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

神经节苷脂是一种重要的鞘糖脂，具有多种生物学功能，如分化信号或细胞间相互作用。偶尔发现新的神经节苷脂，但数量非常低，无法确定结构。糖的结构是神经节苷脂生物活性的基础，因此我们尝试改进LC/ESI-MS方法用于神经节苷脂的微量测定。由于神经节苷脂中含有脂肪酸等疏水成分，因此神经节苷脂是多相分子的混合物，利用其疏水性，采用正相色谱柱进行分离，质谱仪采用负离子电喷雾电离。柱为15 cm x 1 mm i.d.硅胶。溶剂为含有10 mM乙酸铵的2-丙醇/水。以牛脑神经节苷脂为标准样品，牛脑神经节苷脂由神经节苷脂GM 1、GD 1a、GD 1b、GT 1b及少量组分组成，便于作为模型样品进行神经节苷脂分析。测试了HPLC溶剂的各种添加剂，例如四甲基铵盐和铵盐。结果表明，乙酸铵对神经节苷脂的LC/MS表现出良好的性能。含有10 mM乙酸铵的HPLC溶剂对神经节苷脂的LC分离和离子化具有更好的结果。神经节苷脂GM 1a、GD 1a和GD 1b的分子相关离子分别为[M-H]^-、[M-2 H]^<2->和[M-2 H]^<2->。神经节苷脂GT 1b的分子相关离子为[M-3 H]^<3->、[M-2 H]^<2->、[M+Na-3 H]^<2->。当然，同时也发现了少量的鞘糖脂和未知化合物。

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.