Favell, James; Bui, Duong T; Li, Jianing; Han, Ling; Kitova, Elena N.; Schmidt, Edward N.; Brassard, Raelynn; Kitov, Pavel I; St-Pierre, Yves 
ORCID: https://orcid.org/0000-0002-1948-2041; Mahal, Lara K; Lemieux, M Joanne; Macauley, Matthew S. et Klassen, John S.
(2024).
Elusive Protein-Glycosphingolipid Interactions Revealed by Membrane Anchor-Assisted Native Mass Spectrometry
Journal of the American Chemical Society
, vol. ahead
.
DOI: 10.1021/jacs.4c05805.
(Sous Presse)
Résumé
Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging. The present work introduces MEmbrane ANchor-assisted nMS (MEAN-nMS) for the detection of low-affinity GBP-GSL complexes. The assay utilizes a membrane anchor, produced by covalent cross-linking of the GBP and a lipid in the membrane, to localize the GBP on the surface and promote GSL binding. Ligands are identified by nMS detection of intact GBP-GSL complexes (MEAN-nMS) or using a catch-and-release (CaR) strategy, wherein GSLs are released from GBP-GSL complexes upon collisional activation and detected (MEAN-CaR-nMS). To establish reliability, a library of purified gangliosides incorporated into nanodiscs was screened against human immune lectins, and the results compared with affinities of the corresponding ganglioside oligosaccharides. Without a membrane anchor, nMS analysis yielded predominantly false negatives. In contrast, all ligands were identified by MEAN-(CaR)-nMS, with no false positives. To highlight the potential of MEAN-CaR-nMS for ligand discovery, a natural library of GSLs was incorporated into nanodiscs and screened against human and viral proteins to uncover elusive ligands. Finally, nMS-based detection of GSL ligands directly from cells is demonstrated. This breakthrough paves the way for shotgun glycomics screening using intact cells.
| Type de document: | Article |
|---|---|
| Informations complémentaires: | Lemieux, M Joanne Macauley, Matthew S Klassen, John S United States Journal of the American Chemical Society J Am Chem Soc. 2024 Jul 25. doi: 10.1021/jacs.4c05805. |
| Mots-clés libres: | - |
| Centre: | Centre INRS-Institut Armand Frappier |
| Date de dépôt: | 27 juill. 2024 17:27 |
| Dernière modification: | 27 juill. 2024 17:27 |
| URI: | https://espace.inrs.ca/id/eprint/15899 |
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