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

用于检测神经节苷脂、中性糖脂的振动冷却 MALDI、TLC MALDI FTMS

• 批准号: 8170858
• 负责人: Catherine E. Costello
• 金额: $ 1.16万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170858
• 关键词: Affect; Brain; Calibration; Carbon Dioxide; Cells; Computer Retrieval of Information on Scientific Projects Database; Coupling; Detection; Dissociation; Event; Frequencies; Funding; Gangliosides; Gases; Glycolipids; Grant; Home environment; Human Milk; Institution; Ions; Lasers; Leishmania; Lipids; Membrane Microdomains; Methods; Neurons; Nitrogen; Physiologic pulse; Research; Research Personnel; Resolution; Resources; Sampling; Sialic Acids; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Stainless Steel; Surface; System; Techniques; Thin Layer Chromatography; United States National Institutes of Health; Urea; biological systems; ion source; ionization; mass spectrometer; pressure; research study

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. 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 (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 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 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 initial TLC experiments were carried out.

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。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。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。开发的方法正在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乳的糖脂。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，对原有的VC-MALDI系统进行了改造，并进行了初步的TLC实验。