Screening of one-bead-one-peptide combinatorial library using red fluorescent dyes

M. M. MARANI; M. C. MARTINEZ CERON; S. L. GIUDICESSI; E. OLIVEIRA; S. CÔTE; R. ERRA BALSELLS; F. ALBERICIO; O. CASCONE; S. A. CAMPERI

Hotel Portal del Lago, Carlos Paz

Otro; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

Sociedad Argentina de investigación Bioquímica y Biología Molecular

Combinatorial peptide libraries using “one-bead-one-compound” (OBOC) method involves the synthesis of thousands to millions of peptides on beads so that each bead displays only one peptide entity. With the OBOC method, ligands with pharmacological and analytical uses and protein capture agents for their purification have been described. To screen OBOC libraries, tens of thousands to millions of compound-beads are first mixed with a target molecule. The beads that interact with the target molecule will be selected and then isolated for compound structure determination. Herein we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPASTM BIO-BEAD flow sorting equipment to separate fluorescent from non-fluorescent beads. Red dyes investigated were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, positive beads due to peptide-SA interaction and false positive beads due to peptide-fluorescent dye interaction were isolated. Those false positives were a drawback when sorting beads with COPAS. However, a deep analysis of both allows differentiating positive from false positive beads. Using control peptide-beads we realized that false positive had a bright homogeneous fluorescence while positive beads had a heterogeneous fluorescence exhibiting a characteristic halo appearance. Thus, positive from false positive beads could be manually isolated. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe and Tyr.