Heparan Sulfate Separation, Sequencing, and Isomeric Differentiation: Ion Mobility Spectrometry Reveals Specific Iduronic and Glucuronic Acid-Containing Hexasaccharides

Abstract

We describe the resolution of heparan sulfate (HS) isomers by chromatographic methods and their subsequent differentiation by mass spectrometry (MS), ion mobility, and 1 H-NMR analysis. The two purified hexasaccharide isomers produced nearly identical MS spectra, quantitative disaccharide profiles, and partial enzymatic digestions. However, both tandem spectrometry (MS 2 ) and ion mobility spectrometry (IMS) indicated structurally differences existed. All data suggested the distinction between the two hexasaccharides resided in their uronic acid stereochemistries. Glucuronic (GlcA) and iduronic acids (IdoA) were subsequently defined by 1 H-NMR analysis completing the structural analysis and verifying the unique structures initially indicated by MS 2 and IMS. Our results suggest that IMS may be a powerful tool in the rapid differentiation of GlcA and IdoA containing isomers in the absence of prior structural knowledge.

Preparation, Separation, and Conformational Analysis of Differentially Sulfated Heparin Octasaccharide Isomers Using Ion Mobility Mass Spectrometry

“…Both chemically modified heparin octasaccharide and dodecasulfated heparin octasaccharide libraries were separated by strong anion exchange chromatography (SAX) 27, 32 . Briefly, SAX analysis was performed on a Waters Delta 600 system (Waters Corp., Milford, Ma) coupled to a UV-VIS spectrophotometer set at 232nm.…”

Structural Separations by Ion Mobility-MS for Glycomics and Glycoproteomics

“…N-linked and O-linked glycans removed from glycoproteins have also been characterized by IM-MS from purified samples (68, 69) after separation and extensive purification from serum (22) or urine (48). In addition, sulfated glycans were resolved by IM-MS through the differentiation of isomers (70), interpretation of collision cross sections with molecular modeling (71), and interactions with defensin inspired peptides (72). N-glycan structure and glycosylation sites for IgG have been determined using IM-MS/MS (68), and intact glycosylated IgG antibodies have also been analyzed using IM-MS to differentiate IgG1 and two different isoforms of IgG2 (73).…”

Analysis of Glycosaminoglycans Using Mass Spectrometry

“…Heparin lyase III deolymerizes the low degree of sulfation domains while leaving the high degree of sulfation domains intact. An HS samples was partially depolymerized using polysaccharide heparin lyase III, subjected to SEC and then to high resolution strong anion exchange (SAX) chromatography (94). MS analysis showed the presence of the same hexasaccharide composition in two different SAX fractions, indicating the existence of two structural isomers.…”

“…Binding between chemokines and heparan sulfate is essential for formation chemokine gradients. ESI MS has been used to study binding between heparin and heparan sulfate oligosaccharides (94, 125–127, 130, 131). Recently, ESI MS was used to probe competitive interactions between C-C motif chemokine 7 (CCL7), a peptide derived from chemokine receptor CCR2 and the synthetic heparin pentasaccharide Arixtra (see Figure 2 for structures) (132).…”