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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 鞘糖脂和神经节苷脂参与多种生物过程，其生物学作用取决于寡糖和神经酰胺部分的结构。在这里，振动冷却（VC）MALDI-FTMS用于检测不稳定的物种，然后通过SORI-CAD和IRMPD进行有效的片段化。GM 1和GD 1a神经节苷脂分别作为霍乱毒素和相关LTIIb毒素的运输受体。我们假设GD 1a神经节苷脂的人肠细胞是不相关的脂筏，由于其神经酰胺结构的变化，这阻止了LTIIb-GD 1a复合物的内吞作用。毒素受体的神经节苷脂结构被评价为该功能的调节剂，包括神经酰胺链长度、饱和和羟基化水平以及聚糖组成。为了分析这些分子，使用我们先前开发的TLC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高分辨率和质量准确度产生不利影响。碰撞冷却对于稳定和检测完整的神经节苷脂是必要的。我们的早期报告描述了从极化肠上皮细胞系T-84和猴肾Vero细胞中纯化神经节苷脂，并对毒素生物学机制进行了功能研究。将样品以~ 0.2mm的取样步长直接从TLC板表面MALDI解吸。随后通过SORI-CAD和IRMPD进行片段化。观察到唾液酸化和高度岩藻糖基化的鞘糖脂。释放的脂质和聚糖的GC/MS分析与这些观察结果一致。目前正在研究一种在TLC上不迁移的组分，可能是蛋白质片段，因为它也与毒素结合。两份手稿正在编写中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. Here, vibrationally cooled (VC)MALDI-FTMS was used 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. Collisional cooling is necessary for stabilization and detection of intact gangliosides. 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. A fraction, which could be a protein fragment, that does not migrate on TLC is now being studied, since it also binds with the toxin. Two manuscripts are in preparation.