VIBRATIONALLY COOLED MALDI FTMS & TLC MALDI FTMS FOR GANGLIOSIDE ANALYSIS

振动冷却 MALDI FTMS

• 批准号: 7722952
• 负责人: Catherine E. Costello
• 金额: $ 4.41万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722952
• 关键词: Affect; Area; Biological; Brain; Calibration; Carbon Dioxide; Cells; Computer Retrieval of Information on Scientific Projects Database; Coupling; Crystallization; Detection; Dissociation; Event; Frequencies; Funding; Future; Gangliosides; Gases; Glycerol; Grant; Home environment; Institution; Ions; Lasers; Leishmania; Lipids; Membrane Microdomains; Methods; Modification; Natural graphite; Neurons; Nitrogen; Pattern; Personal Satisfaction; Physiologic pulse; Polyacrylamide Gel Electrophoresis; Positioning Attribute; Pulse taking; Pump; Range; Research; Research Personnel; Resolution; Resources; Sampling; Sialic Acids; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Staging; Stainless Steel; Standards of Weights and Measures; Surface; Surface Plasmon Resonance; System; Techniques; Thin Layer Chromatography; Time; United States National Institutes of Health; Urea; analytical method; ion source; ionization; mass spectrometer; pressure; research study; vapor

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们之前开发的薄层色谱 (TLC) 与振动冷却 (VC) MALDI-FTMS 直接耦合的方法可以简单地处理具有 FT 高分辨率的 TLC，且不受 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，该技术的优化可以使直接从 TLC 板解吸的离子具有高灵敏度。此外，在将离子转移到池中之前进行离子积累，可以应用内部校准技术来实现精确的质量分配。神经节苷脂标准品通过 TLC 分离，并在 MS 分析之前确定斑点的位置。将基质溶液喷洒到 TLC 板上，神经节苷脂直接从 TLC 板表面进行 MALDI 解吸。 MALDI-FTMS 实验在我们自制的 FT-ICR 质谱仪上进行，配有 7T 磁铁和 Nd:YAG (UV) 激光器，频率三倍至 355 nm、氮激光器 (337 nm) 和 Er:YAG (IR) 激光器 (2940 nm)。在解吸电离事件期间，碰撞冷却气体提高了离子源中的压力。使用从不锈钢板解吸的标准品作为校准物来执行相邻样品的内部校准 (InCAS)。从 TLC 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，分析物从不平坦的 TLC 板表面解吸不会影响精度，神经节苷脂标准品的质量精度通常为 0.5-1.5 ppm。研究了几个参数对分子稳定的影响，例如冷却气体的类型、与电离-解吸事件相协调的气体脉冲、冷却气体压力、气体脉冲的持续时间、不同的激光强度、聚焦和光束轮廓。以尿素为基质的神经节苷脂的软红外解吸，加上振动冷却源的冷却，消除了唾液酸损失。然而，基质对神经节苷脂碎裂程度的影响是显着的，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很难融入到 TLC 板表面的大部分中。对离子源进行了改进，以减少抽气时间，以允许使用具有高蒸气压的基质，例如甘油。所开发的方法正应用于来自生物系统的样品，例如全脑神经节苷脂混合物、利什曼原虫脂质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（CO2 激光，10.7 um）和 SORI-CAD 被用作补充解离方法。由于大面积 XY 平台可以轻松耦合到许多表面技术，不仅包括 TLC，还包括聚丙烯酰胺凝胶电泳 (PAGE) 和表面等离子共振 (SPR)，未来的研究也将扩展这些分析方法的应用范围。