Disaccharide Analysis of Glycosaminoglycans Using Hydrophilic Interaction Chromatography and Mass Spectrometry

Abstract

Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) participate in many important biological processes. Quantitative disaccharide analysis of HS and CS/DS is essential for the characterization of GAGs and enables modeling of the GAG domain structure. Methods involving enzymatic digestion and chemical depolymerization have been developed to determine the type and location of sulfation/acetylation modifications as well as uronic acid epimerization. Enzymatic digestion generates disaccharides with Δ-4,5-unsaturation at the nonreducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS. This method enables biomedical researchers to determine complete disaccharide profiles on GAG samples using a single LC-MS platform.