Screening of One-Bead-One-Peptide Combinatorial Library Using Red Fluorescent Dyes. Presence of Positive and False Positive Beads Mariela M. Marani, † Marı ´a C. Martı ´nez Ceron, † Silvana L. Giudicessi, ‡ Eliandre de Oliveira, § Simon Co ˆte ´, | Rosa Erra-Balsells, ‡ Fernando Albericio, ⊥,# Osvaldo Cascone, † and Silvia A. Camperi* ,† Ca ´tedra de Microbiologı ´a Industrial y Biotecnologı ´a, Facultad de Farmacia y Bioquı ´mica, UniVersidad de Buenos Aires, Junı ´n 956, (1113) Ciudad Auto ´noma de Buenos Aires, Argentina, CIHDECAR-CONICET, Departamento de Quı ´mica Orga ´nica, Facultad de Ciencias Exactas y Naturales, UniVersidad de Buenos Aires, Pabello ´n II-Ciudad UniVersitaria, (1428) Ciudad Auto ´noma de Buenos Aires, Argentina, Proteomics Platform, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain, Matrix InnoVation Inc., 945 Newton, Suite 132, Que ´bec City, Que ´bec, Canada, Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain, and CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain ReceiVed August 29, 2008 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 this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide- fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However, an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. 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. Introduction Combinatorial peptide libraries using the one-bead-one- peptide method were introduced by Lam et al. 2 and have proven to be a powerful approach for the discovery of binding peptides for various macromolecular targets. The one-bead-one-compound (OBOC) synthetic combinatorial library method, also called the “selectide process”, 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 3-5 and protein capture agents for purification or detection of proteins in complex mixtures 6,7 have been described. To screen these combinatorial libraries, tens of thousands to millions of compound beads are first mixed with the probe molecule. The beads that interact with it are then identified and isolated for compound structure determination. For probe molecules that cannot be detected directly, a reporter group, such as an enzyme or a fluorescent dye, is conjugated to them. If an antibody to the target molecule is readily available, an alternative method uses this antibody conjugated to a reporter group. 8 In its native conception, the selectide process had some bottlenecks: (1) manual isolation of positive beads, (2) discarding of false positives, and (3) analysis of each bead by Edman microsequencing. To reduce the cost and time of Edman microsequencing, in previous studies, we have reported a rapid and inexpensive strategy based on mass spectrometry analysis of peptide beads. 9,10 We used streptavidin (SA) as probe protein, labeled with peroxidase. SA was used as the probe protein because * To whom correspondence should be addressed. E-mail: scamperi@ yahoo.com. † Facultad de Farmacia y Bioquı ´mica, Universidad de Buenos Aires. ‡ Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. § Proteomics Platform, Barcelona Science Park. | Matrix Innovation, Inc. ⊥ Institute for Research in Biomedicine, Barcelona Science Park. # Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park. J. Comb. Chem. 2009, 11, 146–150 146 10.1021/cc800145c CCC: $40.75  2009 American Chemical Society Published on Web 12/11/2008