Yang, K.; Li, S.; Li, B.; Richards, D.; Dong, K.; Seneviratne, U.; Lee, W.; Iannetta, A.; Xu, H.; Gygi, S.; Yu, Q.

Recent advances in activity-based proteome profiling (ABPP) have enabled global mapping of cysteine ligandability, uncovering novel biological insights and opportunities for identifying disease vulnerabilities. While both live cell-based and native lysate-based ABPP have been applied, how cysteine ligandability differs between these systems and what factors influence these measurements remain unclear. Building on our previous development of a high-throughput TMT-ABPP workflow for native lysates, here we adapt the protocol for live cells and systematically compare cysteine ligandability across both platforms. Our analysis reveals three major contributors to the discrepancies: in-cellulo cysteine accessibility, protein abundance changes, and protein relocalization. Notably, we highlight that CDK7 inhibitor THZ1 induces substantial protein relocalization and promotes chromatin binding. Together, these results provide a practical framework for ABPP experimental design and data interpretation, supporting more accurate application of ABPP in functional proteomics and drug discovery.