A solid-phase compatible silane-based cleavable linker enables custom isobaric quantitative chemoproteomics

The human proteome harbors tens of thousands of ligandable or potentially druggable cysteine residues. Consequently, pinpointing the optimal covalent molecule for each cysteine residue is a key challenge for chemical probe and drug discovery campaigns. While chemoproteomic methods have enabled proteome-wide screens of electrophilic molecules, achieving comprehensive proteome-wide structure activity relationship (SAR) maps requires technical innovation in two key areas: (1) streamlined sample preparation workflows and (2) increased sample throughput via multiplexing. Recent inroads in the latter challenge have been made through the incorporation of isobaric tandem mass tags (TMT) into chemoproteomic workflows; the high cost and late-stage isobaric labeling collectively have, however, limited adoption of such MS2/MS3-based quantitation strategies. Here we report the silane-based Cleavable Linkers for Isotopically-labeled Proteomics (sCLIP) method, which harnesses custom isotopically labeled chemoproteomic capture reagents to simplify sample preparation and achieve low cost 6-plex isobaric multiplexing. The sCLIP method is enabled by a high yielding and scalable route to dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc) protected amino acid building blocks, which enable facile synthesis of customizable, isotopically labeled, and chemically cleavable biotin capture reagents. Benchmarking of a panel of fully functionalized sCLIP capture reagents revealed performance comparable to established platforms. Using the diagnostic ion mining of the FragPipe computational pipeline, we identified a characteristic fragment ion with suitable intensity and specificity for tandem mass spectrometry (MS2)-based quantification. By harnessing this unprecedented gas phase chemistry, we established a cost-effective high performance 6-plex isobaric reagent set in which the mass balancer and reporter are encoded in the cysteine-capping and biotin capture reagents, respectively. Application of the sCLIP reagents to chemoproteomic analysis of electrophilic molecules uncovered 4805 total ligandable cysteines, including established and unprecedented cysteine-ligand pairs.