Understanding how fish respond to environmental change requires not only measuring physiological and behavioural responses, but also identifying the cellular processes that underpin them. Proteomics, the identification and quantification of peptides and proteins that determine cellular structure and function, is a powerful tool for investigating these underlying physiological mechanisms. Recent advances in sample preparation, liquid chromatography mass spectrometry (LC-MS), and library-free data-independent acquisition (DIA) have made proteomics increasingly accessible beyond specialist laboratories.
This presentation provides a practical introduction to proteomics, covering what it measures, why protein identity and abundance provide insight into physiological processes, and the workflow from tissue collection through peptide extraction, LC-MS, protein identification, and differential abundance analysis. We outline our pipeline based on PreOmics sample preparation, Bruker timsTOF HT LC-MS, and Spectronaut library-free DirectDIA analysis.
The utility of this approach is demonstrated through case studies spanning diverse reef fishes. Laboratory experiments with the anemone-associated clownfish (Amphiprion ocellaris) and the mesopredatory Malabar grouper (Epinephelus malabaricus) identified physiological responses to simulated marine heatwaves and sedimentation, including shifts in metabolic pathways and cellular stress responses. As genomic and transcriptomic resources become increasingly available, species-specific protein databases can be generated from annotated genomes or assembled transcriptomes, enabling library-free quantitative proteomics in non-model fishes. We further demonstrate this approach using newly annotated genomes for the Indo-Pacific damselfish (Abudefduf vaigiensis) and the stripey (Microcanthus strigatus), illustrating how emerging sequence resources can be readily integrated into quantitative proteomic workflows.
By combining accessible laboratory workflows with high-throughput LC-MS and library-free analysis, modern proteomics provides a practical and scalable approach for studying fish physiology across an increasingly diverse range of model and non-model species, offering researchers new opportunities to uncover the cellular mechanisms underlying responses to rapidly changing environments.