Yin Alexandra Bili, Hawke David, Zhou Dapeng
Undergraduate Program, Rice University, USA.
J Vis Exp. 2013 Apr 16(74):4224. doi: 10.3791/4224.
Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition(1-2). They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Galα4Galβ4Glcβ1Cer) and isoglobotriaosylceramide (Galα3Galβ4Glcβ1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions(3-4). In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases(5). The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions(6). Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells(7-9). This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids(10-15), followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present(9). Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.
糖鞘脂（GSL）属于生物大分子的糖缀合物类别，其携带了诸如胚胎发育、信号转导和免疫受体识别等重要生物学过程的结构信息（1 - 2）。它们包含异构体形式的复杂糖部分，以及具有脂肪酸酰基链长度、不饱和度和羟基化等变化的脂质部分。碳水化合物和神经酰胺部分都可能具有生物学意义。例如，三己糖神经酰胺包括球三糖神经酰胺（Galα4Galβ4Glcβ1Cer）和异球三糖神经酰胺（Galα3Galβ4Glcβ1Cer），它们具有相同的分子量，但碳水化合物部分的糖连接不同，负责完全不同的生物学功能（3 - 4）。再举一个例子，已经证明对α - 半乳糖神经酰胺（一种针对不变自然杀伤T细胞的强效激动剂配体）的神经酰胺部分进行修饰，会改变它们在癌症和自身免疫性疾病动物模型中的细胞因子分泌谱和功能（5）。在免疫器官和细胞中对异构体进行结构分析的困难成为确定许多生物学功能的障碍（6）。在此，我们展示了一种用于相对简单、快速且灵敏地分析免疫细胞中糖鞘脂谱的方法的可视化版本（7 - 9）。该方法基于糖鞘脂的提取和化学修饰（全甲基化，见图5A，全甲基化反应后己糖的所有羟基被甲氧基取代）（10 - 15），随后使用基质辅助激光解吸/电离飞行时间质谱（MALDI - TOF/MS）和离子阱质谱进行后续分析。该方法完成一次完整分析需要5000万个免疫细胞。实验可在一周内完成。通过与合成标准品比较，可以描绘出各种糖鞘脂的相对丰度。当存在2 fmol的总异球三糖神经酰胺/球三糖神经酰胺混合物时，该方法测量球三糖神经酰胺异构体中1%异球三糖神经酰胺的灵敏度为9）。离子阱质谱可用于分析异构体。例如，要分析同一样品中球三糖神经酰胺和异球三糖神经酰胺的存在情况，可以利用糖鞘脂分子的碎片化在结构上区分两者（见图5）。此外，糖部分的化学修饰（通过全甲基化反应）提高了电离和碎片化效率，以实现更高的灵敏度和特异性，并增加了唾液酸残基的稳定性。糖鞘脂的提取和化学修饰可以在经典的认证化学通风橱中进行，质谱分析可以由配备离子阱质谱仪的核心设施完成。
