The proteomics and phosphoproteomics landscape of melanoma under T cell attack.

Understanding how tumor cells interact with tumor-infiltrating lymphocytes (TILs) is crucial for improving immunotherapy, yet protein-level changes remain largely unexplored. To address this, we profile the early responses of patient-derived melanoma cells co-cultured with matched autologous TILs. To distinguish tumor from TIL proteomes without physical sorting, we apply stable isotope labeling by amino acids in cell culture (SILAC) coupled with Orbitrap Astral data-independent acquisition (DIA) mass spectrometry (MS). This approach enables cell type-specific profiling of protein phosphorylation and degradation, alongside bulk analysis of the early newly synthesized proteome during active immune attack. Our analyses resolve interferon-γ-dependent changes in melanoma cells, identify the cytotoxic and regulatory T cell molecule (CRTAM) as a selective marker of reactive TILs, and reveal rapid tumor-intrinsic activation of DNA damage response-associated kinases, exposing potential therapeutic vulnerabilities. Overall, this framework provides a powerful resource for dissecting tumor-immune interactions to guide biomarker discovery and advance immunotherapy.