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 introduceConcentration-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 knownKdvalues. 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 ofN-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 biantennaryN-glycans. Moreover, pharmacological depletion of host complexN-glycans reduces both pseudotyped virions and SARS-CoV-2
cell entry, suggesting that complexN-glycans may
serve as attachment factors.