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

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