VIBRATIONALLY COOLED MALDI FTMS & TLC MALDI FTMS FOR GANGLIOSIDE ANALYSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们以前开发的薄层色谱(TLC)与振动冷却(VC)MALDI-FTMS的直接耦合方法允许以FT高分辨率简单地处理TLC，而不受TLC板不规则表面的影响。使用振动冷却对于神经节苷脂的稳定和检测是必要的。我们现在已经证明，这项技术的优化导致了直接从TLC板上解吸的离子的高灵敏度。此外，在将离子转移到电池中之前，离子的累积允许应用内部校准技术来进行准确的质量分配。用薄层层析法分离神经节苷脂标准品，并在MS分析前确定斑点的位置。将基质溶液喷洒到TLC板上，神经节苷脂直接从TLC板表面MALDI解吸。MALDI-FTMS实验在自行研制的FT-ICR质谱仪上进行，分别采用7T磁体和三倍频的Nd：YAG(UV)激光、337 nm的氮气激光和2940 nm的Er：YAG(IR)激光。在解吸-电离过程中，碰撞冷却气体提高了离子源中的压力。对相邻样品(INCA)进行内部校准，使用从不锈钢板上解吸的标准作为校准剂。从TLC解吸的神经节苷脂的灵敏度远低于1pmole(分离前在TLC板上斑点的总量)。INCAS的使用表明，从不平坦的TLC板表面解吸分析物不会影响准确度，对于神经节苷脂标准，质量准确度通常为0.5-1.5ppm。研究了冷却气体类型、气体脉冲与电离-脱附事件的配合、冷却气体压力、气体脉冲持续时间、不同激光强度、聚焦和光束分布等参数对分子稳定性的影响。以尿素为基质的神经节苷脂的软红外解吸，加上在振动冷却源中的冷却，消除了唾液酸的损失。然而，基质对神经节苷脂碎裂程度的影响是显著的，通过使用尿素作为基质的解吸与直接从石墨涂层不锈钢板上解吸的比较证明了这一点。直接从薄层板层析得到的神经节苷脂IR-MALDI的效率不如UV-MALDI，灵敏度也相对较低，这显然是因为基质由于其结晶模式而不能很好地结合到TLC板表面的主体中。对离子源进行了改进，以减少泵浦时间，以允许使用具有高蒸汽压的基质，如甘油。开发的方法正应用于来自生物系统的样品，如全脑神经节苷脂混合物、利什曼原虫脂类和来自神经元细胞的富含神经节苷脂的脂筏。红外多光子解离(CO_2激光，10.7微米)和SORI-CAD被用作互补的解离方法。由于大面积的XY台可以方便地耦合到许多表面技术，不仅是TLC，而且还有聚丙烯酰胺凝胶电泳(PAGE)和表面等离子体共振(SPR)--未来的研究也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.