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

Recent advances in
targeted covalent inhibitors have aroused significant
interest for their potential in drug development for difficult therapeutic
targets. Proteome-wide profiling of functional residues is an integral
step of covalent drug discovery aimed at defining actionable sites
and evaluating compound selectivity in cells. A classical workflow
for this purpose is called IsoTOP-ABPP, which employs an activity-based
probe and two isotopically labeled azide-TEV-biotin tags to mark,
enrich, and quantify proteome from two samples. Here we report a novel
isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP,
that significantly expands multiplexing power as compared to the original
isoTOP-ABPP. We demonstrate its application in identifying cysteine
on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620.
However, changes in some of these hits can be explained by modulation
at the protein and post-translational levels. Thus, it would be crucial
to interrogate site-level bona fide changes in concurrence to proteome-level
changes for corroboration. In addition, we perform a multiplexed covalent
fragment screening using four acrylamide-based compounds as a proof-of-concept.
This study identifies a diverse set of liganded cysteine residues
in a compound-dependent manner with an average hit rate of 0.07% in
intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds
to demonstrate that the AT-MAPP assay is flexible for noncysteine
functional residues such as tyrosine and lysine. Overall, we envision
that 11plex-AzidoTMT will be a useful addition to the current toolbox
for activity-based protein profiling and covalent drug development.