VIBRATIONALLY COOLED MALDI, TLC MALDI FTMS FOR GANGLIOSIDES, NEUTRAL GLYCOLIPIDS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 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。该技术的优化使直接从TLC板上解吸的离子具有较高的灵敏度。在自制的FT-ICR质谱仪上进行了MALDI-FTMS实验，分别用7T磁体和三倍频的Nd：YAG(UV)激光、337 nm的氮气激光和2.94 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中解吸出来的神经节苷脂的灵敏度为低fmol(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。研究了冷却气体的种类、气体脉冲与电离-脱附事件的配合、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。开发的方法正被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、来自神经元细胞的富含神经节苷脂的脂筏和来自人奶的具有HIV保护作用的糖脂。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。在这一年中，重建了原有的VC-MALDI系统，并正在重新评估其性能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. Optimization of this technique has led to high sensitivity for the ions desorbed directly from the TLC plate. 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 (2.94 um). 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 low fmol (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 was 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. The developed method is being applied to the samples from biological systems such as whole brain ganglioside mix, Leishmania lipids, ganglioside-rich lipid rafts from neuronal cells and HIV-protective glycolipids from human milk. Infrared multiphoton dissociation (CO2 laser, 10.7 um) and SORI-CAD are being used as complementary dissociation methods. During this year the original VC-MALDI system was rebuilt and its performance is being re-evaluated.