Sensitive quantitation of isoglobotriaosylceramide in the presence of isobaric components using electrospray ionization-ion trap mass spectrometry
Sensitive quantitation of isoglobotriaosylceramide in the presence of isobaric components using electrospray ionization-ion trap mass spectrometry
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DOI：
10.1093/glycob/cwm127
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发表时间：
2008-02-01
期刊：
影响因子：
4.3
通讯作者：
Levery, Steven B.
中科院分区：
文献类型：
--
作者：
Li, Yunsen;Zhou, Dapeng;Levery, Steven B.
异血红蛋白三聚神经酰胺（iGb(3)）是一种独特类型的T细胞，自然杀伤T细胞的刺激抗原。iGb(3)由哺乳动物抗原呈递细胞在溶酶体腔室中产生，是为数不多的明确识别的生物受体碳水化合物配体之一。糖缀合物结构多样性的一个主要来源是在相同的单糖单元之间形成不同键的可能性。Globotriaosylceramide （Gb(3)）是iGb(3)的天然异构体，这两种异构体经常同时存在于从哺乳动物细胞膜中提取的鞘糖脂混合物中。利用针对特定碳水化合物表位的单克隆抗体或通过明确的结构表征来区分这些异构体是可行的，这需要从克或几十克生物样品中分离出相对大量的纯化合物。然而，从少量生物样品中精确检测iGb(3)是回答进一步重要的生物学问题（如刺激NKT细胞）的先决条件，其中iGb(3)可能与含量更高的Gb(3)混合。在这里，我们描述了一种基于离子阱质谱的特异性和敏感性方法来区分iGb(3)和Gb(3)。我们还演示了其应用于定量iGb(3)在原型抗原呈递细胞，大鼠RBL-CD1d细胞中的数量，使用化学合成的短n-酰基链iGb(3)作为内标。该方法可能对免疫细胞的功能性鞘糖脂组学和鞘糖脂生物标志物分析具有广泛的意义。
Isoglobotriaosylceramide (iGb(3)) 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, iGb(3) 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 (Gb(3)) exists as a natural isomer for iGb(3), 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 iGb(3) from small amounts of biological samples, where it may be mixed with Gb(3) 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 iGb(3) from Gb(3). We also demonstrate its application to quantifying the amount of iGb(3) in a prototype antigen-presenting cell, rat RBL-CD1d cells, using a chemically synthesized short N-acyl chain iGb(3) as internal standard. This methodology may have wide implications for functional glycosphingolipidomics of immune cells and glycosphingolipid biomarker analysis.