VIBRATIONALLY COOLED MALDI FTMS & TLC MALDI FTMS FOR GANGLIOSIDE ANALYSIS

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 研究中心，而研究中心不一定是研究者所在的机构。 我们先前开发的薄层色谱（TLC）与振动冷却（VC）MALDI-FTMS直接耦合的方法允许简单处理具有FT高分辨率的TLC，其不受TLC板的不规则表面的影响。使用振动冷却对于神经节苷脂的稳定和检测是必要的。我们现在已经表明，这种技术的优化导致直接从TLC板解吸的离子的高灵敏度。此外，在将离子转移到池中之前，离子的积累允许应用内部校准技术以进行准确的质量分配。通过TLC分离神经节苷脂标准品，并在MS分析之前确定斑点的位置。将基质溶液喷雾到TLC板上，并将神经节苷脂直接从TLC板表面MALDI解吸。在自行研制的FT-ICR质谱仪上，采用倍频355 nm的Nd：YAG（UV）激光器、337 nm的氮激光器和2940 nm的Er：YAG（IR）激光器，进行了MALDI-FTMS实验。碰撞冷却气体在解吸-电离事件期间升高离子源中的压力。使用从不锈钢板上解吸的标准品作为校准品，对相邻样品进行内部校准（InCAS）。从TLC中解吸的神经节苷脂的灵敏度远低于1 pmole（分离前在TLC板上点样的总量）。InCAS的使用已证明，分析物从不均匀的TLC板表面解吸后，准确度不受影响，神经节苷脂标准品的质量准确度通常为0.5-1.5 ppm。研究了冷却气体类型、与电离-脱附过程相协调的气体脉冲、冷却气体压力、气体脉冲持续时间、不同的激光强度、聚焦和光束轮廓等参数对分子稳定化的影响。以尿素为基质的神经节苷脂的软IR-解吸，连同在振动冷却源中的冷却，消除了唾液酸损失。然而，基质对神经节苷脂碎片化程度的影响是显著的，如通过使用尿素作为基质的解吸与直接从石墨涂覆的不锈钢板解吸的比较所证明的。直接来自TLC板的神经节苷脂的IR-MALDI不如UV-MALDI有效，并且灵敏度相对较低，显然是因为基质由于其结晶模式而难以掺入TLC板表面的主体中。已对离子源进行了修改，以减少抽气时间，从而允许使用具有高蒸气压的基质，例如甘油。所开发的方法正被应用于生物系统的样品，如全脑神经节苷脂混合物，利什曼原虫脂质，神经元细胞的富含神经节苷脂的脂筏。红外多光子解离（CO2激光，10.7 μ m）和SORI-CAD被用作互补解离方法。由于大面积XY载物台可以轻松耦合到许多表面技术，不仅TLC，而且聚丙烯酰胺凝胶电泳（PAGE）和表面等离子体共振（SPR）-未来的研究将扩大这些分析方法的应用范围。

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. Our previously developed method of direct coupling of thin layer chromatography (TLC) with Vibrationally-Cooled (VC) MALDI-FTMS allows simple handling of TLC with FT high resolution which is not affected by the irregular surface of the TLC plate. Use of vibrational cooling is necessary for ganglioside stabilization and detection. We have now shown optimization of this technique leads to high sensitivity for the ions desorbed directly from the TLC plate. Furthermore, accumulation of the ions prior to transferring them into the cell allows application of an internal calibration technique for accurate mass assignment. The ganglioside standards are separated by TLC and the positions of the spots are determined prior to MS-analysis. The matrix solution is sprayed onto the TLC plate and the gangliosides are MALDI- desorbed directly from the TLC plate surface. MALDI-FTMS experiments have been carried out on our home-built FT-ICR mass spectrometer with 7T magnet and Nd:YAG (UV) laser, frequency tripled to 355 nm, nitrogen laser (337 nm) and Er:YAG (IR) laser (2940 nm). The collision cooling gas raises the pressure in the ion source during the desorption-ionization event. Internal Calibration on Adjacent Samples (InCAS) is performed using standards desorbed from the stainless steel plate as calibrants. The sensitivity the has been obtained for the gangliosides desorbed from TLC is well below 1 pmole (total amount spotted on a TLC plate prior to separation). The use of InCAS has demonstrated that accuracy is not affected upon desorption of the analyte from uneven TLC plate surface and mass accuracy is usually 0.5-1.5 ppm for ganglioside standards. The effect of several parameters on molecule stabilization has been studied, such as the type of the cooling gas, gas pulsing coordinating with ionization-desorption event, cooling gas pressure, duration of gas pulse, different laser intensities, focusing and beam profile. Soft IR-desorption of the gangliosides with urea as the matrix, together with cooling in the Vibrationally Cooled source eliminated sialic acid loss. However, the matrix effect on the extent of the ganglioside fragmentation was significant, as was demonstrated by the comparison of desorption using urea as a matrix versus desorption directly from the graphite-coated stainless steel plate. IR-MALDI of the gangliosides directly from the TLC plate was not as efficient as UV-MALDI and the sensitivity was relatively low, apparently because the matrices were poorly incorporated into the bulk of the TLC plate surface, due to their crystallization patterns. Modifications of the ion source have been made to decrease the pump-down time to allow use of matrices with high vapor pressurre, such as glycerol. The developed method is being applied to the samples from biological systems such as whole brain ganglioside mix, Leishmania lipids, and ganglioside rich lipid rafts from neuronal cells. Infrared multiphoton dissociation (CO2 laser, 10.7 um) and SORI-CAD are being used as complementary dissociation methods. Since the large area XY stage allows facile coupling to many surface techniques, not only TLC, but also polyacrylamide gel electrophoresis (PAGE) and surface plasmon resonance (SPR)- future studies will extend the range of applications to these analytical methods as well.