Protein–protein interaction studies on protein arrays: Effect of detection strategies on signal-to-background ratios

Abstract

Protein arrays hold great promise for proteome-scale analysis of protein–protein interaction networks, but the technical challenges have hindered their adoption by proteomics researchers. The crucial issue of design and fabrication of protein arrays have been addressed in several studies, but the detection strategies used for identifying protein–protein interactions have received little attention. In this study, we evaluated six different detection strategies to identify four different protein–protein interaction pairs. We discuss each detection approach in terms of signal-to-background (S/B) ratio, ease of use, and adaptability to high-throughput format. Protein arrays for this study were made by expressing both the bait proteins (proteins captured at the surface) and prey proteins (probes) in cell-free rabbit reticulocyte lysate (RRL) systems. Bait proteins were expressed as HaloTag fusions that allow covalent capture on a HaloTag ligand-coated glass without any prior protein purification step. Prey proteins were expressed and modified with either tags (protein or peptides) or labels (fluorescent or radiometric) for detection. This simple method for creating protein arrays in combination with our analyses of several detection strategies should increase the usefulness of protein array technologies.