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 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 (Fucalpha2Galbeta4GlcNAcbeta4Galbeta4Glc) pentasaccharide, but not in the MS(2) spectrum of H type 1 pentasaccharide (Fucalpha2Galbeta3GlcNAcbeta4Galbeta4Glc). 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 (Galalpha4Galbeta4Glc) and globotetraose (GalNAcbeta3Galalpha4Galbeta4Glc)) and the isoglobo-series (isoglobotriaose (Galalpha3Galbeta4Glc) and isoglobotetraose (GalNAcbeta3Galalpha3Galbeta4Glc)) 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.
Karlsson
Hasse
H
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology, University of Gothenburg, P.O. Box 440, S-40530 Göteborg, Sweden.
Halim
Adnan
A
Teneberg
Susann
S
D000818:Animals; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003713:Dendritic Cells / Q000737:chemistry; D004912:Erythrocytes / Q000737:chemistry; D006028:Glycosphingolipids / Q000737:chemistry*; D006801:Humans; D007422:Intestines / Q000737:chemistry; D007536:Isomerism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine
D006028:Glycosphingolipids; D011134:Polysaccharides