Screening of one-bead-one-peptide combinatorial library using red fluorescent dyes. Differencing between positive and false positive beads

MARIELA M. MARANI, M. CAMILA MARTÍNEZ CERON, SILVANA L. GIUDICESSI, ELINADRE DE OLIVEIRA, SIMON CÔTÉ, ROSA ERRA BALSELLS, FERNANDO ALBERICIO, OSVALDO CASCONE, SILVIA A. CAMPERI.

J. Comb. Chem.

American Chemicla Society (ACS)

Año: 2009 vol. 11 p. 146 - 150

1520-4766

To screen one-bead-one-compound (OBOC) combinatorial 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 identified 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 differenting positive from false positive.  The false positive had a bright homogeneous fluorescence while positives beads were darker in the centre and brighter in their perimeter. The difference was more evident when using Texas Red instead of ATTO 590.  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 hydrophobic aliphatic amino acids (Val, Leu/Il e), aromatic amino acid (Tyr and Phe) and His.