Silyl Ether Enables High Coverage Chemoproteomic Interaction Site Mapping

Abstract

Most therapeutics are small molecules that function by interacting with specific protein targets. Consequently, delineating the protein targets, including the specific binding sites, for chemical probes and clinical candidates is essential to ensure potent on-target activity and to minimize engagement of unfavorable off-targets. The pairing of mass spectrometry-based chemoproteomics with photoaffinity labeling (PAL) has emerged as a favored approach to generate proteome-wide target engagement maps for reversible compounds. However, a key limitation of most PAL-based proteomic platforms is the absence of strategies that report precise binding sites and enable direct head-to-head comparisons of relative target engagement at these sites by different lead compounds. This gap stems from a confluence of factors including the complex fragmentation patterns for crosslinked peptides, poor recovery of peptides crosslinked with large hydrophobic molecules, and modification masses that differ between molecules of interest (MOI). To address these challenges, here we establish the Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE) approach. SEE-CITE incorporates an unprecedented fully functionalized chemically cleavable crosslinking handle that enables precise site-of-labeling identification and head-to-head comparisons of relative binding site engagement by chemically diverse compounds. To ensure high confidence localization of labeled residues, we also enabled enhanced mapping of photocrosslinked sites by extending the MSFragger algorithm of the FragPipe computational platform to report localization scores customized for PAL and SEE-CITE data. By benchmarking SEE-CITE using scout fragments and analogues of the FDA-approved kinase inhibitors dasatinib and asciminib, we identify known and novel binding sites, including for high impact targets, such as BCR-ABL1, STING, and COX5A.