Small-molecule binding-site discovery using silyl ether-enabled chemoproteomics.

For chemical probe and drug discovery campaigns, the pairing of mass spectrometry-based chemoproteomics with photoaffinity labelling has emerged as a favoured approach for target discovery and mode of action assignment. However, photocrosslinked peptide-compound adducts raise analytic challenges for quantitative binding site discovery. Here, to address these challenges, we establish the Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE) method. SEE-CITE incorporates a fully functionalized chemically cleavable photocrosslinking handle that enables precise site-of-labelling identification and head-to-head comparisons of relative binding site engagement by chemically diverse compounds. To ensure high-confidence localization of labelled residues, we extended the MSFragger algorithm of the FragPipe computational platform to report localization scores customized for photoaffinity labelling and SEE-CITE data. When applied to scout fragments and analogues of select FDA-approved kinase inhibitors, SEE-CITE delineates known drug binding sites and uncovers small-molecule binding sites that affect the protein activity of RTN4 and COX5A.