CAMPERI Silvia Andrea
congresos y reuniones científicas
Screening of one-bead-one-peptide combinatorial library using red fluorescent dyes
M. M. MARANI; M. C. MARTÍNEZ-CERON; S. L. GIUDICESSI; E. DE OLIVEIRA; S. CÔTÉ; R. ERRA-BALSELLS; F. ALBERICIO; O. CASCONE; S. A. CAMPERI
Carlos Paz. Córdoba. Argentina
Congreso; SAIB XLIV Reunión Anual (Sociedad Argentina de Investigación Bioquímica y Biología Molecular); 2008
Sociedad Argentina de Investigación en Química Orgánica
Combinatorial peptide libraries using one-bead-one-compound (OBOC) method involves the synthesis of 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 have been described. To screen OBOC libraries, beads are first mixed with a target molecule and those that interact with the target molecule will be 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-dye conjugate, positive beads due to peptide-SA interaction and false positive beads due to peptide-fluorescent dye interaction were isolated. 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 MALDI-TOF MS. 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.