One-bead-one peptide library two-stage screening followed by MALDI-TOF MS/MS for ligand design

M. C. MARTÍNEZ-CERON; S. L. GIUDICESSI; J.N.KRUSZYN; M.M. MARANI; FERNANDO ALBERICIO; O. CASCONE; S. A. CAMPERI

Cayo Santa María

Simposio; Simposio internacional de Química; 2013

Sociedad Cubana de Química

Short peptides are excellent ligands for affinity chromatography as they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. for the target protein and stability against proteases. Divide-couple-recombine (DCR) method allows obtaining a library with all possible combinations of the amino acids in the form of ?one bead-one peptide?. In this work we designed a two stage library screening method for peptide ligand selection. The combinatorial library was synthesized on hydroxymethylbenzoyl (HMBA)-ChemMatrix resin. The first screening was performed with the target protein (bovine seroalbumin-BSA.) coupled with Texas Red. Fluorescent beads were automatically isolated using Complex Object Parametric Analyzer and Sorter (COPAS). Isolated beads were washed and the second screening was performed with BSA coupled with biotin and then streptavidin-peroxidase and revealed with 3,3`-diaminobenzidine. Those peptides showing more consensuses were synthesized and immobilized on agarose. All peptide-agarose matrices showed affinity for BSA with Kd values between 10E-5 and 10E-6 M. The two stage screening avoids false positive selection.