We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.

We here describe the online liquid chromatography (LC) electrospray ionization mass spectrometry (MS) of underivatized glycans using a nanoscale normal-phase amide column at a flow rate of 300 nL/min. Retention on the amide column is based on polar interactions of the oligosaccharide hydroxyl groups with the stationary phase, and thus, the retention time predictably increases with elongation of the oligosaccharide chain. The system is characterized by its high chromatographic resolution, which routinely allows the separation of isobaric structures. Separation of oligosaccharide mixtures over a 1-h range permits the detailed characterization of the different species by multiple ion selection and fragmentation steps using ion trap MS. The here presented miniaturization of the online-LC system to the nanoscale in combination with ion trap MS allows the detection of oligosaccharide species in a mixture at low-femtomole sensitivity. Online normal-phase nano-LC-MS of complex oligosaccharide mixtures further facilitates the sensitive and detailed structural analysis of oligosaccharides by overcoming the need for cumbersome and time-consuming derivatization procedures such as reductive amination for labeling with hydrophobic fluorophores or labeling with tritium. The method should be useful for the sensitive and quick analysis of glycosylation patterns and individual oligosaccharides from biotechnologically produced glycoproteins as well as scarcely available biological samples.