Screening Glycolipids Against Proteins in Vitro Using Picodiscs and Catch-and-Release Electrospray Ionization-Mass Spectrometry

This work describes the application of the catch-and-release electrospray ionization-mass spectrometry (CaR-ESI-MS) assay, implemented using picodiscs (complexes comprised of saposin A and lipids, PDs), to screen mixtures of glycolipids (GLs) against water-soluble proteins to detect specific interactions. To demonstrate the reliability of the method, seven gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD2, and GT1b) were incorporated, either individually or as a mixture, into PDs and screened against two lectins: the B subunit homopentamer of cholera toxin (CTB5) and a subfragment of toxin A from Clostridium difficile (TcdA-A2). The CaR-ESI-MS results revealed that CTB5 binds to six of the gangliosides (GM1, GM2, GM3, GD1a, GD1b, and GT1b), while TcdA-A2 binds to five of them (GM1, GM2, GM3, GD1a, and GT1b). These findings are consistent with the measured binding specificities of these proteins for ganglioside oligosaccharides. Screening mixtures of lipids extracted from porcine brain and a human epithelial ce...