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Australian Journal of Chemistry Australian Journal of Chemistry Society
An international journal for chemical science
RESEARCH ARTICLE (Open Access)

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

Jack W. Klose https://orcid.org/0000-0002-4084-1870 A B # , Yezhou Yu https://orcid.org/0009-0007-9173-549X A B # , Giovanna Di Trapani https://orcid.org/0000-0003-2583-5832 B , Kathryn F. Tonissen https://orcid.org/0000-0002-1018-2798 A B , Louise M. Sternicki https://orcid.org/0000-0001-6158-663X A B * and Sally-Ann Poulsen https://orcid.org/0000-0003-4494-3687 A B *
+ Author Affiliations
- Author Affiliations

A Institute for Biomedicine and Glycomics, Griffith University, Gold Coast, Qld 4222, Australia.

B School of Environment and Science, Griffith University, Brisbane, Qld 4111, Australia.


Handling Editor: Mibel Aguilar

Australian Journal of Chemistry 78, CH25081 https://doi.org/10.1071/CH25081
Submitted: 16 May 2025  Accepted: 28 July 2025  Published online: 29 August 2025

© 2025 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY)

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, which is not possible with intact denaturing MS. This study demonstrates the utility of using nMS and the advantages when compared to intact denaturing MS for the discovery and characterisation of new covalent ligands.

Keywords: acrylamide, carbonic anhydrase, carbonic anhydrase inhibitor, chloroacetamide, covalent drug discovery, cysteine, electrophilic fragments, fragment based drug discovery, native mass spectrometry, thiocyanate.

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