Development and Application of a Targeted Phosphoproteomics Method for Analysing the mTOR Pathway Dynamics in Zebrafish PAC2 Cell Line

The mechanistic target of rapamycin (mTOR) signalling pathway plays a crucial role in regulating cellular growth and proliferation. While extensively studied in mammals, the phosphorylation dynamics of this pathway in non-mammalian model organisms remain largely unexplored, often due to the scarcity of suitable antibodies to measure (phosphorylated) proteins of interest. To address this gap, we developed an antibody-independent targeted phosphoproteomics method applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the abundance and phosphorylation levels of mTOR pathway-related proteins in zebrafish (Danio rerio), using the permanent cell line PAC2 as a model system. With optimized sample processing and data analysis strategies, we could successfully quantify 10 endogenous phosphosites and 15 endogenous proteins at different cell culture growth phases, revealing complex phosphorylation dynamics for both the upstream regulators (e.g., AKT, AMPK) and downstream effectors (e.g., eIF4EBP1, RPS6) of the mTOR pathway, which reflected transition from exponential growth to stationary subsistence. Our findings confirm the overall similarity of the mTOR pathway structure and functionality between zebrafish and mammals. Furthermore, this work demonstrates the high potential of the LC-MS/MS-based analytical approaches for studying phosphorylation-governed signalling in diverse organisms of interest, thus paving the way for further investigations in comparative physiology and toxicology across species.