Differentiation of glycosphingolipid-derived glycan structural isomers by liquid chromatography/mass spectrometry

作者：Karlsson Hasse; Halim Adnan; Teneberg Susann^*

来源：Glycobiology, 2010, 20(9): 1103-1116.

DOI：10.1093/glycob/cwq070

• endoglycoceramidase
• glycosphingolipid characterization
• graphitized carbon columns
• liquid chromatography
• mass spectrometry
• oligosaccharide isomers

Isolation and characterization of glycosphingolipids is of importance in many aspects of glycobiology, but is difficult to achieve due to the high degree of heterogeneity and isomerism present in these compounds. In this study, oligosaccharides obtained from non-acid glycosphingolipids by enzymatic digestion with endoglycoceramidase II of Rhodococcus sp. were analyzed by liquid chromatography/electrospray ionization mass spectrometry using graphitized carbon columns. Resolution of isomeric oligosaccharides was achieved, and the MS(2) analyses gave complete sequence information and allowed differentiation of linkage positions. Diagnostic cross-ring (0,2)A-type fragments have previously been described for GlcNAc substituted on C-4 and for 4-substituted Glc. Diagnostic cross-ring (0,2)A-type fragments were present in the MS(2) spectrum of the H type 2 (Fuc alpha 2Gal beta 4GlcNAc beta 4Gal beta 4Glc) pentasaccharide, but not in the MS(2) spectrum of H type 1 pentasaccharide (Fuc alpha 2Gal beta 3GlcNAc beta 4Gal beta 4Glc). Cross-ring (0,2)A-type fragments were also obtained from the 4-substituted Glc at the reducing end of the glycosphingolipid-derived oligosaccharides. Oligosaccharides of the globo-series (globotriaose (Gal alpha 4Gal beta 4Glc) and globotetraose (GalNAc beta 3Gal alpha 4Gal beta 4Glc)) and the isoglobo-series (isoglobotriaose (Gal alpha 3Gal beta 4Glc) and isoglobotetraose (GalNAc beta 3Gal alpha 3Gal beta 4Glc)) were also chromatographically resolved on the graphitized carbon column. Furthermore, diagnostic fragment ions from cross-ring (0,2)A-type cleavages were present in the MS(2) spectra of the globo-series oligosaccharides, having a Gal substituted on C-4. The applicability of this method on tissue-derived samples was demonstrated using a non-acid glycosphingolipid fraction from human gastric epithelium and a partially purified non-acid glycosphingolipid fraction from 8 x 10(7) bone marrow-derived mouse dendritic cells. Here, liquid chromatography/mass spectrometry of the oligosaccharides released by endoglycoceramidase allowed tentative identification of a number of glycosphingolipids ranging from tri- to nonaglycosylceramides.

• 出版日期2010-9