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

DuongT.Bui,JamesFavell,ElenaN.Kitova,ZhixiongLi,KelliA.McCord,EdwardN.Schmidt,FahimaMozaneh,MohamedElaish,AmrEl-Hawiet,YvesSt-Pierre,TomC.Hobman,MatthewS.Macauley,LaraK.Mahal,MorrisR.Flynn,JohnS.Klassen
ACS Central Science Pub Date : 06/15/2023 00:00:00 , DOI:10.1021/acscentsci.3c00294
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 (Kd) 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 Kd 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.
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