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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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。LTIIb在人肠细胞中没有活性，因为LTIIb- gd1a复合物不会从质膜逆行进入内质网。我们已经测试了GD1a脂质锚的结构变化解释了这种神经节苷脂作为运输受体的失败的想法。为了解决这个问题，我们正在使用我们之前开发的TLC板与振动冷却（VC） MALDI-FTMS直接耦合的方法(2)。这允许直接TLC- maldi - ftms，而不会因TLC板的表面不规则性而对FT高分辨率和质量精度产生不利影响。碰撞冷却对于稳定和检测完整的神经节苷脂是必要的（3-5）。先前的实验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(6)。我们描述了VC MALDI-FTMS的神经节苷脂分离和仪器参数(2)。在本研究中，将整个HP-TLC板或从TLC板上剪下的含有样品轨迹的条带附着在MALDI样品探针上，并将基质溶液沉积在分析物上。样品以~0.5 mm的取样步骤直接从TLC板表面进行MALDI解吸。在解吸/电离过程中，碰撞冷却气体将离子源中的压力提高到1-10毫巴区域。随后通过SORI-CAD和IRMPD技术（10.6 m CO2激光，200-400 ms）进行碎片化。采用Finnigan/Thermoquest GCQ离子阱质谱仪进行气相色谱-质谱分析。将神经节苷脂进行甲醇分解，然后进行n -乙酰化，最后加入TMS试剂。将GC峰进行整合，根据标准品的检测灵敏度进行调整，并以标准化量（摩尔）绘制。甲醇解后氯仿提取脂肪酸甲酯，分别进行分析。结果表明，两种细胞系神经节苷脂的神经酰胺结构除了寡糖头基组成不同外，还表现出明显的异质性。HP-TLC板的高分离效率允许在每个扫描步骤后观察许多同源物。这是通过分析纯？市售和合成神经节苷脂和全脑提取物。通过FTMS和GC-MS观察到T-84中神经节苷脂的集中化水平高于Vero细胞系，与传统的神经节苷脂结构相比，两者都具有延长的聚糖部分。振动冷却导致不稳定的唾液酸和集中的糖苷键稳定，这一特征对异质混合物的分析非常有利。不均匀薄层板表面解吸对质量精度和分辨率没有影响。通过应用InCAS技术可以克服空间电荷对质量精度的影响(7)。与SORI-CAD相比，IRMPD对GM1和T-84神经节苷脂的亲本离子和产物离子的裂解效率更高。需要从长度、不饱和程度、羟基化和分支等方面对神经酰胺部分进行结构解析，因为这些因素被认为是受感染细胞中毒素运输的原因。两种细胞系也表达了不同的脂肪酸分布。进一步的分析将集中在单个长链碱基和脂肪酸的表征上。先前的实验表明，通过优化SORICAD/IRMPD技术和/或在样品中掺杂碱性盐，神经酰胺内部的裂解是可行的(8)，现在通过Li盐增强脑侧蛋白的裂解证明了这一特性。1) Fujinaga， Y., Wolf， A.A., Rodigherio， C., Wheeler， H., Tsai， B., Allen， L., Jobling， M.G., Rapoport， T., Holmes， R.K., Lencer， W.I.。与脂链相关的神经节内酯介导霍乱和相关毒素从质膜到内质网Mol生物细胞的运输（2001）Ivleva V.B, Elkin， Y.N., Budnik B.A, Moyer， S.C， O'Connor， P.B, Costello，耦合薄层色谱与振动冷却基质辅助激光解吸/电离傅里叶变换质谱分析神经节苷脂混合物[j]。[3]张建军，张建军，张建军，等。激光解吸/电离傅里叶变换质谱法离子源在生物分子热稳定研究中的应用。化学通报，2004,36(6):684 - 612。4)张建军，张建军，张建军，等。高压基质辅助激光解吸/电离傅里叶变换质谱法测定神经节苷脂碎片的研究进展[j] .质谱学报，2001,15(5):182 -1868。Soc。[5]张建军，张建军，张建军，等。中国生物医学工程学报，2002,13(4):462 - 467。[6]吴志强，刘志强，刘志强，刘志强，刘志强。霍乱毒素对细胞信号转导的影响及其与极化上皮小泡样膜结构域的关系[j] .细胞生物学杂志，1998,14(1):917-927。8) micorodskaya， E., Costello， C.E.，第48届美国质谱学会会刊，2000

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. GM1 and GD1a gangliosides serve as trafficking receptors for cholera toxin and the related LTIIb toxin, respectively. LTIIb is not active in human intestinal cells because the LTIIb-GD1a complex does not move retrograde from the plasma membrane into the endoplasmic reticulum. We have tested the idea that structural variation in the GD1a lipid anchor explains the failure of this ganglioside to act as a trafficking receptor. To address this problem, we are using our previously developed method of direct coupling of TLC plates with vibrationally cooled (VC) MALDI-FTMS (2). 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 (3-5). Previous experiments have descibed ganglioside purification from polarized intestinal epithelial cell line T-84 and monkey kidney Vero cells and functional studies on the mechanism of toxin biology (6). We have described ganglioside separations and instrumental parameters for VC MALDI-FTMS (2). In the current study, the whole HP-TLC plate or the strip cut from TLC plate containing the sample track was attached to the MALDI sample probe and matrix solution was deposited on top of the analyte. The samples were MALDI- desorbed directly off TLC plate surfaces with ~0.5 mm sampling steps. Collisional cooling gas raises the pressure in the ion source up into the 1-10 mbar region during the desorption/ ionization event. Fragmentation was subsequently performed by SORI-CAD and IRMPD techniques (10.6 ¿m CO2 laser, 200-400 ms). GC-MS studies were performed on Finnigan/Thermoquest GCQ ion trap mass spectrometer. Gangliosides were subject to methanolysis, followed by N-acetylation, and addition of TMS reagent. GC peaks were integrated, adjusted according to the detection