A Liquid Chromatography-Mass Spectrometry Method for Screening Disulfide Tethering Fragments

Abstract

AbstractWe report the refinement of a high-throughput, liquid-chromatography/mass spectrometry (LC/MS)-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 over a 24-month period. The method is applicable to protein targets of various classes and a range of molecular masses. 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’). An example pipeline has been made available through Biovia’s ScienceCloud Protocol Exchange. 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.