sCIP-ing towards streamlined chemoproteomics

Mapping the ligandability or potential druggability of all proteins in the human proteome is a central goal of mass spectrometry-based chemoproteomics. Achieving this ambitious objective requires high throughput and high coverage sample preparation and LC-MS/MS analysis for hundreds to thousands of reactive compounds and chemical probes. Conducting chemoproteomic screens at this scale benefits from technical innovations that achieve increased sample throughput. Multiplexed analysis using commercially available amine-reactive isobaric reagents (e.g. tandem mass tags or TMT) is a favored strategy to decrease instrument acquisition time. These reagents are ideally suited for protein-based quantification applications, with efficient capping and pooling of peptides after sequence specific digestion. The added enrichment steps in nearly all chemoproteomic sample preparation workflows reveals a still largely untapped opportunity for isobaric labeling, namely incorporation of the TMT label into the chemoproteomic enrichment handle for early sample pooling and increased sample preparation throughput. Here we realize this vision by establishing the silane-based Cleavable Linkers for Isotopically-labeled Proteomics (sCIP)-TMT proteomic platform. sCIP-TMT pairs a custom click-compatible sCIP capture reagent that is readily functionalized in high yield with commercially available TMT tags. Synthesis and benchmarking of a 10-plex set of sCIP-TMT reveals a 1.5-fold decrease in sample preparation time together with high coverage and high accuracy quantification. By screening a focused library of cysteine-reactive electrophiles, we demonstrate the utility of sCIP-TMT for chemoproteomic target hunting, identifying 789 total liganded cysteines. Distinguished by its compatibility with established enrichment and quantification protocols, we expect sCIP-TMT will readily translate to a wide range of chemoproteomic applications.