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

Abstract

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.

Pervasive aggregation and depletion of host and viral proteins in response to cysteine-reactive electrophilic compounds

“…Thus, the ramifications of our findings extend to the many cysteine chemoproteomic cell based screens 57,60,67,70,73,[141][142][143][144][145][146][147][148][149][150][151][152][153] . In reanalysis of targets identified by prior studies 64,85 including our own 76 , a marked number of shared targets emerge, including RANBP2 and NUP153, which we interrogated here. Given the recent uptick in covalent degrader modalities, we expect that the pervasive protein aggregation should extend to recently reported covalent molecular glues 60,64 .…”

sCIP-ing towards streamlined chemoproteomics

“…Enabled by our previously described solid-phase compatible https://doi.org/10.26434/chemrxiv-2023-0szhj ORCID: https://orcid.org/0000-0001-8541-1404 Content not peer-reviewed by ChemRxiv. License: CC BY-NC-ND 4.0 dialkoxydiphenyl silane (DADPS) building block 75 , solid-phase peptide synthesis proceeded smoothly, yielding the final capture reagent (sCIP-Gly-NH2) in 53% yield and high purity (Scheme 1A). Of note, glycine was included as a spacer to minimize steric hindrance and to facilitate high yield conjugation with costly isobaric reagents.…”

“…Having demonstrated highly efficient formation of sCIP-TMTzero, we next assessed reagent performance in chemoproteomics. We selected our established cysteine profiling workflow for benchmarking 11,50,51,75,[78][79][80][81] . Following the workflow shown in Figure 1B, cell lysates were capped with the pan-cysteine reactive iodoacetamide alkyne (IAA) probe followed by click conjugation to the preformed sCIP-TMTzero conjugate.…”

“…As illustrated by our own silane-based cleavable isotopically labeled proteomics (sCIP) method 75 and the recently reported azidoTMT method 76 , an alternative strategy is to introduce the isobaric label earlier in sample preparation via a fully functionalized "clickable" handle that features the built-in capacity for sample enrichment. The key advance of the sCIP platform was our fully functionalized enrichment reagents that contain biotin, a chemically cleavable DADPS group, an azide for copper catalyzed azide-alkyne cycloaddition (CuAAC or "click") enrichment, and an isobaric label.…”

A Highly Efficient Click Linker for Enrichment of Alkyne‐Tagged Proteins in Living Cells

“…To release the enriched labeled peptides from the solid support, the click linker needs to be cleavable under mild conditions. A variety of click linkers have been reported, such as DADPS azide biotin ( Scheme 1 c ) in which the biotin handle is used for anchoring the clicked peptides onto streptavidin‐coated supports while the dialkoxydiphenylsilane (DADPS) group can be subsequently cleaved under mild acidic conditions [12–16] . Herein, we report the design and synthesis of a new click linker ( APA biotin , Scheme 1 c ) and demonstrate its high efficiency in enriching labeled proteome of living yeast cells with unprecedented coverage.…”