The human proteome harbors tens of thousands of ligandable or potentially druggable cysteine residues. Consequently, pinpointing the optimal covalent molecule for each cysteine residue represents an exciting means to close the druggability gap, namely the ~96% of human proteins not yet targeted by an FDA approved drug. Realizing the full therapeutic potential of the cysteineome will require comprehensive proteome-wide cysteine-compound structure activity relationship (SAR) analysis. While mass spectrometry-based chemoproteomic platforms have made significant inroads into this challenge, achieving comprehensive cysteine-SAR necessitates technical innovation in two key areas: (1) streamlined sample preparation workflows and (2) high throughput and high coverage data acquisition. Here we report the silane-based Cleavable Linkers for Isotopically labeled Proteomics (sCLIP) method. sCLIP streamlines sample preparation with unparalleled early-stage isobaric labeling and sample pooling, allowing for high coverage and increased sample throughput via customized low cost 6-plex sample multiplexing. The sCLIP method is distinguished by its unprecedented click-assembled isobaric tags, in which the reporter group is encoded in the sCLIP capture reagent and balancer in the pan cysteine-reactive probe. When paired with a custom FragPipe data analysis workflow and applied to cysteine-reactive fragment screens, sCLIP proteomics revealed established and unprecedented cysteine-ligand pairs, including the discovery that the mitochondrial uncoupling agent FCCP acts as a covalent-reversible cysteine-reactive electrophile.
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