Abstract
Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. Nonetheless, the extent to which such residue-directed ABPP platforms fully assess the protein targets of electrophilic compounds in human cells remains unclear. Here, we introduce a complementary approach that directly identifies proteins showing stereoselective reactivity with focused libraries of stereochemically-defined, alkynylated electrophilic compounds. Integration of protein- and cysteine-directed ABPP data from compound-treated human cancer cells revealed generally well-correlated target maps and highlighted specific features, such as protein size and the proteotypicity of cysteine-containing peptides, that help to explain gaps in each ABPP platform. The integrated ABPP strategy furnished stereoselective, high-engagement covalent ligands for > 300 structurally and functionally diverse human proteins, including compounds that modulate enzymes by canonical (active-site cysteine) and non-canonical (isotype-restricted and non-catalytic cysteines) mechanisms.
Content

Supplementary materials
Supplementary Table 1
Glutathione reactivity rates of stereoprobes
Supplementary Table 2
Reagents used in the manuscript
Supplementary Table 3
Genes and primers used in the manuscript
Supplementary Dataset 1
Proteomic data related to studies in the manuscript

Supplementary Synthetic Chemistry Information
Information on synthesis and characterization of compounds used in this study