De Novo Sequencing of Enoxaparin Oligosaccharides through HILIC-Q-cIM-MS: A Strategy for Resolving Complex Glycans

In comparison to the advances in genome and proteome biology, decoding the structures and functions of glycans remains a significant challenge, mainly due to the complexity and heterogeneity of biological glycans. In this study, we have established a HILIC-Q-cIM-MS platform to advance the in-depth structural characterization of saccharides. The approach is designed to separate molecules through liquid chromatography and ion mobility dimensions prior to MS analysis. Based on conventional cIM, we introduced slicing, slicing/slicing, and peak-dissecting procedures followed by MS/MS experiments to elucidate the sequences of individual species, which significantly leveraged the separation power. Using enoxaparin oligosaccharides as examples, we demonstrated the feasibility of resolving complicated isomer compositions and sequencing each component in the oligosaccharide mixtures. On the basis of our experimental results, a universal strategy for sequencing heterogeneous oligosaccharides is proposed. This systematic approach provides a novel concept and methodology for characterizing complicated natural and synthetic glycans, which can be applied to unveil structural-functional correlations.