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

M.M. MARANI; M.C. MARTÍNEZ CERON; S. GIUDICESSI; E. OLIVEIRA; R. ERRA-BALSELLS; F. ALBERICIO; O. CASCONE; S.A. CAMPERI

Villa Carlos Paz, Córdoba

Congreso; XLIV Reunión Nacional de SAIB; 2008

SAIB

 Combinatorial peptide libraries using "one-bead-one-compound" (OBOC) method involves the synthesis of millions of peptides onmethod 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 usesthat each bead displays only one peptide entity. With the OBOC method, ligands with pharmacological and analytical useswith pharmacological and analytical uses and protein capture agents have been described.protein capture agents have been described. To screen OBOC libraries, beads are first rnixed with a targetscreen OBOC libraries, beads are first rnixed with a target nlolecule and those that interact with the target molecule rvill be isolated for compound shucture cletenninationi. Hc.rein rve describeand those that interact with the target molecule rvill be isolated for compound shucture cletenninationi. Hc.rein rve describefor compound shucture cletenninationi. Hc.rein rve describe an OBOC peptide library screening using streptavidin (SA) aslibrary screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using theprotein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equ\trncnt to separ.ateBIO-BEAD flow sorting equ\trncnt to separ.ate fluorescent frorn non-fluorescent beads. Red dyes investigated we¡e A'l''lO 590 and Texas Red. After incubating the library withfrorn non-fluorescent beads. Red dyes investigated we¡e A'l''lO 590 and Texas Red. After incubating the library withA'l''lO 590 and Texas Red. After incubating the library with the SA-dye conjugate, positive beads due to peptide-SA interactionSA-dye conjugate, positive beads due to peptide-SA interaction and false positive beads due to peptide-fluorescent dye interactionpositive beads due to peptide-fluorescent dye interaction rvere isolated. Using control peptide-beads we realized that falseUsing control peptide-beads we realized that false positive had a bright homogeneous fluorescence while positivebright homogeneous fluorescence while positive beads had a heterogeneous fluorescence exhibiting a characteristic halo appearance. Thus, positive from false positive beads could bea heterogeneous fluorescence exhibiting a characteristic halo appearance. Thus, positive from false positive beads could beappearance. 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-Glnbeads were analyzed by MALDI-TOF MS. Sequences obtained from positive beads had the His-pro-Glnobtained from positive beads had the His-pro-Gln rnotif. Peptides from false positive beads were rich in Leu/Ileu, His,Peptides from false positive beads were rich in Leu/Ileu, His, Phe andTyr.andTyr.