Abstract
Specific recognition of glycans by proteins is important in many biological processes and immune responses. Here we present a general approach for derivatizing free glycans with a novel linker MTZ (3-(methoxyamino)-propylamine added to a bioorthogonal-functional tetrazine tag) that exploits click chemistry to generate multiple platforms of glycan coupling. This derivatization preserves glycan integrity, is reversible and quantifiable, and incorporates a bioorthogonal tetrazine tag for click coupling. A library of ABO-(H) blood group MTZ-glycans was efficiently conjugated to avidin Luminex beads through a Biotin-PEG11-TCO (trans-cyclooctene) spacer, generating a multiplex array that was reproducibly interrogated in a high-throughput Luminex approach with multiple lectins and antibodies. We also rapidly profiled antiglycan IgG, IgM, and IgA antibodies in multiple, serially diluted human serum samples, revealing unique repertoires of antiglycan responses in each sera. Glycans were efficiently coupled to bovine serum albumin (BSA) at a high density (∼19-24 glycans/BSA) to generate a neoglycoprotein library that was useful in microarray formats that provided results equivalent to those obtained from the Luminex approach. Neoglycoproteins have many uses, including serving as acceptors for glycosyltransferases, as we demonstrate for assays of ST6Gal1 sialyltransferase. These facile and efficient technologies significantly expand the toolbox available to explore glycan-GBP interactions.