摘要Proteins are indispensable to all biological systems and drive life processes through activities that are intricately linked to their three-dimensional(3D)structures.Traditional proteomics often provides static snapshots of protein expression,leaving unanswered questions about how proteins respond to stimuli and affect cellular functions.Limited proteolysis coupled with mass spectrometry(LiP-MS)has emerged as a powerful technique for exploring protein structure and function under near-natural conditions.Studies have revealed that LiP-MS is invaluable for structural and functional proteomics because it offers novel insights into protein dynamics.In this review,we summarise the current applications of LiP-MS in diverse areas such as the discovery and identification of drug targets,metabolite action mechanisms,proteome dynamics,protein interactions,and disease biomarkers.We also address the critical challenges in ongoing research and discuss their broader implications for advancing our understanding of protein biology and drug discovery.LiP-MS holds significant promise for accelerating biomarker and therapeutic target development as well as advancing molecular biology research in animals,plants,and microorganisms.