Analysis of Confounding Factors in Reactive Cysteine Profiling Reveals Enhanced Chromatin-Protein Association via CDK7 Inhibition by THZ1

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.