Screening of one-bead-one-peptide combinatorial library using red fluorescent dyes. Presence of positive and false positive beads.
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.
Marani
Mariela M
MM
Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina.
Martínez Ceron
María C
MC
Giudicessi
Silvana L
SL
de Oliveira
Eliandre
E
Côté
Simon
S
Erra-Balsells
Rosa
R
Albericio
Fernando
F
Cascone
Osvaldo
O
Camperi
Silvia A
SA
D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005456:Fluorescent Dyes; D008863:Microspheres; D019151:Peptide Library
D005456:Fluorescent Dyes; D019151:Peptide Library