Electrospray ionization-mass spectrometry (ESI-MS) is extensively employed to detect and quantify protein-carbohydrate interactions in vitro and is increasingly used to screen carbohydrate libraries against target proteins. However, current ESI-MS methods are limited to carbohydrate ligands that are relatively soluble in water and are, therefore, not generally suitable for studying protein interactions with glycolipids, an important class of cellular receptors. Here, we describe a catch-and-release (CaR)-ESI-MS assay, which exploits nanodiscs (NDs) to solubilize glycolipids and mimic their natural cellular environment, suitable for screening libraries of glycosphingolipids (GSL) against proteins to identify specific interactions and to rank their relative affinities. Using the B subunit homopentamers of cholera toxin and heat labile toxin as model GSL-binding proteins, the CaR-ESI-MS was applied to NDs containing mixtures of gangliosides. The results demonstrate that the CaR-ESI-MS assay can simultaneously detect both high and low affinity GSL ligands using either a library of NDs that each contains one GSL or incorporating a mixture of GSLs into a single ND. Moreover, the relative abundances of the released ligands appear to reflect their relative affinities in solution. Application of the CaR-ESI-MS assay using NDs containing gangliosides extracted from porcine brain led to the discovery of a neolacto GSL as a cholera toxin ligand, highlighting the power of the assay for identifying specific protein-glycolipid interactions from biologically relevant mixtures.
ASJC Scopus subject areas
- Analytical Chemistry