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

Hasse Karlsson 1 Adnan Halim 0 Susann Teneberg 1 0 Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine, University of Gothenburg , SE 413 45 Gteborg, Sweden 1 Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology, University of Gothenburg , P.O. Box 440, S-405 30 Gteborg, Sweden The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 1103 - Received on December 21, 2009; revised on March 29, 2010; accepted on May 3, 2010 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 (Fuc2Gal4GlcNAc4Gal4Glc) pentasaccharide, but not in the MS2 spectrum of H type 1 pentasaccharide (Fuc2Gal3GlcNAc4Gal4Glc). 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 (Gal4Gal4Glc) and globotetraose (GalNAc3Gal4Gal4Glc)) and the isoglobo-series (isoglobotriaose (Gal3Gal4Glc) and isoglobotetraose (GalNAc3Gal3Gal4Glc)) 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 glycosphingo1To whom correspondence should be addressed: Tel: +46-31-786-34-92; Fax: +46-31-413-190; e-mail: lipid fraction from human gastric epithelium and a partially purified non-acid glycosphingolipid fraction from 8 107 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. Introduction Glycosphingolipids are found in the outer leaflet of the cell membrane of virtually all vertebrate cells. These amphipathic compounds consist of a hydrophilic oligosaccharide chain and a hydrophobic ceramide moiety. The oligosaccharide part exhibits a great complexity due to variation of the constituent monosaccharides, carbohydrate sequence, binding position, anomeric configuration and branching. The expression of glycosphingolipids varies both quantitatively and qualitatively between different species, individuals of the same species, organs and cells, and undergoes specific changes in an age-dependent as well as disease-specific manner. Thus, glycosphingolipids represent potential biomarkers during development and for various diseases (Schnaar et al. 2008). Therefore, there is a need for sensitive and specific methods for detection and characterization of potential glycosphingolipid biomarkers in small amounts of biological samples. This study explores the use of liquid chromatography/electrospray ionization mass spectrometry (LC-ESI/MS) using graphitized carbon columns for analysis of oligosaccharides derived from non-acid glycosphingolipids by enzymatic digestion by endoglycoceramidase II of Rhodococcus sp. The chromatographic behavour and the fragmentation patterns in MS2 were first investigated using oligosaccharides from well-characterized pure non-acid reference glycosphingolipids. Resolution of isomeric oligosaccharides was obtained, and the MS2 analyses gave complete sequence information and allowed differentiation of linkage positions. The method was applied on mixtures of non-acid glycosphingolipids of human gastric epithelium and a partially purified non-acid glycosphingolipid fraction from mouse dendritic cells, which allowed identification of a number of glycosphingolipids ranging from H Karlsson et al. tri- to nonaglycosylceramides. Thus, this method gives valuable data about the glycan part of glycosphingolipids and is suitable for analyses of glycosphingolipid mixtures. Results Endoglycoceramidase digestion Reference glycosphingolipids were incubated with endoglycoceramidase II from Rhodococcus sp. at 37C for 48 h. After 3 and 48 h, 4 g from the incubation mixtures was analyzed by thin-layer chromatography and chemical detection. As shown in Figure 1A, lane 1, no iGb3 was detected in the sample incubated with endoglycoceramidase II after 48 h. In contrast, considerable amounts of Gb3 remained (Figure 1A, lane 2) in line with the reported relative resistance of globo-series glycosphingolipids to hydrolysis by the Rhodococcus endoglycoceramidase II (Ito and Yamagata 1989; Li et al. 2009). |----48 h---||----0 h---| |----------0 h-----------| |-----------3 h------------| |----------48 h------------| Fig. 1. Hydrolysis of reference glycosphingolipids by endoglycoceramidase II from Rhodococcus sp. The glycosphingolipids were incubated with Rhodococcus endoglycoceramidase II at 37C as described under Materials and methods. After 3 and 48 h, 8 L was taken off the incubation mixtures and analyzed by thin-layer chromatography and anisaldehyde detection. The glycosphingolipids were separated on silica gel plates, using chloroform/ methanol/water 60:35:8 (by volume) as solvent system. The lanes on the chromatogram in (A) were: lane 1, isoglobotriaosylceramide (iGb3; Gal3Gal4Glc1Cer) after 48 h endoglycoceraminidase digestion, 4 g; lane 2, globotriaosylceramide (Gb3; Gal4Gal4Glc1Cer) after 48 h endoglycoceraminidase digestion, 4 g; lane 3, reference iGb3, 4 g; lane 4, reference Gb3, 4 g. The lanes on the chromatograms in (BD) were: lane 1, globotetraosylceramide (Gb4; GalNAc3Gal4Gal4Glc1Cer), 4 g; lane 2, isoglobotetraosylceramide (iGb4; GalNAc3Gal3Gal4Glc1Cer), 4 g; lane 3, blood group A type 1 heptaglycosylceramide (GalNAc3(Fuc2) Gal3(Fuc4)GlcNAc3Gal4Glc1Cer), 4 g; lane 4, blood group A type 2 heptaglycosylceramide (GalNAc3(Fuc2)Gal4(Fuc3) GlcNAc3Gal4Glc1Cer), 4 g; lane 5, non-acid glycosphingolipids of human gastric epithelium, 40 g. The reference glycosphingolipids are shown on the chromatogram in (B), while the chromatograms in (C) and (D) show the glycosphingolipid samples after 3 and 48 h endoglycoceraminidase digestion, respectively. The arrows to the right of the chromatograms denote the mobil (...truncated)