Sensitive quantitation of isoglobotriaosylceramide in the presence of isobaric components using electrospray ionization-ion trap mass spectrometry.

Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA.
Glycobiology (Impact Factor: 3.75). 03/2008; 18(2):166-76. DOI: 10.1093/glycob/cwm127
Source: PubMed

ABSTRACT Isoglobotriaosylceramide (iGb3) is a stimulatory antigen for a unique type of T cell, Natural Killer T cells. Produced in the lysosomal compartment by mammalian antigen-presenting cells, iGb3 is one of the few clearly identified carbohydrate ligands for biological receptors. A major source of glycoconjugate structural diversity arises from the possibility of forming different linkages between the same monosaccharide units. Globotriaosylceramide (Gb3) exists as a natural isomer for iGb3, and both isomers are frequently found together in mixtures of glycosphingolipids extracted from mammalian cell membranes. Discriminating these isomers has been feasible using monoclonal antibodies raised against specific carbohydrate epitopes, or by unambiguous structural characterization, which requires relatively large amounts of pure compounds isolated from grams, or tens of grams, of biological samples. However, the precise detection of iGb3 from small amounts of biological samples, where it may be mixed with Gb3 present in much higher abundance, is a prerequisite for answering further important biological questions such as stimulation of NKT cells. Here we describe a specific and sensitive method based on ion trap mass spectrometry to discriminate iGb3 from Gb3. We also demonstrate its application to quantifying the amount of iGb3 in a prototype antigen-presenting cell, rat RBL-CD1d cells, using a chemically synthesized short N-acyl chain iGb3 as internal standard. This methodology may have wide implications for functional glycosphingolipidomics of immune cells and glycosphingolipid biomarker analysis.

Download full-text


Available from: Dapeng Zhou, Dec 18, 2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: A method for generation of novel fluorocarbon derivatives of glycosphingolipids (GSLs) with high affinity for fluorocarbon phases has been developed, and their potential applications to mass spectrometry (MS)-based methodologies for glycosphingolipidomics have been investigated. Sphingolipid ceramide N-deacylase (SCDase) is used to remove the fatty acid from the ceramide moiety, after which a fluorocarbon-rich substituent (F-Tag) is incorporated at the free amine of the sphingoid. In initial trials, a neutral GSL, globotriaosylceramide (Gb(3)Cer), three purified bovine brain gangliosides, and four fungal glycosylinositol phosphorylceramides (GIPCs) were de-N-acylated, derivatized by prototype F-Tags, and recovered by solid phase extraction on fluorocarbon-derivatized silica (F-SPE). The efficacy of SCDase treatment of GIPCs was here demonstrated for the first time. Compatibility with subsequent per-N,O-methylation was established for the F-tagged Gb(3) Cer and purified gangliosides, and extensive mass spectra (MS(1) and MS(2)) consistent with all of the expected products were acquired. The potential use of F-tagged derivatives for a comprehensive MS based profiling application was then demonstrated on a crude ganglioside mixture extracted from bovine brain. Finally, a simple trial in microarray format demonstrated fixation of F-tagged G(M1) ganglioside to a fluorous glass surface, with the glycan intact and available for interaction with a fluorescent derivative of cholera toxin B chain. The methods described thus provide a new avenue for rapid GSL recovery or cleanup, potentially compatible with a variety of platforms for mass spectrometric profiling and structure analysis, as well as parallel analysis of functional interactions.
    Journal of Mass Spectrometry 05/2010; 45(5):504-19. DOI:10.1002/jms.1734 · 2.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell-to-cell or cell-to-protein interactions. In this study, qualitative and quantitative analyses of GSL-derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL-derived glycans from the endothelial cells and islets, respectively, were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and collision-induced fragmentation using positive-ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the alpha-Gal-terminated GSL was not detected but NeuGc-terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL-derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI-TOF MS. The structural information of the GSL-derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation.
    Journal of Mass Spectrometry 10/2009; 44(10):1489-99. DOI:10.1002/jms.1638 · 2.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.
    The Journal of Lipid Research 03/2011; 52(4):618-34. DOI:10.1194/jlr.M010819 · 4.73 Impact Factor