This project seeks to establish a workflow for targeted analysis of phosphoprotein-based signaling dynamics in fish cell lines (of zebrafish and rainbow trout origin) as well as in zebrafish embryos. The pipeline will include protein extraction and digestion followed by enrichment of phosphopeptides and mass spectrometric assays to quantify phosphorylated and dephosphorylated protein forms using heavy-labeled peptide counterparts. The established workflow will provide a valuable tool to study phosphorylation-based molecular signaling in fish, where this mechanistic aspect has been so far largely neglected due to challenges with obtaining species-specific antibodies for (de)phosphorylated proteins of interest.
Currently, we focus on the phosphorylation-dependent signaling within the mechanistic target of rapamycin (mTOR) pathway, selected because of its hypothesized involvement in mediation of chemical effects on growth in fish. Phosphoprotein dynamics upon intentional modulation of mTOR pathway (i.e., using specific pharmacological inhibitors or activators of this pathway), as well as in response to chemical exposures, will be measured and correlated to growth-related outcomes and other relevant physiological parameters in fish cells and embryos.