Development of micro-scale saccharide chain analysis of glycosphingolipids by liquid chromatography/mass spectrometry.

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Gangliosides are well known glycosphingolipids that have many biological functions such as the signals of differentiation or cell-cell interaction. Novel gangliosides have been found occasionally, however the amount is very low to determine the structure. The sugar structure is essential for biological activity of gangliosides, then we tried to improve the method of LC/ESI-MS for micro-assay of gangliosides. The ganglioside is the mixture of heterogeneous molecules due to the hydrophobic component such as fatty acids, and then we used normal phase column which separate depending upon the hydrophobicity.Ionization of mass spectrometer was negative ESI. Column was 15 cm x 1 mm i.d. silica gel. Solvent was 2-propanol/water containing 10 mM ammonium acetate. The bovine brain ganglioside was used as standard sample.The bovine brain gangilioside consist of ganglioside GM1, GD1a, GD1b, GT1b and some minor components, and then this is convenient to the ganglioside analysis as the model sample. Various additives of HPLC solvent such as tetramethylammonium salts and ammonium salts were tested. As a result, ammonium acetate showed good performance for LC/MS of gangliosides. The HPLC solvent with 10 mM ammonium acetate gave better results on the LC separation and the ionization of gangliosides. The ganglioside GM1a, GD1a and GD1b showed [M-H]^-, [M-2H]^<2-> and [M-2H]^<2-> as the molecular related ions, respectively. The molecular related ions from ganglioside GT1b were observed as [M-3H]^<3->, [M-2H]^<2->, [M+Na-3H]^<2->. Of course, a few minor glycosphingolipids and unknown compounds were found simultaneously.