Screening of One-Bead-One-Peptide Combinatorial Library Using Red Fluorescent Dyes. Presence of Positive and False Positive Beads

Abstract

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.

Other Applications of Combinatorial Peptide Libraries

“…The use of red fluorescent dyes as a direct method for sorting out positive beads was further investigated by Marani et al (2009). Streptavidin was used as a probe protein conjugated with either Atto 590 or Texas Red dyes.…”

Process Automation toward Ultra-High-Throughput Screening of Combinatorial One-Bead-One-Compound (OBOC) Peptide Libraries

“…The false-positive beads can be generated by the interaction of fluorescent dyes with peptides and nonspecific interactions between peptides on beads and target proteins. 23,27 The inclusion of false-positive beads due to nonspecific interactions between peptides and target proteins is not the only problem with COPAS sorting. Usually, a competing protein such as bovine serum albumin (BSA) and nonfat milk in the target protein solution has been used to minimize the nonspecific interactions between peptides and target proteins.…”

Combinatorial/Library Peptide Synthesis

“…The success of this technique can be attributed to the use of highly porous solid support with minimal autofluorescence. Alternatively, the library can be first presorted to eliminate beads with high autofluorescence [274][275][276] or a different fluorescent labeling agent (such as quantum dots) with a different fluorescence emission wavelength can be used [277]. Validation of the selected (resynthesized) structures has to be performed in any case (as discussed, for example, by Kodadek and Bachhawat-Sikder [275]) to eliminate hits resulting from artifactual interactions.…”

“…Fluorescence-based detection of positive beads is complicated by the autofluorescence of the bead material or the synthesized compounds, which can be higher than the fluorescence of the bound target macromolecule [276,277]. However, the fact that the macromolecular fluorescently labeled target does not penetrate into the interior of the polymeric bead can be used for distinguishing between autofluorescence and fluorescence from the molecules bound to the surface of the bead.…”