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• W3129562318 abstract "Interactions between carbohydrates (glycans) and glycan-binding proteins (GBPs) regulate a wide variety of important biological processes. However, the affinities of most monovalent glycan–GBP complexes are typically weak (dissociation constant (Kd) > μM) and difficult to reliably measure with conventional assays; consequently, the glycan specificities of most GBPs are not well established. Here, we demonstrate how electrospray ionization mass spectrometry (ESI-MS), implemented with nanoflow ESI emitters with inner diameters of ∼50 nm, allows for the facile quantification of low-affinity glycan–GBP interactions. The small size of the droplets produced from these submicron emitters effectively eliminates the formation of nonspecific glycan–GBP binding (false positives) during the ESI process up to ∼mM glycan concentrations. Thus, interactions with affinities as low as ∼5 mM can be measured directly from the mass spectrum. The general suppression of nonspecific adducts (including nonvolatile buffers and salts) achieved with these tips enables ESI-MS glycan affinity measurements to be performed on C-type lectins, a class of GBPs that bind glycans in a calcium-dependent manner and are important regulators of immune response. At physiologically relevant calcium ion concentrations (2–3 mM), the extent of Ca2+ nonspecific adduct formation observed using the submicron emitters is dramatically suppressed, allowing glycan affinities, and the influence of Ca2+ thereon, to be measured. Finally, we show how the use of submicron emitters and suppression of nonspecific binding enable the quantification of labile (prone to in-source dissociation) glycan–GBP interactions." @default.
• W3129562318 created "2021-03-01" @default.
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• W3129562318 date "2021-02-25" @default.
• W3129562318 modified "2023-09-29" @default.
• W3129562318 title "Submicron Emitters Enable Reliable Quantification of Weak Protein–Glycan Interactions by ESI-MS" @default.
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• W3129562318 doi "https://doi.org/10.1021/acs.analchem.0c05003" @default.
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• W3129562318 hasPublicationYear "2021" @default.
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