Glycosaminoglycan glycomics using mass spectrometry

Abstract

The fact that sulfated glycosaminoglycans (GAGs) are necessary for the functioning of all animal physiological systems drives the need to understand their biology. This understanding is limited, however, by the heterogeneous nature of GAG chains and their dynamic spatial and temporal expression patterns. GAGs have a regulated structure overlaid by heterogeneity but lack the detail necessary to build structure/function relationships. In order to provide this information, we need glycomics platforms that are sensitive, robust, high throughput, and information rich. This review summarizes progress on mass-spectrometry-based GAG glycomics methods. The areas covered include disaccharide analysis, oligosaccharide profiling, and tandem mass spectrometric sequencing.

Fig. 1.

Workflow for analysis of HS/heparin polysaccharides. Biochemically isolated HS chains are subjected to disaccharide analysis. A typical procedure is to digest samples exhaustively using heparin lyases I, II, and III. Modeling of the HS domain structure is most effective when disaccharide analysis is performed on lyase digests performed in series and on deaminative cleavage products.

Fig. 2.

Methods for the depolymerization of heparan sulfate. A representative HS hexasaccharide is shown with sulfate groups highlighted in green, glucuronic acid carboxyl groups in yellow, iduronic acid carboxyl groups in red, and acetate groups in blue. Heparin lyase digestion produces disaccharides with Δ^4,5-unsaturated uronic acid for which information on the stereochemistry of C5 is lost. Deaminative cleavage produces disaccharides that contain anhydromannose (for CS GAGs, anhydrotalose is produced). Such disaccharides retain information on the stereochemistry of uronic acid C5 but lose information regarding the substitution of the hexosamine amino group.

Fig. 3.

The tendency for the loss of sulfate group equivalents during vibrational excitation depends on the charge and the nature of the ion pair. A, protonated sulfate groups are readily lost as SO₃ during vibrational excitation. B, deprotonated sulfate groups are much more stable. Thus, the higher the density of negative charge, the lower the extent of SO₃ loss, and the greater the abundances of ions from glycosidic bond dissociation. C, pairing of a metal cation (X) also stabilizes sulfate groups and enables glycosidic bond cleavage.

Fig. 4.

Comparison of methods for CAD tandem MS of highly sulfated HS/heparin oligosaccharides. A, CAD tandem MS of the octasulfated pentasaccharide Arixtra, subjected to the replacement of N-sulfate groups with d₃-N-acetate groups as a 4− ion. Ions were supercharged with sulfolane (60). The data were acquired using an Agilent 6520 QTOF instrument. The ion labeled with a diamond is the precursor, and that with a star resulted from the loss of SO₃ from the precursor. Product ions are labeled using the widely accepted Domon–Costello nomenclature (65). B, CAD tandem MS of a synthetic octasulfated hexasaccharide adducted with 7 equivalents of Na as a 3− ion (59). Data were acquired using a Fourier transform ion cyclotron resonance mass spectrometer. The CAD product ion is indicated with a slash (/), open circles indicate SO₃ loss, and solid circles indicate the loss of two or more equivalents of SO₃.