title: Detecting Protein-Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS.
abstract: This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB5) and Shiga toxin type 1 B (Stx1B5) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB5exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B5does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB5or Stx1B5and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB5with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB5and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB5to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B5reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB5with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein-GL interactions is prone to false positives and false negatives and must be used with caution. Graphical Abstract <!-- [INSERT GRAPHICAL ABSTRACT TEXT HERE] -->.
authors.affiliation: Alberta Glycomics Center and Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada.
authors.forename: Ling
authors.initials: L
authors.lastname: Han
authors.affiliation: Alberta Glycomics Center and Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada.
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.affiliation: Alberta Glycomics Center and Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada. john.klassen@ualberta.ca.
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
keywords: Affinity; Electrospray ionization; Glycolipid; Interactions; Mass spectrometry; Micelles; Protein