Mass spectrometric analysis of glycosphingolipid antigens.
Mass spectrometric analysis of glycosphingolipid antigens.
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DOI：
10.3791/4224
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发表时间：
2013-04-16
期刊：
影响因子：
--
通讯作者：
Zhou D
中科院分区：
文献类型：
--
作者：
Yin AB;Hawke D;Zhou D
鞘糖脂（GSL）属于生物大分子的糖缀合物类，其承载重要生物过程（例如胚胎发育、信号转导和免疫受体抑制）的结构信息1 -2。它们含有异构体形式的复杂糖部分和具有变化的脂质部分，包括脂肪酰基链长、不饱和度和羟基化。碳水化合物和神经酰胺部分可能是生物学意义的基础。例如，三己糖神经酰胺包括三己糖神经酰胺（Galα4Galβ4Glcβ1Cer）和异三己糖神经酰胺（Galα3Galβ4Glcβ1Cer），它们具有相同的分子量，但碳水化合物部分的糖键不同，导致完全不同的生物学功能3 -4。在另一个实例中，已经证明，α-半乳糖基神经酰胺的神经酰胺部分（一种用于不变NKT细胞的有效激动剂配体）的修饰改变了它们在癌症和自身免疫疾病的动物模型中的细胞因子分泌谱和功能5。对免疫器官和细胞中的异构体进行结构分析的困难是确定许多生物功能的障碍6。在这里，我们提出了一种相对简单，快速和灵敏的免疫细胞中鞘糖脂谱分析方法的可视化版本7 -9。该方法基于鞘糖脂的提取和化学修饰（全甲基化，见下图5A，全甲基化反应后己糖的所有OH基团均被MeO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。这种方法需要5000万个免疫细胞才能进行完整的分析。实验可以在一周内完成。各种鞘糖脂的相对丰度可以通过与合成标准品进行比较来描绘。当存在2 fmol的总iGb 3/Gb 3混合物时，该方法具有测量Gb 3异构体中的1% iGb 3的灵敏度9。离子阱质谱法可用于分析异构体。例如，为了分析相同样品中globotriaosylceramide和isobotriaosylceramide的存在，可以使用鞘糖脂分子的片段化来在结构上区分两者（参见下图5）。此外，糖部分的化学修饰（通过全甲基化反应）提高了电离和片段化效率以获得更高的灵敏度和特异性，并增加了唾液酸残基的稳定性。鞘糖脂的提取和化学修饰可以在经典认证的化学罩中进行，质谱分析可以通过具有离子阱MS仪器的核心设施进行。
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 recognition1-2. 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 functions3-4. 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 diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6. Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. 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 glycosphingolipids10-15, 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 present9. 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.
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影响因子：
4.4
作者：
Li Y;Thapa P;Hawke D;Kondo Y;Furukawa K;Furukawa K;Hsu FF;Adlercreutz D;Weadge J;Palcic MM;Wang PG;Levery SB;Zhou D
通讯作者：
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通讯作者：
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