Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• Covalent peptide binders are crucial as activity-based probes and irreversible therapeutic inhibitors.
• Existing methods lack rapid, label-free, and tunable affinity selection for identifying these binders from synthetic libraries.

Purpose of the Study:

• To develop a novel, rapid, label-free, and tunable platform for identifying covalent peptide binders.
• To enable the enrichment and sequencing of covalent peptide binders against native protein targets.

Main Methods:

• Development of the ReAct-ASMS (Reversible Affinity Selection-Mass Spectrometry) platform.
• Utilizing mixed disulfide-containing peptides for reversible peptide-protein conjugate formation.
• Employing tandem high-resolution mass spectrometry (MS/MS) for sequencing after reduction.

Main Results:

• Successful identification of covalent peptide binders against HPV16 E6 and Pin1 oncoproteins.
• Demonstrated efficient and selective cross-linking of Cys58 of E6 and Cys113 of Pin1.
• Validated the platform's capability to identify selective covalent binders for diverse targets.

Conclusions:

• ReAct-ASMS provides a robust platform for discovering covalent peptide binders.
• The platform facilitates preclinical therapeutic development by enabling identification of targeted inhibitors.
• This approach enhances the discovery pipeline for covalent therapeutics.