AzidoTMT Enables Direct Enrichment and Highly Multiplexed Quantitation of Proteome-Wide Functional Residues

Recent advances intargeted covalent inhibitors have aroused significantinterest for their potential in drug development for difficult therapeutictargets. Proteome-wide profiling of functional residues is an integralstep of covalent drug discovery aimed at defining actionable sitesand evaluating compound selectivity in cells. A classical workflowfor this purpose is called IsoTOP-ABPP, which employs an activity-basedprobe and two isotopically labeled azide-TEV-biotin tags to mark,enrich, and quantify proteome from two samples. Here we report a novelisobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP,that significantly expands multiplexing power as compared to the originalisoTOP-ABPP. We demonstrate its application in identifying cysteineon- and off-targets using a KRAS G12C covalent inhibitor ARS-1620.However, changes in some of these hits can be explained by modulationat the protein and post-translational levels. Thus, it would be crucialto interrogate site-level bona fide changes in concurrence to proteome-levelchanges for corroboration. In addition, we perform a multiplexed covalentfragment screening using four acrylamide-based compounds as a proof-of-concept.This study identifies a diverse set of liganded cysteine residuesin a compound-dependent manner with an average hit rate of 0.07% inintact cell. Lastly, we screened 20 sulfonyl fluoride-based compoundsto demonstrate that the AT-MAPP assay is flexible for noncysteinefunctional residues such as tyrosine and lysine. Overall, we envisionthat 11plex-AzidoTMT will be a useful addition to the current toolboxfor activity-based protein profiling and covalent drug development.

Automated summary

Recent advancements in targeted covalent inhibitors have garnered considerable interest for their potential applications in drug development for challenging therapeutic targets. In this study, a novel isobaric 11plex-AzidoTMT reagent and a new workflow called AT-MAPP were introduced, greatly enhancing the multiplexing ability compared to the traditional isoTOP-ABPP method. These techniques were successfully applied to identify cysteine on- and off-targets using a covalent inhibitor, demonstrating their potential in activity-based protein profiling and covalent drug development.