STRUCTURAL CHARACTERIZATION OF TOXIN-BINDING GANGLIOSIDES BY TLC/VC-FTMS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 鞘糖脂和神经节苷脂参与多种生物学过程，它们的生物学作用依赖于寡糖和神经酰胺部分的结构。我们使用了振动冷却(VC)MALDI-FTMS来检测不稳定物种，然后用Sori-CAD和IRMPD对它们进行有效的碎裂。GM1和GD1a神经节苷脂分别作为霍乱毒素和相关LTIIb毒素的转运受体。我们假设人类肠道细胞的GD1a神经节苷脂与脂筏无关，因为它的神经酰胺结构变化阻止了LTIIb-GD1a复合体的内吞作用。毒素受体的神经节苷脂结构被评估为调节这一功能的因素，包括神经酰胺链长度、饱和程度和羟化程度以及多糖组成。为了分析这些分子，我们使用了我们以前开发的薄层板与VC-MALDI-FTMS直接偶联的方法。这允许直接TLC-MALDI-FTMS，而不会因TLC板的表面不规则性而对FT的高分辨率和质量准确度产生不利影响。我们早期的报道描述了从极化的肠上皮细胞系T-84和猴肾Vero细胞中分离纯化神经节苷脂，并对毒素的生物学机制进行了功能研究。样品直接从TLC板表面以~0.2 mm的采样步长进行MALDI解吸。随后用Sori-CAD和IRMPD进行碎裂。观察到唾液酸化和高度岩藻糖化的鞘糖脂。对释放的脂类和多糖进行的GC/MS分析与这些观察结果一致。在最近的实验中，GM1被修饰了荧光标记，并对其在细胞内的交通进行了跟踪。Lencer的团队现在已经确定，只有带有不饱和酰基链的GM1才能有效地从PM到ER进行排序。毒素结合是必需的，但其他膜成分也是必需的，包括胆固醇和脂筏蛋白Flotillin。在BUSM质谱库中，GM1或荧光标记的GM1基于N-酰基脂肪链、饱和度等的差异正在通过高效液相色谱与电喷雾电离(ESI)QStar Pulsar I飞行时间(TOF)MS相结合进行研究，该MS结合了速度、灵敏度、高质量精度和高分辨率。MALDI和ESI MS也被用于验证荧光标记在细胞条件下是否稳定。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glycosphingolipids and gangliosides participate in diverse biological processes, and their biological roles are dependent on the structures of both the oligosaccharide and the ceramide portions. We have used vibrationally cooled (VC)MALDI-FTMS for the detection of labile species followed by their efficient fragmentation by SORI-CAD and IRMPD. GM1 and GD1a gangliosides serve as trafficking receptors for cholera toxin and the related LTIIb toxin, respectively. We assume that GD1a ganglioside of human intestinal cells is not associated with lipid rafts due to its ceramide structural variation, which prevents endocytosis of the LTIIb-GD1a complex. The toxin receptors' ganglioside structures were evaluated as a moderator of this function, including ceramide chain length, level of saturation and hydroxylation, as well as glycan composition. To analyze these molecules, our previously developed method of direct coupling of TLC plates with VC-MALDI-FTMS was used. This allows direct TLC-MALDI-FTMS without adversely affecting the FT high resolution and mass accuracy by the surface irregularity of the TLC plate. Our earlier reports have described ganglioside purification from polarized intestinal epithelial cell line T-84 and monkey kidney Vero cells and functional studies on the mechanism of toxin biology. The samples were MALDI-desorbed directly off TLC plate surfaces with ~0.2 mm sampling steps. Fragmentation was subsequently performed by SORI-CAD and IRMPD. Both sialylated and highly fucosylated glycosphingolipids were observed. GC/MS analysis of released lipids and glycans were consistent with these observations. In recent experiments, GM1 has been modified with a fluorescent label and its traffic within the cells has been followed. Lencer's group has now determined that only GM1 with unsaturated acyl chains sorts efficiently from PM to the ER. Toxin binding was required, but other membrane components were also required, including cholesterol and the lipid raft protein flotillin. In the BUSM Mass Spectrometry Resource, the difference based on N-acyl fatty acid chains, degree of saturations, etc. of GM1 or fluorescent labeled GM1 is being investigated by high performance liquid chromatography coupled to an electrospray ionization (ESI) QStar Pulsar i time-of-flight (TOF) MS which combines, speed, sensitivity, high mass accuracy and high resolution. MALDI and ESI MS are also being used to verify that the fluorescent label is stable under the cellular conditions.