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Glycobiology Advance Access published online on November 28, 2007
Glycobiology, doi:10.1093/glycob/cwm127
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Sensitive Quantitation of Isoglobotriaosylceramide in the Presence of Isobaric Components Using Electrospray Ionization-Ion Trap Mass Spectrometry

Yunsen Li1, Dapeng Zhou2,*, Chengfeng Xia3, Peng G. Wang3 and Steven B. Levery1,*

1 Department of Chemistry, University of New Hampshire, Durham, NH 03824
2 Department of Melanoma Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX 7455
3 Department of Biochemistry, The Ohio State University, Columbus, OH 43210

* To whom correspondence should be addressed: Dr. Steven B. Levery, Phone: 603-862-2529, email: slevery{at}cisunix.unh.edu

Received on June 21, 2007; accepted on November 19, 2007

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 pre-requisite 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.

Key words: glycosphingolipid / globotriaosylceramide / NKT cell / CD1d / thymocyte


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