Glycobiology Advance Access originally published online on March 6, 2007
Glycobiology 2007 17(6):646-654; doi:10.1093/glycob/cwm024
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Lex in mice
2 Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK
3 Imperial College Genetics and Genomics Research Institute, London SW7 2AZ, UK
4 M-SCAN Ltd, Wokingham, Berks RG41 2TZ, UK
1 To whom correspondence should be addressed; Tel: +44 2075945219; fax: +44 2072250458; e-mail: a.dell{at}imperial.ac.uk
Received on December 14, 2006; revised on February 21, 2007; accepted on February 22, 2007
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
The current interest in applying systems biology approaches to studying an organism's form or function promises to reveal further insights into the role of glycosylation in cells and whole organisms. This has prompted the development of a rapid, sensitive method of profiling the glycan component of both glycosphingolipids and glycoproteins from a single sample. Here we report a new mass spectrometric screening strategy for characterizing glycosphingolipid-derived oligosaccharides, which can be integrated into an existing highly sensitive glycoprotein glycomics strategy. Using ceramide glycanase to release the glycans from glycosphingolipids, this method provides a reliable profile of the glycosphingolipid-derived glycans present in a sample and has revealed new glycan structures. Glycoproteins are also efficiently recovered using this method, allowing the subsequent analysis of glycoprotein-derived glycans by mass spectrometry. The high sensitivity of this glycomic screening method allowed us to directly characterize the sialyl Lex epitope from mouse brain for the first time, where it was observed on an O-mannose structure. Thus, we present a mass spectrometric method that allows glycomic screening of N- and O-glycans as well as glycosphingolipid-derived glycans from a single tissue.
Key words: glycan / glycosphingolipid / glycoprotein / mass spectrometry / method
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
The availability of entire genome sequences and the advent of transcriptomic and proteomic technologies have led to expression profiling of many genes and proteins within biological systems. Although the recent surge in this type of systems biology has yielded tremendous insights into cellular function and response to stimuli, it is becoming increasingly apparent that other components of the system must also be screened to elucidate the entire structural repertoire within the cell. Glycomics, the study of all the glycan structures produced by a defined cell, tissue, or organism, promises to be a major feature of this emerging field. Mass spectrometry (MS) has already established itself as a powerful tool in the study of glycosylation because of its sensitivity of detection and ability to analyze complex mixtures of glycans derived from a variety of organisms and cell lines (Dell 1987
; Sutton-Smith et al. 2000; Haslam et al. 2002; Zamfir and Peter-Katalinic 2004; Levery 2005; Haslam et al. 2006; Parry et al. 2006). Although these methods are very powerful and have considerably advanced our understanding of glycan structure and function, they tend to define the glycans from a single type of glycoconjugate such as glycoproteins. For glycomics to be integrated into systems biology initiatives, medium-to-high-throughput profiling methods that analyze glycans derived from more than one type of glycoconjugate must be developed. Manzi et al. (2000) have reported an NMR-based methodology that profiles all of the major glycan classes. However, NMR spectroscopy is relatively insensitive with respect to the amount of sample required for high-quality structural information (von der Lieth et al. 2004).
Glycolipids comprise a very important class of glycoconjugate and it is desirable to integrate their analysis into any system-scale glycomics initiative. Glycolipids are implicated in a wide range of cellular functions such as receptors, modulators of cellular differentiation and growth, and oncogenic transformation (Ozkara 2004). The ability of microbial glycolipids presented by CD1d to modulate the cytokine response of natural killer T cells promises enormous therapeutic potential for the treatment of human autoimmune diseases such as multiple sclerosis and rheumatoid arthritis (Kronenberg 2005; Kronenberg and Rudensky 2005; Van Kaer 2005). Furthermore, mutations in genes that encode enzymes involved in the catabolic pathway of glycolipids have been associated with human disorders such as Fabry disease, Gaucher disease, and Tay Sachs disease (Ozkara 2004; Raas-Rothschild et al. 2004; Jeyakumar et al. 2005). In these instances, a block in the breakdown of the glycan portion of the glycolipid causes accumulation of substrate in the lysosome and leads to a disease state and finally clinical symptoms.
