Absolute Affinities from Quantitative Shotgun Glycomics Using Concentration-Independent (COIN) Native Mass Spectrometry
Duong T. Bui, James W. 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, M. R. Flynn, John S. Klassen
Abstract
High Resolution Image Download MS PowerPoint Slide 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 Co ncentration- In dependent (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.