Abstract
We report the refinement of a high-throughput, liquid-chromatography mass spectrometry-based screening method for the identification of covalent small-molecule binders to proteins. Using a custom library of 1600 disulfide-capped fragments targeting surface cysteine residues, we optimize sample preparation, chromatography, and ionization conditions to maximize the reliability and flexibility of the approach. Data collection at a rate of 90 seconds per sample balances speed and reliability for sustained screening over multiple, diverse projects run in 24 months. The method is applicable to protein targets of various class and molecular mass. Data are processed in a custom pipeline that calculates a % bound value for each compound and detects false-positives by calculating significance of detected masses (‘signal significance’). Data collection and analysis methods for the screening of covalent adducts of intact proteins are now fast enough to screen the largest covalent compound libraries in 1-2 days.