TLC & MALDI TOF MS ANALYSIS OF GLYCOLIPIDS AT UV & INFRARED WAVELENGTHS

薄层色谱法

• 批准号: 6123241
• 负责人: Catherine E. Costello
• 金额: --
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6123241
• 关键词: biological products; biomaterials; biomedical equipment development; biomedical resource; carbohydrates; lipids; nervous system; spectrometry; structural biology; technology /technique

Thin-layer chromatography is a well-established means for the separation and partial characterization of glycolipids. Over the last several years, successful structural determinations of glycolipids separated by TLC have been carried out by using liquid secondary ionization mass spectrometry (LSI MS) for direct analysis of samples on the thin layer plates or for analysis of samples transferred to polyvinylidene difluoride (PVDF) membranes. The technique amplifies the information content of the mobility data and the specific structure/activity relationships evidenced by overlay assays, wherein the separated glycolipids are allowed to interact with antibodies or other agents with known binding specificities. Because matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS is readily amenable to surface analysis and has been used for direct analysis of proteins on electrophoretic gels and transfer membranes, it seemed likely that an analogous approach could b e suitable for samples separated by thin layer chromatography, and would be especially useful in combination with high sensitivity overlay assays.. We have found that glycolipids may indeed be detected by MALDI-TOF MS. Neutral compounds may be observed both on TLC plates and on transfer membranes. Detection of acidic glycolipids is possible only on the transfer membranes. The sensitivity observed thus far exceeds that reported for LSIMS experiments, even though experimental conditions for MALDI-TOF MS analysis are still being optimized. Under the MALDI desorption conditions, using either ultraviolet (337 nm) or infrared (2.94 um) irradiation, little excess energy is imparted to the molecules; for all compounds examined, including sialylated species (gangliosides), stable molecular ions can be detected. Post-source decay time-of-flight analysis of desorbed ions provides fragmentation to allow assignment of structural details. Fragmentation follows the patterns we have previously repor ted for standard MALDI sample preparations.

薄层色谱法是一种行之有效的方法 糖脂的分离和部分表征。 过去的 多年来，成功测定糖脂的结构 采用二级液体进行薄层色谱分离 用于直接分析样品的电离质谱 (LSI MS) 在薄层板上或用于分析转移到的样品 聚偏二氟乙烯 (PVDF) 膜。 该技术放大了 移动数据的信息内容和具体 通过重叠测定证明结构/活性关系，其中 分离的糖脂可以与抗体相互作用或 具有已知结合特异性的其他试剂。 因为 基质辅助激光解吸/电离 (MALDI) 飞行时间 (TOF) MS 很容易进行表面分析，并已用于 在电泳凝胶和转印上直接分析蛋白质 膜，似乎可以采用类似的方法 适用于通过薄层色谱分离的样品，并且会 与高灵敏度叠加结合使用特别有用 化验..我们发现糖脂确实可以通过以下方法检测到 MALDI-TOF MS。 在 TLC 板上均可观察到中性化合物 以及转移膜上。 酸性糖脂的检测是 只能在转移膜上进行。 观察到的灵敏度 迄今为止，远远超过了 LSIMS 实验的报告，尽管 MALDI-TOF MS 分析的实验条件仍在研究中 优化。 在 MALDI 解吸条件下，使用 紫外线（337 nm）或红外线（2.94 um）照射，少量过量 能量被传递给分子；对于所有检查的化合物， 包括唾液酸化物质（神经节苷脂），稳定的分子离子可以 被检测到。 解吸的源后衰减飞行时间分析 ions 提供碎片以允许分配结构细节。 碎片遵循我们之前报告的模式 标准 MALDI 样品制备。