蛋白质组
计算生物学
化学
半胱氨酸
药物发现
共价键
克拉斯
组合化学
蛋白质组学
生物化学
生物
酶
基因
有机化学
突变
作者
P. Taylur,Anita Izrael-Tomasevic,Rana Mroue,Hanna G. Budayeva,Sushant Malhotra,Ryan Raisner,Marie Evangelista,Christopher M. Rose,Donald S. Kirkpatrick,Kebing Yu
标识
DOI:10.1021/acs.jproteome.2c00703
摘要
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.
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