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

High-diversity genetically-encoded combinatorial libraries (108-1013 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 ~ 106 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 108 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 106-108. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/ß-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 ß-amino acids in facilitating a key binding contact.

Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins.

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-ß (IFN-ß), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.