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

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 MS² analyses gave complete sequence information and allowed differentiation of linkage positions. Diagnostic cross-ring ⁰,²A-type fragments have previously been described for GlcNAc substituted on C-4 and for 4-substituted Glc. Diagnostic cross-ring ⁰,²A-type fragments were present in the MS² spectrum of the H type 2 (Fucα2Galβ4GlcNAcβ4Galβ4Glc) pentasaccharide, but not in the MS² spectrum of H type 1 pentasaccharide (Fucα2Galβ3GlcNAcβ4Galβ4Glc). Cross-ring ⁰,²A-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 (Galα4Galβ4Glc) and globotetraose (GalNAcβ3Galα4Galβ4Glc)) and the isoglobo-series (isoglobotriaose (Galα3Galβ4Glc) and isoglobotetraose (GalNAcβ3Galα3Galβ4Glc)) were also chromatographically resolved on the graphitized carbon column. Furthermore, diagnostic fragment ions from cross-ring ⁰,²A-type cleavages were present in the MS² 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 10⁷ 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.