High-Throughput Screening of One-Bead–One-Compound Peptide Libraries Using Intact Cells

Abstract

Screening approaches based on one-bead-one-compound (OBOC) combinatorial libraries have facilitated the discovery of novel peptide ligands for cellular targeting in cancer and other diseases. Recognition of cell surface proteins is optimally achieved using live cells, yet screening intact cell populations is time-consuming and inefficient. Here, we evaluate the Complex Object Parametric Analyzer and Sorter (COPAS) large particle biosorter for high-throughput sorting of bead-bound human cell populations. When a library of RGD-containing peptides was screened against human cancer cells that express αvβ3 integrin, it was found that bead-associated cells are rapidly dissociated when sorted through the COPAS instrument. When the bound cells were reversibly cross-linked onto the beads, however, we demonstrated that cell/bead mixtures can be sorted quickly and accurately. This reversible cross-linking approach is compatible with matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based peptide sequence deconvolution. This approach should allow one to rapidly screen an OBOC library and identify novel peptide ligands against cell surface targets in their native conformation.

Microarray Based Screening of Peptide Nano Probes for HER2 Positive Tumor

“…26,27 Nowadays, new generations of OBOC screening have been developed to accelerate the peptide screening processes. 28−31 With magnetic conjugation assay, OBOC peptide beads could be trapped by the magnetic field instead of being picked out manually.…”

Viral nanoparticles decorated with novel EGFL7 ligands enable intravital imaging of tumor neovasculature

“…To achieve this, we employed two sequential approaches: (1) a magnetic/fluorescent ‘beads on a bead’ screening approach, 29 and (2) a cell-based positive/negative selection screen 30 (Fig. 1).…”

“…In our search for novel ligands targeting EGFL7, we have undertaken a stringent combinatorial peptide library screen that integrates several complementary approaches that we developed previously. 29,30 This has resulted in the discovery of a novel peptide ligand, E7p72, with high affinity and specificity for human EGFL7 that labels endothelial cells in an EGFL7-dependent manner. To demonstrate the utility of this EGFL7-directed ligand for molecular targeting, we have engineered fluorescent viral nanoparticles decorated with E7p72 peptides and evaluated their capacity to specifically label tumor neovasculature in two animal models.…”

“…A secondary cell-based screen was performed using EGFL7-overexpressing HT1080-tdTomato human fibrosarcoma cells (red), and EGFL7-low MDA-MB-435-GFP human breast cancer cells (green) as previously described. 30 Beads were visualized under an Olympus IX70 inverted fluorescent microscope, and the ones that associated with the red (high EGFL7 expression) HT1080-tdT cells, but not the green (low EGFL7 expression) MDA-MB-435-GFP cells, were manually isolated using a micropipette. Hits were collected, washed and the peptides were sequenced on-bead using MALDI TOF MS/MS as described.…”

Design of a Microfluidic Chip for Magnetic-Activated Sorting of One-Bead-One-Compound Libraries

“…Automated sorting based on measuring optical parameters using the Complex Object Parametric Analyzer and Sorter (COPAS) instrument (Union Biometrica) has been adopted to improve the precision of the conventional manual screening method. 7,8 Nonetheless, the utility of this instrument is limited to sorting library beads based on fluorescence. Furthermore, the COPAS large-particle sorter is exceedingly costly and remains unaffordable to many laboratories.…”