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Electrophilic fragment screening using native mass spectrometry to identify covalent probes for surface cysteines

Jack Klose , Yezhou Yu , Giovanna Di Trapani , Kathryn Tonissen , Louise Sternicki , Sally-Ann Poulsen

Abstract

Covalent chemical probes form a covalent bond with a target protein of interest to elicit an effect and methods to identify and characterise them are needed. We developed a native mass spectrometry (nMS) method to screen an electrophilic covalent fragment library and identified specific novel binders for the surface exposed cysteine residues of carbonic anhydrase III (CA III). The nMS method was extended to determine the site of protein modification and measure simultaneous binding of an active site noncovalent inhibitor and covalent fragment hit, not possible with intact denaturing MS. This study demonstrates the utility of using nMS and the advantages compared to intact denaturing MS for the discovery and characterisation of new covalent ligands.

CH25081  Accepted 28 July 2025

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