Abstract
Mass spectrometry (MS)-based proteomics has emerged as a versatile platform in modern drug discovery, enabling system-wide interrogation of protein abundance, posttranslational modifications (PTMs), turnover, structural stability, and interactions with small molecules in native biological contexts. In this review, we introduce recent advances in MS and summarize how scalable, high-throughput technologies support target identification and mechanism-of-action studies. We discuss global profiling of the proteome and PTMs under drug perturbation, as well as proteome-wide stability assays such as thermal proteome profiling and limited proteolysis–MS. We further discuss activity-based protein profiling, fully functionalized and photoaffinity probes, and affinity-matrix approaches that expand covalent and noncovalent ligand discovery. Finally, we highlight multiple proteomics-driven strategies in targeted protein degradation. Together, these integrated MS-based approaches in drug discovery, often termed chemoproteomics, provide a coherent framework for linking chemical perturbations to functional proteome remodeling, expanding the druggable proteome and accelerating therapeutic development.