Glycosphingolipids (GSLs) are a functionally important class of glycolipid found in the membranes of nearly all living cells, including some bacteria (Kolter and Sandhoff 1999; Olsen and Jantzen 2001), and consist of a glycoside of ceramide or myo-inositol-(1-O)-phosphoryl-(O-1)-ceramide (Levery 2005). Although the glycoside component is synthesized according to certain biosynthetic pathway limitations, a diverse range of structures is still possible. Effective protocols, such as thin-layer chromatography (TLC), have been extremely useful for analyzing GSLs, but there is still a requirement for a methodology that routinely delivers high-level structural information of the glycan component of the GSLs. In this study, we describe the development of a highly sensitive and rapid MS screening strategy for defining the glycosylation repertoire of GSLs from a wide range of tissues from mice, including brain, liver, kidney, and testes. Importantly, this method can be combined with existing MS protocols for N- and O-glycan analysis to yield a powerful means of investigating the action of a broad range of glycogenes in a systems biology context.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
Strategy for purifying and analyzing glycans from GSLs and glycoproteins
The overall strategy for extracting GSLs and glycoproteins from tissues and analyzing the glycome of these glycoconjugates is shown in Figure 1. Glycoconjugates are extracted from cells or tissues by homogenization and lysis with methanol and chloroform. Under these conditions, GSLs are soluble, whereas (glyco)proteins form precipitates and are pelleted by centrifugation. GSLs from the supernatant are further fractionated by phase partitioning into upper phase (relatively polar) and lower phase (relatively nonpolar) fractions, and these are separately digested with ceramide glycanase, an enzyme that cleaves the ß-glucosyl bond between the glycan and the ceramide moiety of the GSL. Cleavage of specific glycans occurs at various rates (Zhou et al. 1989
), so extended incubation times in the presence of excess enzyme have been employed to mitigate this issue. Separation of the glycans from the lipids is then achieved on the basis of differential hydrophobicity.
|
To obtain glycans from the glycoproteins, the pellet from the first centrifugation step is treated essentially the same as reported previously, except that guanidine–HCl is used to assist in the solubilization of the pellet (Haslam et al. 2006
; Parry et al. 2006). Glycans from GSLs or glycoproteins are permethylated and then analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS to obtain a profile of the glycans in the mixture. Additional structural information can be obtained by tandem MS (MS/MS) and linkage analysis.
Some GSLs from mouse tissues exhibit novel glycan structures
To demonstrate the versatility of the GSL glycome profiling method, a range of mouse tissues was analyzed. Figure 2 shows the MALDI-TOF mass spectra of GSL-derived glycans from mouse testes, liver, and kidney. To assist in the interpretation of MS/MS fragmentation, the reducing ends of the testes and kidney glycans were specifically tagged by deuteroreduction.
|
, galactose; 
, glucose; 
, GalNAc; 
, fucose; 
, NeuAc; 
, NeuGc.In mouse testes, a wide range of glycans was observed in the upper phase fraction after deuteroreduction and permethylation (Figure 2A). An abundant series of putative fucosylated glycans was present at m/z 1117, 1321, 1362, 1566, 1740, 1770, and 1811; as well as sialylated versions at m/z 1478, 1682, 1723, and 1927. Gangliosides GM3, GM1, and GD1 and a derivative of GM1 with a HexNAc extension (IV-HexNAc-GM1) were present in relatively minor amounts. The remaining series of ions in the MALDI profile at m/z 943 and 1188 were shown by electrospray ionization MS/MS (ESI-MS/MS) to be Gb4 and IV-HexNAc-Gb4 of the (iso)globo family of GSL (data not shown).
