On-Bead Screening of Combinatorial Libraries: Reduction of Nonspecific Binding by Decreasing Surface Ligand Density

Abstract

On-bead screening of one-bead-one-compound (OBOC) libraries provides a powerful method for the rapid identification of active compounds against molecular or cellular targets. However, on-bead screening is susceptible to interference from nonspecific binding, which results in biased screening data and false positives. In this work, we have found that a major source of nonspecific binding is derived from the high ligand loading on the library beads, which permits a macromolecular target (e.g., a protein) to simultaneously interact with multiple ligands on the bead surface. To circumvent this problem, we have synthesized a phosphotyrosyl (pY)-containing peptide library on spatially segregated TentaGel microbeads, which feature a 10-fold reduced peptide loading on the bead surface but a normal peptide loading in the bead interior. The library was screened against a panel of 10 Src homology 2 (SH2) domains including those of Csk and Fyn kinases and adaptor protein SLAP, and the specific recognition motif(s) was successfully identified for each of the domains. In contrast, when the SH2 domains were screened against a control library that contained unaltered (high) ligand loading at the bead surface, six of them exhibited varying degrees of sequence biases, ranging from minor perturbation in the relative abundance of different sequences to the exclusive selection of false positive sequences that have no measurable affinity to the target protein. These results indicate that reduction of the ligand loading on the bead surface represents a simple, effective strategy to largely eliminate the interference from nonspecific binding, while preserving sufficient amounts of materials in the bead interior for compound identification. This finding should further expand the utility of OBOC libraries in biomedical research.

Other Applications of Combinatorial Peptide Libraries

“…This high density favors the docking of proteins by mechanisms already described and probably influences what is qualified as nonspecific for the process of selecting ligand for affinity separations. As said above, to reduce this phenomenon, Chen et al (2009) found first that the major reason for false positives comes from high-ligand density; they consequently proposed to reduce the ligand density by a factor of 10. However, when reducing the ligand density, the peptide sequencing becomes more challenging due to the very low availability of material.…”

“…In fact, the high-ligand density may induce simultaneous interactions with more than one peptide ligand at different amino acid locations. In order to resolve this Other Applications of Combinatorial Peptide Libraries question, Chen et al (2009) proposed to screen a library where the peptide ligand density was reduced by a factor of 10 and tested against a high-density control library. The work was constructed around the identification of a specific ligand against several SH2 domains of Csk and Fyn kinases using a phosphotyrosyl-containing peptide library.…”

Combinatorial/Library Peptide Synthesis

“…They found that actually 99.9% of the ligands had to be eliminated from the surface to prevent bidentate binding. It was later shown and explained by Chen et al [268] that the decrease of the surface substitution is the best way to reduce the nonspecific binding of the macromolecular targets. High surface loading with the test compound was probably the reason for the earlier reported unsuccessful results from screening OBOC libraries.…”

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

“…To facilitate in-line quality assessment, MRBLEs were first functionalized with an acid-labile rink amide linker within the bead core and a non-labile glycine linker on the outer bead shell ( Fig. S1) 24 ; varying the ratio of extendible to nonextendible glycine linkers in the bead shell allows tuning of displayed peptide density 19,25 . Next, peptides were synthesized on both linkers via standard Fmoc SPPS.…”