Absolute Affinities from Quantitative Shotgun Glycomics Using Concentration-Independent (COIN) Native Mass Spectrometry

Duong T Bui; James Favell; Elena N Kitova; Zhixiong Li; Kelli A McCord; Edward N Schmidt; Fahima Mozaneh; Mohamed Elaish; Amr El-Hawiet; Yves St-Pierre; Tom C Hobman; Matthew S Macauley; Lara K Mahal; Morris R Flynn; John S Klassen

doi:10.1021/acscentsci.3c00294

Absolute Affinities from Quantitative Shotgun Glycomics Using Concentration-Independent (COIN) Native Mass Spectrometry

ACS Cent Sci. 2023 Jun 15;9(7):1374-1387. doi: 10.1021/acscentsci.3c00294. eCollection 2023 Jul 26.

Authors

Duong T Bui¹, James Favell¹, Elena N Kitova¹, Zhixiong Li¹, Kelli A McCord¹, Edward N Schmidt¹, Fahima Mozaneh¹, Mohamed Elaish^{2

3}, Amr El-Hawiet⁴, Yves St-Pierre⁵, Tom C Hobman^{2

6

7}, Matthew S Macauley^{1

6}, Lara K Mahal¹, Morris R Flynn⁸, John S Klassen¹

Affiliations

¹ Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Alberta, Canada.
² Department of Cell Biology, University of Alberta, Edmonton T6G 2H7, AB, Canada.
³ Poultry Diseases Department, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.
⁴ Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria 21561, Egypt.
⁵ Institut National de la Recherche Scientifique (INRS), INRS-Centre Armand-Frappier Santé Biotechnologie, Laval H7 V 1B7, QC, Canada.
⁶ Department of Medical Microbiology and Immunology, University of Alberta, Edmonton T6G 2E1, AB, Canada.
⁷ Li Ka Shing Institute of Virology, University of Alberta, Edmonton T6G 2E1, Alberta, Canada.
⁸ Department of Mechanical Engineering, Faculty of Engineering, University of Alberta, Edmonton T6G 1H9, Alberta, Canada.

Abstract

Native mass spectrometry (nMS) screening of natural glycan libraries against glycan-binding proteins (GBPs) is a powerful tool for ligand discovery. However, as the glycan concentrations are unknown, affinities cannot be measured directly from natural libraries. Here, we introduce Concentration-Independent (COIN)-nMS, which enables quantitative screening of natural glycan libraries by exploiting slow mixing of solutions inside a nanoflow electrospray ionization emitter. The affinities (K_d) of detected GBP-glycan interactions are determined, simultaneously, from nMS analysis of their time-dependent relative abundance changes. We establish the reliability of COIN-nMS using interactions between purified glycans and GBPs with known K_d values. We also demonstrate the implementation of COIN-nMS using the catch-and-release (CaR)-nMS assay for glycosylated GBPs. The COIN-CaR-nMS results obtained for plant, fungal, viral, and human lectins with natural libraries containing hundreds of N-glycans and glycopeptides highlight the assay's versatility for discovering new ligands, precisely measuring their affinities, and uncovering "fine" specificities. Notably, the COIN-CaR-nMS results clarify the sialoglycan binding properties of the SARS-CoV-2 receptor binding domain and establish the recognition of monosialylated hybrid and biantennary N-glycans. Moreover, pharmacological depletion of host complex N-glycans reduces both pseudotyped virions and SARS-CoV-2 cell entry, suggesting that complex N-glycans may serve as attachment factors.