modulated in the environment and derived from proteins likely involved in the environmental stress response. Overall, this study proposes a breakthrough approach to screen and identify potential proteins responding to relevant environmental conditions in sentinel species.