Sandhoff et al. (2005) recently demonstrated the presence of eight fucosylated GSLs (FGSLs) in mouse testes, which contained glycans corresponding to the ions at m/z 1117, 1321, 1362, and 1566 and their monosialylated versions at m/z 1478, 1682, 1723, and 1927, respectively (Figure 2A). In the current study, we also observed several additional molecular ions with compositions consistent with FGSLs. These glycans were analyzed by MS/MS to gain structural information. Figure 3 shows the ESI-MS/MS spectrum for the glycan at m/z 1740, and the fragmentation pattern is consistent with a difucosylated structure.
|
, fucose.The MALDI-TOF fingerprints of the upper phase GSL-derived glycans from rat and guinea pig testes also contain fucosylated species (data not shown). The rat testes GSLs contain fucosylated glycans with molecular ions at m/z 1566 and 1770, with their sialylated counterparts at m/z 1927 and 2131. In contrast, only one FGSL-derived glycan was detected in guinea pig (m/z 1478), corresponding to a glycan with composition NeuAcFucHex3HexNAc.
C57 mouse liver contained GM2 (NeuGc, N-glycolylneuraminic acid) as a major ganglioside in the upper phase GSL fraction with minor amounts of GM3 (NeuAc and NeuGc), GM2 (NeuAc, N-acetylneuraminic acid), and GM1 (NeuGc) (Figure 2B). The structures of other monosialogangliosides are likely to be HexNAc-GM1b (NeuAc and NeuGc) and Hex-HexNAc-GM1b (NeuGc) on the basis of composition, knowledge of biosynthetic pathways, and MS/MS analysis (data not shown). MS/MS confirmed the presence of Gb4 (data not shown).
The kidney GSLs were deuteroreduced, permethylated, and analyzed by MALDI-MS (Figure 2C). In the lower phase fraction, glycans with a composition Hex3-4HexNAc1-2 were observed, whereas in the upper phase fraction, ions corresponding to glycans Fuc0–1Hex5HexNAc2 were detected. MS/MS analysis of the ions revealed that the glycans belonged to the (iso)globo series and demonstrated that the most abundant glycan (m/z 1770) (FucHex5HexNAc2) contained a Lex epitope. Trace levels of GM3 (m/z 855; Figure 2C inset) were also confirmed by ESI-MS/MS.
MALDI-TOF analysis of GSL- and glycoprotein-derived glycans from mouse brain
An important feature of the method being described is its ability to be integrated into existing glycomics screening strategies. To demonstrate the effectiveness of the integrated glycomics method, the mouse brain was profiled for glycans derived from glycoproteins as well as GSLs. Figure 4 shows the MALDI-TOF profile of the upper phase GSL-derived glycans from the mouse brain after permethylation and indicates their putative structures. The glycan profile is consistent with the ganglioside series GM1, GD1, and GT1 with lower amounts of GQ1. Additional gangliosides GM2, GM3, and GD3 and globoside Gb4 were minor components of the mixture. All of the glycans were confirmed by ESI-MS/MS, and from this analysis, the positions of the sialic acids were determined.
|
The brain protein pellet obtained during the first steps of the GSL protocol (Figure 1) contains the N- and O-linked glycoproteins. The pellet was resuspended in a denaturing buffer and the proteins subsequently reduced, alkylated, and digested with trypsin. N-glycans were removed from the proteins by peptide N-glycosidase F (PNGase F), permethylated, and analyzed by MALDI-TOF MS. Figure 5A shows the N-glycan profile, which consists of predominantly high mannose-type glycans and bisected, core fucosylated bi-, tri-, and tetra-antennary glycans with sialic acid and fucose present on the antennae.
