Development of proteome-derived cyclic peptide libraries via ortho-phthalaldehyde-assisted cyclization

Cyclic peptides possess unique structural features and binding capabilities that make them attractive scaffolds for molecular recognition. However, strategies for generating functionally diverse libraries directly from natural proteomes remain comparatively underdeveloped. Here, we present a proteome-informed approach for constructing cyclic peptide libraries by integrating cysteine-enrichment protocols with ortho-phthalaldehyde (OPA)-mediated macrocyclization. This method enables direct library generation from proteome digests and is compatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based annotation and downstream functional screening. We systematically identify key parameters governing OPA-mediated cyclization topology and develop a rule-based pipeline for automated library annotation of OPA-cyclized peptides. Applying this strategy, we generate a library of over 15,000 unique cyclic peptides and perform target-specific screening against human IgG. This yielded cyclic peptide binder candidates, including one lead peptide showing cyclization-dependent IgG-binding potential. Our study establishes an OPA-enabled, proteome-informed workflow for cyclic peptide library construction and annotation.