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 non-reducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography (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.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring

“…A likely explanation for this phenomenon is anomeric interconversion. Similar findings have been reported previously [13]. These authors found that interconversion between the α and β forms occurred with one form predominating at increased temperature.…”

“…Depolymerization can be achieved using specific lyases which cleave the glycosidic linkage between a hexosamine and a hexuronic acid thus introducing an unsaturated bond (absorbing at 232 nm) between the C4 and C5 of the hexuronic acid. GAGs can also be depolymerized chemically by mild hydrazinolysis and subsequent deaminative cleavage with nitrous acid [13]. This method retains the original epimeric nature of the hexuronic acid but loses information about the N-sulfation or N-acetylation because the resulting disaccharide comprise a hexuronic acid linked to a 2,5-anhydrohexose bearing an aldehyde group [13].…”

“…GAGs can also be depolymerized chemically by mild hydrazinolysis and subsequent deaminative cleavage with nitrous acid [13]. This method retains the original epimeric nature of the hexuronic acid but loses information about the N-sulfation or N-acetylation because the resulting disaccharide comprise a hexuronic acid linked to a 2,5-anhydrohexose bearing an aldehyde group [13].…”

“…However, co-elution of disaccharides has been reported [13]. To overcome these issues, GAG disaccharides may be derivatized with a hydrophobic fluorophore which increases their retention on RPLC and may improve MS sensitivity.…”

“…The analysis of underivatized GAG disaccharides either by direct MS infusion or with specialized separation techniques including strong anion exchange [17], graphitized carbon LC [26] and HILIC columns [13] has been reported previously. The key advantage of these methods is that loss of disaccharides during derivatization is avoided but there are several disadvantages associated with direct analysis.…”