Disaccharide analysis of glycosaminoglycan mixtures by ultra-high-performance liquid chromatography-mass spectrometry

Abstract

Glycosaminoglycans are a family of polysaccharides widely distributed in all eukaryotic cells. These polyanionic, linear chain polysaccharides are composed of repeating disaccharide units that are often differentially substituted with sulfo groups. The diversity of glycosaminoglycan structures in cells, tissues and among different organisms reflect their functional an evolutionary importance. Glycosaminoglycan composition and structure also changes in development, aging and in disease progression, making their accurate and reliable analysis a critical, albeit, challenging endeavor. Quantitative disaccharide compositional analysis is one of the primary ways to characterize glycosaminoglycan composition and structure and has a direct relationship with glycosaminoglycan biological functions. In this study, glycosaminoglycan disaccharides, prepared from heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate and neutral hyaluronic acid using multiple polysaccharide lyases, were fluorescently labeled with 2-aminoacridone, fractionated into 17 well-resolved components by reverse-phase ultra-performance liquid chromatography, and analyzed by electrospray ionization mass spectrometry. This analysis was successfully applied to cell, tissue, and biological fluid samples for the picomole level detection of glycosaminoglycan composition and structure.

Figure 1

Fluorophore derivatization reaction of unsaturated disaccharides with AMAC. A generalized GAG disaccharide is shown that can either be 1,3 or 1,4 linked and can contain a GalN or GlcN residue at the reducing end where W = H or glycosidic, X = H or SO₃⁻, Y = H or SO₃⁻ or glycosidic, and Z = Ac or SO₃⁻.

Figure 2

Effect of the different ammonium acetate concentrations on the retention time by UV detection at 255 nm. (A) Constant ammonium acetate concentration with methanol gradient. Eluent A was water/methanol (88/12, v/v), and eluent B was water/methanol (12/88, v/v). Both eluents contained the same concentration ammonium acetate either 20 mM (a), 40 mM(b), 60 mM(c), 80 mM(d) or 100 mM(e); (B) Ammonium acetate and methanol gradient. Eluent A was ammonium acetate solution either 20 mM (a), 40 mM (b), 60 mM (c), 80 mM (d), or 100 mM (e). Eluent B was methanol. 1. Tri S_HS, 2. NS6S_HS, 3. NS2SHS, 4. Tri S_CS, 5. NS_HS, 6. SB_CS, 7. 2S6S_HS, 8. SD_CS, 9. 6S_HS, 10. SE_CS, 11. 2S_HS, 12. 2S_CS, 13. 4S_CS, 14. 0S_HS, 15. 6S_CS, 16. 0S_HA, and 17. 0S_CS.

Figure 3

Mass specta of heparin/HS-derived disaccharide: (A) Tri S_HS, (B) NS6S_HS, (C) NS2S_HS (D) Tri S_CS, (E) NS_HS, (F) SB_CS, (G) 2S6S_HS, (H) SD_CS, (I) 6S_HS, (J) SE_CS, (K) 2S_HS, (L) 2S_CS, (M) 4S_CS, (N) 0S_HS, (M) 6S_CS, (O) 0S_HA, and (P) 0S_CS.

Figure 4

RP-UPLC-MS chromatograms of 17 AMAC-tagged disaccharide standards with TIC (A), UV_{255 nm} (B), and EIC (C) detection.

Figure 5

EIC of AMAC-tagged disaccharide analysis of GAGs from different biological sources. (A) disaccharide standards; (B) disaccharides from camel liver; (C) disaccharides from camel urine. (D) disaccharides from CHO cells.