Phosphoproteome profiling of HEK 293 cells under osmotic stress using SILAC

High NaCl causes numerous damaging and protective cellular responses, the signaling pathways not always being clear. We used Stable Isotope Labeling by Amino acids in Cell culture (SILAC) coupled to mass spectrometry to identify phosphorylation based signaling pathways in HEK293 cells. We identified more than 13,000 phosphopeptides in three biological replicate samples with 1% FDR. Of ~6,000 unique phosphopeptides quantified, 80% have a single phosphate group and 20% two or more phosphate groups. High NaCl significantly changes the abundance of ~300 phosphopeptides. The proteins containing them are enriched in the following functional categories: cellular response to stress, cytoskeleton organization, cell cycle, regulation of protein complex disassembly, and nucleocytoplasmic transport. In particular, high NaCl increases phosphorylation of p38 alpha (Y182) and c‐Jun (S63), consistent with previous reports. We also found novel increased phosphorylation of c‐Jun (S73), UBA1 (S46), and CDK13 (S437, S439) and decreased phosphorylation of G3BP1 (S232), GPS1 (T479), and STK10 (S438). Systematic profiling of phosphorylation changes in response to osmotic stress advances understanding of the hypertonic cellular stress response and identifies previously uncharacterized signaling pathways. Supported by DIR NHLBI.