|
, mannose; 
, GlcNAc; 
, fucose; O-glycans were released from O-glycopeptides by reductive elimination, derivatized, and then analyzed by MALDI-MS (Figure 5B). The MALDI-TOF spectrum of the O-glycans from mouse brain revealed two series of ions corresponding to short core 1 and 2 mucin-type structures. The core 1 structures comprised the Tn antigen (m/z 534) and sialylated versions of this epitope (m/z 895 and 1256). The main core 2 structure (m/z 1157) carried a putative Lex epitope. The O-glycan structures were confirmed by MS/MS (data not shown). The new methodology showed a significant increase in sensitivity in the detection of O-mannose structures. In the MALDI-TOF spectrum of the permethylated O-glycans, intense ions were observed at m/z 534, 912, and 1099, corresponding to potential O-mannose glycans with composition HexHexNAc, FucHex2HexNAc, and NeuAcHex2HexNAc, respectively. An additional minor component was detected at m/z 1273 and corresponded to a novel putative O-mannose glycan with the composition NeuAcFucHex2HexNAc. MALDI-TOF/TOF analysis of the ion at m/z 1273 revealed diagnostic ions at m/z 275, 646 (double cleavage), and 1021, revealing that this glycan contained a potential sialyl Lex epitope (Figure 6). The ion at m/z 1067, which corresponds to the elimination of fucose, is evidence for this residue being attached at the 3'-position of the GlcNAc. Further support for this structure was obtained by deuteropermethylating the O-glycans from the same mouse brain. The ion at m/z 1273 was shifted to the expected m/z 1331, and ESI-MS/MS analysis also resulted in the expected fragment ions at m/z 670 and 1063 (data not shown). Linkage analysis was also carried out on the O-glycan sample and, consistent with O-mannose glycans, a 2-linked mannitol was observed (data not shown). This is the first direct evidence for the sialyl Lex epitope in mice.
|
R, mannitol; 
, fucose; |
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
We have developed a highly sensitive and rapid MS methodology to investigate the glycosylation of GSLs and glycoproteins from a single tissue or cell line. Previously, we demonstrated a method that profiles the N- and O-glycans of a sample but the GSLs were not characterized (Haslam et al. 2006
; Parry et al. 2006). The method described here fractionates the glycoproteins from the GSLs, allowing the glycome of both glycoconjugates to be analyzed from the same sample. In both cases, glycomic profiling of the glycoconjugates is based on the analysis of derivatized glycans which yield molecular ions at high sensitivity, using MALDI-TOF MS. Putative structures are assigned to each molecular ion on the basis of their usually unique composition for a given mass, knowledge of the biosynthetic pathways, and where possible, MS/MS analysis. Further analyses such as gas chromatography-MS and exoglycosidase digestions would be required for gaining information on the linkages, the identity of the sugars, and the anomeric configuration of the linkages.
The GSL method was demonstrated on mouse brain, kidneys, liver, and testes (Figures 2 and 4). High-quality MALDI data were obtained from <5% of the glycans purified from a single tissue or cell preparation. The increased sensitivity of the MS method identified some new GSL glycan structures as well as profiling of the major and minor components that have been seen previously using alternate technologies such as high-performance TLC or normal-phase high-performance liquid chromatography of fluorescently labeled glycans (Nakamura et al. 1988; Lanne et al. 1995; Muthing and Kemminer 1996; Neville et al. 2004). Using the MALDI-TOF profiling also enables information to be gained on the relative abundance of the glycans in a sample, especially when considering ions of similar m/z values. Hence it is extremely useful for detecting glycosylation changes even in minor components when comparing the same tissue from two different animals from the same species. Absolute quantitation could be achieved, if required, by supplementation of the glycomics profiling methodology with rigorous chromatographic profiling.
Although the method described here provides increased sensitivity in the characterization of GSL-derived glycan structures and allows quantitation, in the case of brain, the ratio of the gangliosides differed slightly from that observed by TLC. This is likely to be due to the partial desialylation of the NeuAc-NeuAc glycosidic bond caused by the low-pH of the ceramide glycanase buffer. Desialylation was not observed in stability tests using di-sialylated structures where the sialic acids were not linked to each other (data not shown).
