Sequencing analysis of heparin reducing terminals with orthogonal chromatographic approaches

Heparin is a linear sulfated polysaccharide with a complex structure. It is important to figure out the sequences at the terminals of the sugar chains, as it will help us understand the heparin structure deeper and control its quality properly. The tetrasaccharide linkage region (LR) could be a tag to help us find out heparin terminals after digestion by different combinations of heparinases. In this work, orthogonal chromatographic approaches including SAX, SEC-MS and 2D-LC-MS were applied to qualitatively and quantitatively analyze the heparinase released LR-terminals. The disaccharides next to LR are those ones with low or non-sulfation, UA-GlcNAc and UA-GlcNAc6S, and then they are extended with the highly sulfated disaccharides, IdoA2S-GlcNS and IdoA2S-GlcNS6S. It is suggested that the sulfo transferases did not work at the sugar residues next to LR terminal, especially the 2-O-sulfo and N-sulfo transferases, which could be affected by steric hindrance from LR, when heparin is biosynthesized. This conclusion will be theoretical fundamental to help us understand heparin's structure deeper. The methods provided in this work could be potential ways to control heparin's quality and monitor the production processes of heparin properly.