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

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.