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.
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, realizing 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 those labeled by covalent-reversible electrophilic modalities.
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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