Purification of natural neutral N-glycans by using two-dimensional hydrophilic interaction liquid chromatography × porous graphitized carbon chromatography for glycan-microarray assay.

Glycan microarray for studying carbohydrate-protein interactions requires diverse classes of well-defined glycan standards. In this study, a purification strategy was established based on two-dimensional hydrophilic interaction liquid chromatography and porous graphitized carbon chromatography (HILIC × PGC) for the acquisition of neutral N-glycan standards from natural source. A total of thirty-one N-glycan compounds including seven pairs of isomers with the amounts from 0.7 to 230.0 nmol were isolated from ovalbumin as the model glycoconjugate. The purified N-glycans covered high-mannose, hybrid as well as multi-antenna asymmetric complex types. The purity of majority of these N-glycans was higher than 90%. Detailed structures of the N-glycan compounds were verified via negative ion tandem MS analysis, in which specific diagnostic ions including D- and E-ions were used to identify isomeric and terminal fine structures. The tag-free glycan compounds with well-defined structures, purity and amounts were finally assembled on the glass slide through neoglycolipid technology. Microarray binding assay of purified glycans with WGA lectin indicated the potential of the established strategy in glycan library expansion and functional glycomics.