An important feature of this method is that it can be integrated into existing MS screening strategies so that N- and O-glycans can be analyzed in conjunction with GSLs from a single tissue. Here, we purified the N- and O-glycans from mouse brain and analyzed them by MALDI-TOF MS. The N- and O-glycan profiles observed were comparable with data obtained previously, with the single exception that O-mannose structures are more prominent in the O-glycan profile achieved with the new strategy (Sutton-Smith et al. 2002). The applicability of this protocol has also been demonstrated using other tissues and other organisms.
As mentioned earlier, one significant improvement offered by the current method is in the recovery of O-mannose structures. This improved detection allowed the first direct characterization of sialyl Lex (m/z 1273 in Figures 5B and 6) in the mouse. Sialyl Lex, which has the structure NeuAc
2-3Galß1-4(Fuc1-3)GlcNAc, is important in the leukocyte rolling/neutrophil recruitment, as it acts as a ligand for the selectins which mediate the primary interactions of leukocytes to the endothelium wall during inflammation (Gawaz et al. 2005; Afshar-Kharghan 2006). The role of selectins has been described in other physiological events such as allergy, autoimmunity, and cancer (Steinhoff et al. 2006; Witz 2006). Current evidence for the presence of sialyl Lex on murine glycans is based on indirect experiments such as antibody binding and abolition of function by enzymatic digestion (Kobzdej et al. 2002; Lowe 2003). In addition, when the fucosyltransferase (FucT-VII) that forms most of the sialyl Lex in neutrophils was knocked out in a mouse model, this resulted in neutrophils that no longer bind to E- and P-selectin (Maly et al. 1996).
The identification of sialyl Lex on O-mannose contrasts with its apparent absence on other forms of glycan. Despite the high levels of sialylation and fucosylation on the N-glycans and mucin-type O-glycans, MALDI-TOF/TOF analyses found no evidence of sialyl Lex on these structures from mouse brain (data not shown). Indeed, it would appear that formation of sialyl Lex is an unfavorable event as Lex and the VIM-2 epitope [NeuAc-Gal-GlcNAc-Gal-(Fuc)-GlcNAc] were preferentially observed on N-glycans (data not shown).
The method documented here will facilitate future studies of normal and diseased tissues or cell lines by enabling the glycome of GSLs and glycoproteins to be easily screened from a single tissue. The high sensitivity and ability to gain considerable structural information from minor components in a mixture make this method an exciting advance in systems biology glycomics.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
Animals
CD1 and C57 black strains of mice, Sprague–Downey rats, and Dunkin Hartley guinea pigs were used in this study. Animals were kept under standard maintenance conditions on 12-h light/dark cycle. All experiments were carried out in accordance with national guidelines. Tissues were harvested, snap-frozen in liquid nitrogen, and stored at –80 °C.
GSL and glycoprotein preparation
Figure 1 summarizes the overall strategy for the structural studies employed. The extraction conditions are based on the work of Schnaar (1994). Tissues (typically 1 g) or cells (105–107 dependent on cell type) were disrupted using a Homogenizer X120 (Ingenieurbüro CAT, Staufen, Germany) in the presence of four volumes of ice-cold water. Methanol (2.67 volumes of aqueous volume, assuming 80% tissue weight is water) was added and vortexed. Chloroform (1.33 volumes of aqueous volume) was added, mixed, and then centrifuged (1550g, 10 min). The protein pellet was subsequently used for N- and O-glycan analysis, whereas the supernatant containing the GSLs was transferred to a fresh tube and water (0.173 volumes of supernatant) added. After mixing and centrifugation (1550g, 10 min), two phases were obtained. The lower phase was extracted twice with water and dried under nitrogen. The upper phase was loaded onto a conditioned tC18 Plus Sep-Pak cartridge (Waters, Elstree, Herts, UK) and washed with 50% (v/v) aqueous methanol. GSLs were eluted with methanol, followed by 50% (v/v) chloroform in methanol. The eluants were combined and dried under nitrogen.
