Profiling Protein-Peptide Interactions by Yeast Surface Display

Protein-peptide interactions are central to many biological processes and form the basis for many techniques in basic research and therapeutic discovery. A common research challenge is to identify specific peptide ligands or binding motifs where no initial hit is known. Here we describe a protocol for discovering protein-binding peptides using a very large, target-agnostic yeast surface display library containing approximately 6.1 × 10⁹ unique clones and providing broad coverage of short peptide sequence space. To facilitate its use, this library has been made commercially available at low cost. The described workflow entails thawing and expanding the library, validating target protein reagents, enriching target-binding cells through iterative rounds of cell sorting, monitoring enrichment by analytical flow cytometry, and analyzing selected pools by next-generation sequencing. We emphasize practical considerations that affect selection outcomes, including target labeling strategy, appropriate counterselections, and cell sorting parameters. A selection campaign using these protocols can be completed in a matter of weeks, and will typically result in the identification of thousands of candidate target-binding peptide sequences. These protocols are broadly applicable to many diverse protein targets, provided that the target can be purified and labeled in a suitable format. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Selecting protein-binding peptides from the naïve peptide library using MACS and FACS sorting Support Protocol 1: Thawing, expanding, and cryopreserving the peptide library Support Protocol 2: Preparing and validating target protein reagents Support Protocol 3: Performing analytical flow cytometry to assess bulk library binding Support Protocol 4: Extracting plasmid DNA and preparing NGS libraries from post-selection libraries.