A versatile strategy for high-resolution separation of reducing glycan mixtures as hydrazones by two-dimensional high-performance liquid chromatography

Glycomics analysis has been undermined by the lack of structurally defined individual glycans as model compounds. However, it is challenging to prepare individual glycans from natural resources, mainly due to separation difficulties caused by highly diverse structure, complicated mixture form and chromophore-free property of naturally-existing glycans. In this study, we report a simple, universal and low-cost glycan separation strategy, glycoselection, which allows preparation of individual reducing glycans from their mixtures through reversible chromogenic derivatization by hydrazide chemistry in combination with two-dimensional high-performance liquid chromatography (2D-HPLC). Investigations on reaction conditions using lactose and maltodextrin as model glycans showed the feasibility of reversible hydrazide labeling and one-pot hydrazone conversion under mild conditions, the good stability of hydrazone-form derivatives of glycans in solution and the difference among seven selected hydrazine-carrying chromogenic reagents in product yields during glycan labeling and post-column detagging. The 2D-HPLC separation conditions were established on maltodextrin, from which fourteen highly-purified individual reducing oligo-glucans were ultimately obtained. Using this strategy, we also successfully prepared and identified eleven individual neutral reducing N-glycans from chicken ovalbumin and thirteen individual neutral reducing oligosaccharides and eight individual sialylated ones from human milk, demonstrating its good applicability to different types of reducing glycans as well as biological samples. Given the compatibility of individual reducing glycans with almost all of glycan derivatization protocols and analytical techniques of glycans and the potential of the method for larger scale application, this work provides a universal approach to compound-specific analysis of natural glycans and has great significance for glycomics studies.