Ceramide glycanase digestion and glycan purification
GSLs were resuspended in 50-mM sodium acetate buffer, pH 5.0, containing 0.1% (w/v) sodium cholate, and incubated with ceramide glycanase (100 mU; Calbiochem, Beeston, Nottingham, UK) at 37 °C for 24 h. The reaction mixture was extracted with 1-butanol and the aqueous phase loaded onto a conditioned C18 Sep-Pak column. The glycans in the unbound fraction were purified by Hypercarb chromatography (Packer et al. 1998) and freeze-dried. Permethylation of glycans was performed using the sodium hydroxide procedure (Dell et al. 1993).
Deuteroreduction of GSL-derived glycans
Glycans were deuteroreduced by 10 mg/mL sodium borodeuteride in 2 M ammonia solution (2 h, 25 °C). After neutralization with acetic acid, the borates were removed by coevaporation with 10% (v/v) acetic acid in methanol. Glycans were permethylated as described earlier.
Analysis of N- and O-glycans
The pelleted proteins from the first centrifugation step described earlier were reduced in 4 M guanidine–HCl (Pierce, Cramlington, Northumberland, UK), 0.6 M Tris–HCl buffer, pH 8.5, containing 2 mg/mL dithiothreitol. Reduction was performed at 50 °C for 2 h, and carboxymethylation was carried out by the addition of iodoacetic acid (5-fold molar excess over dithiothreitol) at room temperature for 1 h. Carboxymethylation was terminated by dialysis against 50 mM ammonium bicarbonate, pH 8.5, at 4 °C for 48 h, followed by lyophilization. N- and O-glycans were prepared from the alkylated glycoproteins, as described previously (Parry et al. 2006). Permethylation of glycans was performed as described earlier.
Mass spectrometry
MALDI-TOF data were acquired on a Voyager-DE sSTR mass spectrometer (PerSeptive Biosystems, Framingham, MA) in the reflectron mode with delayed extraction. Permethylated samples were dissolved in 10 µL of 80% (v/v) methanol in water, and 1 µL of dissolved sample was premixed with 1 µL of matrix [10 mg/mL 2,5-dihydroxybenzoic acid (DHB) in 80% (v/v) aqueous methanol] before loading onto a metal plate. ESI-MS/MS spectra were acquired using a quadrupole-TOF (Micromass, Manchester, UK) instrument. The permethylated glycans were dissolved in methanol before loading into a nanospray capillary coated with a thin layer of gold/palladium, inner diameter 2 µm (Proxeon, Odense, Denmark). A potential of 1.5 kV was applied to a nanoflow tip to produce a flow rate of 10–30 nL/min. The drying gas used was N2 and the collision gas was argon, with the collision gas pressure maintained at 10–4 mbar. Collision energies varied depending on the size of the carbohydrate, typically between 30 and 90 eV. The MALDI-TOF/TOF experiments were performed on a 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA) operated in reflectron positive ion mode. Both DHB and -cyano-4-hydroxycinnamic acid matrices [10 mg/mL in 50% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid] were used in conjunction with setting the potential difference between the source acceleration voltage and the collision cell at 1 kV to obtain different degrees and patterns of fragmentation.
|
Supplementary data |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/).
|
Conflict of interest statement |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
None declared.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
Special thanks to Ron Schnaar for advice and useful discussions. This work was funded by the Wellcome Trust Functional Genomics Initiative (Biological Atlas of Insulin Resistance), Consortium for Functional Glycomics (grant number GM62116), and the Biotechnology and Biological Sciences Research Council (BBSRC). B.T. is funded by the RCUK Basic Technology Programme (UK GlycoArrays Consortium grant). A.D. is a BBSRC Professorial Research Fellow.
|
Abbreviations |
|---|
DHB, 2,5-dihydroxybenzoic acid; ESI-MS/MS, electrospray ionization-MS/MS; FGSL, fucosylated glycosphingolipid; GSL, glycosphingolipid; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; PNGase F, peptide N-glycosidase F; TLC, thin-layer chromatography.
