Protein–Glycosphingolipid Interactions Revealed Using Catch-and-Release Mass Spectrometry

Abstract

Glycosphingolipids (GSL) on the surface of cells are important receptors in antigen/microbial recognition and cell adhesion. However, their functional characterization is often challenging. We have developed a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for the identification of specific interactions between water-soluble proteins or protein complexes with GSL incorporated into nanodiscs. The specificity and sensitivity of the assay is demonstrated for interactions involving cholera toxin and Shiga toxin, with their natural GSL receptors, the ganglioside GM1, and the globotriaosylceramide Gb3, respectively. The detection of binding between cholera toxin and GM1 within a mixture of lipids extracted from cell membranes highlights the potential of this assay for the discovery of biologically relevant protein-GSL interactions.

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

“…The favorability of fragmentation at D-and E-residues is the result of transfer of their acidic protons from their side chains to the polypeptide backbone, resulting in a weakening of the amide bond [36,37]. As expected, the most abundant fragment ions in Figure 4a (y 46 , y 53 , y 54 ) are the result of polypeptide backbone fragmentation on the C-terminal side of D residues (except for those proximal to the N-or C-termini). Lesser abundant fragments ions are the result of fragmentation on the C-terminal side of E residues (y 43 , y 55 , y 61 , b 57 , b 64 ) and occasionally lysine residues (y 65 ).…”

“…They also noted that other reseachers had implicated the aspartic acid at residue 24 as responsible for differences in holotoxin/receptor binding [9]. The issue of Stx2 holotoxin/Gb3 receptor binding is beyond the scope of the current work; however, native state mass spectrometry [45,46], which involves analysis of higher order protein complexes by electrospray ionization mass spectrometry (ESI-MS), may be used to confirm the importance of residues 16 and 24 on holotoxin stability and Gb3 binding. In any case, MALDI-TOF-TOF-MS/MS-PSD is particularly well-suited for detecting the presence (or absence) of amino acid substitutions involving D-residues (and other residues) in a polypeptide sequence.…”

Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

“…An alternative approach is to incorporate the GLs in a lipid monolayer or bilayer, such that the protein-GL interactions can be studied in a more native-like environment [10]. For such studies, a variety of different model membranes have been used to solubilize the GLs, including supported lipid bilayers, liposomes, micelles, bicelles, nanodiscs, and picodiscs [11][12][13][14], and the protein-GL interactions probed using diverse analytical techniques (e.g., fluorescence, nuclear magnetic resonance (NMR), and SPR spectroscopy) [15][16][17][18].between water-soluble lectins and GLs, solubilized using nanodiscs (NDs), have been detected using the catch-andrelease (CaR)-ESI-MS assay [19,20]. Nanodiscs are discoidal phospholipid bilayers surrounded by two copies of an amphipathic membrane scaffold protein [12,13].…”

Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

“…The ability to measure multiple binding equilibria simultaneously makes the ESI-MS assay well suited for screening libraries of compounds against target proteins [38,39]. Where direct detection of the protein-ligand complexes by ESI-MS is not possible due to factors such as high molecular weight (MW) or protein heterogeneity, the catch-and-release (CaR) ESI-MS assay can be employed [40][41][42][43][44][45][46][47]. In this assay, ligand binding is established by releasing (as ions) ligands bound to the protein in the gas phase using activation methods such as collisioninduced dissociation (CID).…”

“…Typically, ligands can be identified based on their measured MW; in some cases, fragmentation of the released corresponding ligand ion or ion mobility separation (IMS) may be required for positive identification. The CaR-ESI-MS assay has been used to screen carbohydrate, peptide, and small molecule libraries against target proteins [40][41][42][43][44][45][46][47]. However, the reliability of the method has only been rigorously tested in the case of carbohydrate libraries [40,41,[45][46][47].…”