biostudies-literatureHu HNational Natural Science Foundation of ChinaNational Natural Science Foundation of China (National Science Foundation of China)1465https://www.ebi.ac.uk/biostudies/studies/S-EPMC10874396biostudies-literatureUnknown15(1)Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner. By integrating enzymatic PTM-mechanisms, and genetically encoding residue-selective photo-crosslinker into PTM-enzymes, our strategy expands the substrate profileNature communicationsSpatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells.PMC108743969205310622177120Ma STan MHu WZhou BSNie HJGuo ADLiu TYao XLiu XHu HZhai LJia XChen XHfalseSpatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells.Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner. By integrating enzymatic PTM-mechanisms, and genetically encoding residue-selective photo-crosslinker into PTM-enzymes, our strategy expands the substrate profile2024-01-01T00:00:00Z2024 Feb2026-05-29T11:40:43.93Z2025-04-19T07:34:59.738ZS-EPMC108743963836841910.1038/s41467-024-45765-3