Ultra-large chemical libraries for the discovery of high-affinity peptide binders

High-diversity genetically-encoded combinatorial libraries (10(8)−10(13) members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 10(6) compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 10(8) members—a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 10(6)–10(8). These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3–19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact.