Clinical Applications of Phosphoproteomics: Illuminating Cancer Signaling and Enabling Rational Therapeutic Strategies

ABSTRACT Protein phosphorylation is a central post‐translational modification regulating cellular signaling, frequently dysregulated in cancer. Mass spectrometry (MS)‐based phosphoproteomics has emerged as a powerful approach to systematically profile phosphorylation events, thereby revealing aberrant kinase activity and therapeutic vulnerabilities that are not captured by genomic or transcriptomic analyses. Recent advances across the workflow—including optimized sample preparation and phosphopeptide enrichment, isotope‐ or label‐free quantitative strategies, high‐resolution mass spectrometry platforms, specialized algorithms for site identification and quantification, and integrative informatics analyses—have enabled the detection of tens of thousands of phosphorylation sites even from small clinical specimens. These developments have facilitated the characterization of signaling pathways across diverse cancer types, leading to the identification of targetable kinases and informing therapeutic strategies. In this review, we highlight studies that employed phosphoproteomic analyses of clinical specimens or patient‐derived cancer cells to delineate signaling characteristics and to propose and validate therapeutic targets. Collectively, MS‐based phosphoproteomics is poised to become a cornerstone of precision oncology. By enabling comprehensive and quantitative mapping of phosphorylation events, this technology allows mechanistic dissection of cancer signaling pathways and uncovers therapeutic vulnerabilities that may be exploited with targeted agents.