sensitivity for standards, and plotted as normalized amounts in moles. Fatty acid methyl esters also were analyzed separately after extraction with chloroform following the methanolysis. It was shown that, in addition to a variety of oligosaccharide headgroup compositions, the ceramide structure in gangliosides from both cell lines exhibit substantial heterogeneity. The high separation efficiency of the HP-TLC plate allowed for observation of numerous homologs following each scanning step. This was demonstrated by analysis of the ?pure? commercially available and synthetic gangliosides and whole brain extract. A higher level of ganglioside fucosylation was observed in the T-84 than in the Vero cell line by both FTMS and GC-MS, with both having an extended glycan moiety compared to traditional ganglioside structures. Vibrational cooling resulted in stabilization of the labile sialic acid and fucose glycosidic linkages, and this feature was highly advantageous for the analysis of the heterogenous mixtures. Mass accuracy and resolution were not affected by desorption from the uneven TLC plate surface. The effect of space charge on mass accuracy can be overcome by applying the InCAS technique (7). Compared to SORI-CAD, IRMPD demonstrated more efficient fragmentation of both parent and product ions for the GM1 and T-84 gangliosides. Structural elucidation of the ceramide moiety in terms of length, degree of unsaturation, hydroxylation and branching is required, as these factors are assumed to be responsible for the toxin traffic in an infected cell. The two cell lines also expressed different distributions of fatty acids. Further analysis will focus on characterization of individual long-chain bases and fatty acids. Previous experiments have shown that cleavage within the ceramide is feasible by optimization of SORICAD/IRMPD techniques and/or doping the samples with alkaline salts (8), and this property was now demonstrated by an enhancement of the cerebroside fragmentation with Li salt. 1) Fujinaga, Y., Wolf, A.A., Rodigherio, C., Wheeler, H., Tsai, B., Allen, L., Jobling, M.G., Rapoport, T., Holmes, R.K., and Lencer, W.I. Ganglioisides that Associate with Lipid Rafts Mediate Transport of Cholera and Related Toxins from the Plasma Membrane to Endoplasmic Reticulum Mol Biol Cell 2001, 14, 4783-4793 2) Ivleva V.B., Elkin, Y.N., Budnik B.A., Moyer, S.C., O'Connor, P.B., Costello, C.E. Coupling Thin Layer Chromatography with Vibrational Cooling Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry for the Analysis of Ganglioside Mixtures Anal. Chem. 2004, 76, 6484-6512 3) O'Connor, P.B., Costello, C.E. A High Pressure Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry Ion Source for Thermal Stabilization of Labile Biomolecules Rapid Commun. Mass Spectrom. 2001, 15, 1862-1868 4) O'Connor P.B., Mirgorodskaya, E., Costello, C.E. High Pressure Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry for Minimization of Ganglioside Fragmentation J. Am. Soc. Mass Spectrom. 2002, 13, 402-407 5) Mirgorodskaya, E., Costello, C.E. TLC/MALDI-FT-ICR MS and SORI-CAD Analysis of Glycosphingolipids Proc. of 47th ASMS Conf. on Mass Spectrom. and Allied Topics 1999 6) Wolf, A.A., Jobling, M.G., Wimer-Mackin, S., Madara, J.L., Holmes, R.K., and Lencer, W.I. Ganglioside Structure Dictates Signal Transduction by Cholera Toxin and Association with Caveolae-like Membrane Domains in Polarized Epithelia J. Cell Biol. 1998, 141, 917-927 7) O'Connor, P. B., Costello, C.E. Internal Calibration on Adjacent Samples (InCAS) with Fourier Transform Mass Spectrometry Anal. Chem. 2000, 72, 5881-5885 8) Mirgorodskaya, E., Costello, C.E. Proc. of 48th ASMS Conf. on Mass Spectrometry and Allied Topics 2000