Rapid-Throughput Competitive Colorimetric Assay Based on Monosaccharide-Capped Gold Nanoparticles for Detecting Lectin–Protein Interactions

Abstract

Identification of protein binding partners is one of key challenges in proteomics. A rapid-throughput competitive colorimetric assay is presented that uses gold nanoparticles capped by sugars such as mannopyranoside (Man–GNPs), N-acetylglucosamine (GlcNAc–GNPs), glucose (Glc–GNPs), or N-acetylgalactosamine (GalNAc–GNPs). The assay expediently detects protein–protein interactions in solution, particularly protein–lectin interactions. The competitive assays were conducted in microtiter plates; ten proteins (two glycoproteins and eight lectins) and three sugar-binding lectins (concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA₁₂₀)) were combinatorially arranged in a 30-well plate, and constant concentrations of the monosaccharide-capped GNPs (sugar–GNPs) were added to each well. If interactions occurred between the proteins, the sugar–GNPs retained their burgundy color. If no interactions occurred between the proteins, the sugar–GNPs were agglomerated by the corresponding binding lectin, thus producing a blue color. Several new binding pairs were identified for the first time by using this assay, and the binding constants and stoichiometric ratios were determined on the basis of the wavelength shifts. The results were further verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence resonance energy transfer (FRET) spectroscopy. The assay is very sensitive, requiring only nanomolar protein concentrations.