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
|---|
Afshar-Kharghan V. Leukocyte adhesion and thrombosis. Curr Opin Hematol (2006) 13:34–39.[ISI][Medline]
Dell A. F.A.B.-mass spectrometry of carbohydrates. Adv Carbohydr Chem Biochem. (1987) 45:19–72.
Dell A, Khoo KH, Panico M, McDowell RA, Etienne AT, Reason AJ, Morris HR. FAB-MS and ES-MS of glycoproteins. In: Glycobiology: a practical approach—Fukuda M, Kobata A, eds. (1993) Oxford: Oxford University Press. 187–222.
Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. J Clin Invest (2005) 115:3378–3384.[CrossRef][ISI][Medline]
Haslam SM, Gems D, Morris HR, Dell A. The glycomes of Caenorhabditis elegans and other model organisms. Biochem Soc Symp (2002) 69:117–134.[Medline]
Haslam SM, North SJ, Dell A. Mass spectrometric analysis of N- and O-glycosylation of tissues and cells. Curr Opin Struct Biol (2006) 16:584–591.[CrossRef][ISI][Medline]
Jeyakumar M, Dwek RA, Butters TD, Platt FM. Storage solutions: treating lysosomal disorders of the brain. Nat Rev Neurosci (2005) 6:713–725.[Medline]
Kobzdej MM, Leppanen A, Ramachandran V, Cummings RD, McEver RP. Discordant expression of selectin ligands and sialyl Lewis x-related epitopes on murine myeloid cells. Blood (2002) 100:4485–4494.
Kolter T, Sandhoff K. Sphingolipids—their metabolic pathways and the pathobiochemistry of neurodegenerative diseases. (1999) 38, Angewandte Chemie International Edition. 1532–1568.
Kronenberg M. Toward an understanding of NKT cell biology: progress and paradoxes. Annu Rev Immunol (2005) 23:877–900.[CrossRef][ISI][Medline]
Kronenberg M, Rudensky A. Regulation of immunity by self-reactive T cells. Nature (2005) 435:598–604.[CrossRef][Medline]
Lanne B, Olsson BM, Jovall PA, Angstrom J, Linder H, Marklund BI, Bergstrom J, Karlsson KA. Glycoconjugate receptors for P-fimbriated Escherichia coli in the mouse. An animal model of urinary tract infection. J Biol Chem (1995) 270:9017–9025.
Levery SB. Glycosphingolipid structural analysis and glycosphingolipidomics. Meth Enzymol (2005) 405:300–369.[ISI][Medline]
Lowe JB. Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr Opin Cell Biol (2003) 15:531–538.[CrossRef][ISI][Medline]
Maly P, Thall A, Petryniak B, Rogers CE, Smith PL, Marks RM, Kelly RJ, Gersten KM, Cheng G, Saunders TL, et al. The alpha(1,3) fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell (1996) 86:643–653.[CrossRef][ISI][Medline]
Manzi AE, Norgard-Sumnicht K, Argade S, Marth JD, van Halbeek H, Varki A. Exploring the glycan repertoire of genetically modified mice by isolation and profiling of the major glycan classes and nano-NMR analysis of glycan mixtures. Glycobiology (2000) 10:669–689.
Muthing J, Kemminer SE. Nondestructive detection of neutral glycosphingolipids with lipophilic anionic fluorochromes and their employment for preparative high-performance thin-layer chromatography. Anal Biochem (1996) 238:195–202.[CrossRef][ISI][Medline]
Nakamura K, Hashimoto Y, Yamakawa T, Suzuki A. Genetic polymorphism of ganglioside expression in mouse organs. J Biochem (Tokyo) (1988) 103:201–208.
