MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v08/MSV000095804/primary200OKProteomicsJin WangOrbitrap Fusion LumosOrbitrap AscendHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=73e24ac0d5444635be540238c18fce71wangj@bcm.eduBaylor College of Medicine, Houston TXMassIVE62UNIMOD:2016 - "TMTpro 16plex Tandem Mass Tag"Covalent inhibitors are an emerging class of therapeutics, but methods to comprehensively profile their binding kinetics and selectivity across the proteome have been limited. Here we introduce COOKIE-Pro (COvalent Occupancy KInetic Enrichment via Proteomics), an unbiased method for quantifying irreversible covalent inhibitor binding kinetics on a proteome-wide scale. COOKIE-Pro uses a two-step incubation process with mass spectrometry-based proteomics to determine k_inact and K_I values for covalent inhibitors against both on-target and off-target proteins. We validated COOKIE-Pro using BTK inhibitors spebrutinib and ibrutinib, accurately reproducing known kinetic parameters and identifying both expected and unreported off-targets. The method revealed that spebrutinib has over 10-fold higher potency for TEC kinase compared to its intended target BTK. To demonstrate the method's utility for high-throughput screening, we applied a streamlined two-point strategy to a library of 16 covalent fragments. This approach successfully generated thousands of kinetic profiles, enabling the quantitative decoupling of intrinsic chemical reactivity from binding affinity at scale and validating the method's broad applicability. By providing a comprehensive view of covalent inhibitor binding across the proteome, COOKIE-Pro represents a powerful tool for optimizing the potency and selectivity of covalent drugs during preclinical development.COOKIE-Pro: covalent inhibitor binding kinetics profiling on the proteome scale.Lin Hanfeng H, Yang Bin B, Ding Lang L, Yang Yen-Yu YY, Holt Matthew V MV, Jung Sung Yun SY, Zhang Bing B, Wang Meng C MC, Wang Jin JfalseCOOKIE-Pro: Covalent Inhibitor Binding Kinetics Profiling on the Proteome ScaleCOOKIE-Pro mass spec dataset collected from Orbitrap Lumos for spebrutinib SB2 (20231129_Speb_SB2_TMT18_ScaleUp_MS2_8ul.mzML; Ascend_VQneo_ES75500_SP2_X.mzML) and ibrutinib IB-C8-DTB (Lang_HFL_IB_COOKIE_X). Two-point COOKIE-Pro data are collected form Orbitrap Ascend, named as "Ascend_VQneo_ES75500_FAIMS_MS2_COOKIEX_Y". Two-point COOKIE-Pro data are searched with dynamic modification of unimod:1 (Carbamidomethyl) and unimod:2062 (DBIA) on cysteine.MSV00009580441027871