Reversibly reactive affinity selection mass spectrometry enables maturation of covalent peptides

22 December 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Covalent peptides have found widespread applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and highly tunable affinity selection method to enrich covalent reactive peptides from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform enabled by tandem high resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfides to build reversible peptide-protein conjugates that can enrich crosslinked peptides that after reduction can be sequenced with MS/MS. Using this platform, we achieved maturation of covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1). The resulting peptides selectively covalently crosslink Cys58 of E6 with 96% yield in 4 hours at 37 °C and Cys113 of Pin1 with >99% yield in 1 hour at room temperature, respectively. This approach enables the identification of highly selective covalent peptide inhibitors for diverse molecular targets, introducing an applicable method to assist pre-clinical therapeutic development pipelines.


Affinity Selection
HPV16 E6
Covalent binders
Peptide binders
Protein inhibitors

Supplementary materials

Supporting Information
Supplementary figures, materials and methods, analytical characterization, library sequencing results.


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