Neville DC, Coquard V, Priestman DA, te Vruchte DJ, Sillence DJ, Dwek RA, Platt FM, Butters TD. Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling. Anal Biochem (2004) 331:275–282.[CrossRef][ISI][Medline]
Olsen I, Jantzen E. Sphingolipids in Bacteria and Fungi. Anaerobe (2001) 7:103–112.[CrossRef][ISI]
Ozkara HA. Recent advances in the biochemistry and genetics of sphingolipidoses. Brain Dev (2004) 26:497–505.[CrossRef][ISI][Medline]
Packer NH, Lawson MA, Jardine DR, Redmond JW. A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj J (1998) 15:737–747.[CrossRef][ISI][Medline]
Parry S, Hadaschik D, Blancher C, Kumaran MK, Bochkina N, Morris HR, Richardson S, Aitman TJ, Gauguier D, Siddle K, et al. Glycomics investigation into insulin action. Biochim Biophys Acta (2006) 1760:652–668.[Medline]
Raas-Rothschild A, Pankova-Kholmyansky I, Kacher Y, Futerman AH. Glycosphingolipidoses: beyond the enzymatic defect. Glycoconj J (2004) 21:295–304.[CrossRef][ISI][Medline]
Robinson S, Routledge A, Thomas-Oates J. Characterisation and proposed origin of mass spectrometric ions observed 30 Th above the ionised molecules of per-O-methylated carbohydrates. Rapid Commun Mass Spectrom (2005) 19:3681–3688.[CrossRef][ISI][Medline]
Sandhoff R, Geyer R, Jennemann R, Paret C, Kiss E, Yamashita T, Gorgas K, Sijmonsma TP, Iwamori M, Finaz C, et al. Novel class of glycosphingolipids involved in male fertility. J Biol Chem (2005) 280:27310–27318.
Schnaar RL. Isolation of glycosphingolipids. Meth Enzymol (1994) 230:348–370.[ISI][Medline]
Steinhoff M, Steinhoff A, Homey B, Luger TA, Schneider SW. Role of vasculature in atopic dermatitis. J Allergy Clin Immunol (2006) 118:190–197.[CrossRef][ISI][Medline]
Sutton-Smith M, Morris HR, Dell A. A rapid mass spectrometric strategy suitable for the investigation of glycan alterations in knockout mice. Tetrahedron Asymmetry (2000) 11:363–369.[CrossRef][ISI]
Sutton-Smith M, Morris HR, Grewal PK, Hewitt JE, Bittner RE, Goldin E, Schiffmann R, Dell A. MS screening strategies: investigating the glycomes of knockout and myodystrophic mice and leukodystrophic human brains. Biochem Soc Symp (2002) 69:105–115.[Medline]
Van Kaer L. alpha-Galactosylceramide therapy for autoimmune diseases: prospects and obstacles. Nat Rev Immunol (2005) 5:31–42.[CrossRef][ISI][Medline]
von der Lieth C-W, Bohne-Lang A, Lohmann KK, Frank M. Bioinformatics for glycomics: status, methods, requirements and perspectives. Brief Bioinform (2004) 5:164–178.
Witz IP. The involvement of selectins and their ligands in tumor-progression. Immunol Lett (2006) 104:89–93.[CrossRef][ISI][Medline]
Zamfir A, Peter-Katalinic J. Capillary electrophoresis–mass spectrometry for glycoscreening in biomedical research. Electrophoresis (2004) 25:1949–1963.[CrossRef][ISI][Medline]
Zhou B, Li SC, Laine RA, Huang RT, Li YT. Isolation and characterization of ceramide glycanase from the leech, Macrobdella decora. J Biol Chem (1989) 264:12272–12277.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Tissot, N. Gasiunas, A. K Powell, Y. Ahmed, Z.-l. Zhi, S. M Haslam, H. R Morris, J. E Turnbull, J. T Gallagher, and A. Dell Towards GAG glycomics: Analysis of highly sulfated heparins by MALDI-TOF mass spectrometry Glycobiology, September 1, 2007; 17(9): 972 - 982. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||