Covalent inhibitors, with irreversible target engagement, have become pivotal in drug discovery. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is uniquely suited for high-throughput screening of these molecules, enabling label-free detection of native proteins and covalent adducts, precise quantification of target engagement through exact mass analysis, rapid characterization of binding kinetics (e.g., kinact/KI values) via intact protein analysis, and further binding site identification by peptide mapping analysis.
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