biostudies-literatureZheng ZScience, Technology and Innovation Commission of Shenzhen MunicipalityNational Natural Science Foundation of ChinaNational Key Research and Development Program of China6064-6075https://www.ebi.ac.uk/biostudies/studies/S-EPMC11040650biostudies-literatureUnknown15(16)The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs <i>in vitro</i> using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals <i>ortho</i>-phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by InChemical scienceProtein painting for structural and binding site analysis <i>via</i> intracellular lysine reactivity profiling with <i>o</i>-phthalaldehyde.PMC1104065022150610470JCY202001091408144082021YFA130260322074060Zheng ZLai KTan CSHLiao BLi PZeng YfalseProtein painting for structural and binding site analysis <i>via</i> intracellular lysine reactivity profiling with <i>o</i>-phthalaldehyde.The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs <i>in vitro</i> using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals <i>ortho</i>-phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by In2024-01-01T00:00:00Z2024 Apr2026-06-01T21:55:33.607Z2026-05-23T03:08:18.869ZS-EPMC110406503866552210.1039/d4sc00032c