Protocol for covalent ligand discovery via library-versus-proteome screening

Here, we present a dynamic combinatorial library-versus-proteome activity-based protein profiling (DCL-ABPP) workflow for high-throughput covalent ligand discovery. We describe steps for integrating a dynamic combinatorial library with proteome-wide mass spectrometry to generate and screen hundreds of ligands in situ, eliminating the need for pre-synthesis. We detail procedures for two complementary modes: competitive screening, which identifies enzyme inhibitors (e.g., serine hydrolases) through family-wide probe competition; and direct screening, which maps covalent ligand binding sites on cysteines. For complete details on the use and execution of this protocol, please refer to Huang et al.¹.