Wendy A. Lea Ph.D. NIH

This literature review discusses a paper where researchers used ESI-MS with nanodiscs to screen glycolipids against proteins.

Protein–glycan interactions represent important events in many infectious diseases. Understanding the binding interactions between glycans and proteins can help researchers decode glycan structural information and aid in the identification of functional glycan receptors. Protein–glycan interactions are commonly featured by small glycan binding sites spanning ∼2.5 sugar residues.

Despite recognition of specific glycans present on a cell surface by proteins, such as bacterial adhesin, toxin, and plant lectin, the nature of the interaction is usually weak with binding affinities in the micromolar range (Otto et al., Anal Biochem 2011;411:261–270). Typical methods applied to study protein–glycan interactions are X-ray diffraction, nuclear magnetic resonance, calorimetry, equilibrium dialysis, and enzyme-linked immunosorbent assay. However, limited solubility of glycolipids and low affinities of the interaction pose a significant experimental challenge to the aforementioned approaches.

One current dominant technique for the study of protein–glycan interactions is glycan microarrays that utilize immobilized glycolipids as chip-format libraries for the screen against glycan binding proteins (Rillahan et al., Annu Rev Biochem 2011;80:797–823). Although it revolutionized glycobiology research for over a decade, the platform does suffer from the required alteration of glycolipids during surface immobilization. More recently, electrospray ionization mass spectrometry (ESI-MS) became an emerging tool for in vitro measurement of protein–ligand complexes. Herein, the article by Leney and co-workers* reported a catch-and-release (CaR)-ESI-MS assay in which nanodiscs (NDs) were utilized for solubilization of glycolipids. 

Nanodiscs are composed of a lipid bilayer solubilized by two molecules of membrane scaffold protein. Thus, nanodiscs, when properly prepared, represent a homogeneous model with appreciable stability and native-like environment for in vitro studies. The design of the (CaR)-ESI-MS assay is based on the principle that protein–glycolipid–ND complexes that are preformed in aqueous solution and subsequently subjected to ESI for the release of protein–glycolipid complex and separation from NDs (see figure). Protein–glycolipid interaction is subsequently broken through collision-induced dissociation (CID) with glycolipids released as ions, the mass measurements of which are used to establish glycolipid receptors.

Using cholera toxin B subunit homopentamers (CTB5) and B subunits of heat labile toxin (HLTB5) as model systems, the authors illustrated feasibility of the (CaR)-ESI-MS platform for the screen of specific glycan-binding proteins. The assay has been shown to be versatile because it is capable of screening NDs containing one or multiple glycosphingolipids (GSLs) and GSL mixtures extracted from cell cultures. In addition, the assay was also sensitive to differentiate NDs with different percentages of gangliosides and gangliosides with different binding affinities through the relative ion abundances of specific complex species. This study exemplifies and further extends the power of ESI-MS for the detection and quantification of protein–ligand interactions when combined with the use of NDs for glycolipid solubilization.


Cartoon representation of the CaR-ESI-MS assay for screening glycosphingolipids (GSLs) against target proteins. The protein (red) and ND (lipid bilayer [blue/black] with surrounding membrane scaffold protein [white]) containing GSLs (purple) are incubated to form a complex. Using mild in-source dissociation conditions, the protein–GSL complexes are stripped out of the ND. The GSL ligands are then released (as ions) from the protein using collision-induced dissociation (CID) and mass analyzed.

*Abstract from Analytical Chemistry 2014, Vol. 86: 5271–5277

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.

Wendy A. Lea, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Analytical Chemistry titled "Nanodiscs and electrospray ionization mass spectrometry: a tool for screening glycolipids against proteins." Authors of the paper are Leney AC, Fan X, Kitova EN, and Klassen JS.