Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry

Abstract

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin (∼25 μg) onto porous glass beads in a tubing column (9.8 μl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acα2-6Galβ1-4Glc/GlcNAc containing oligosaccharides with Kd values near 10 μM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.