Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods-strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology.