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

薄层色谱法是一种成熟的方法， 糖脂的分离和部分表征。 在过去 几年来，糖脂的成功结构测定 采用液相色谱法进行了分离， 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 聚偏二氟乙烯（PVDF）膜。 这项技术放大了 移动性数据的信息内容和特定的 通过重叠测定证明的结构/活性关系，其中 使分离的糖脂与抗体相互作用， 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飞行时间 (TOF)MS易于进行表面分析，并已用于 在电泳凝胶上直接分析蛋白质和转移 膜，这似乎是可能的，类似的方法可以B e 适用于通过薄层色谱法分离的样品， 特别适用于与高灵敏度覆盖组合 检测.. 我们发现糖脂确实可以通过 MALDI-TOF MS。在TLC板上均可观察到中性化合物 和转移膜上。 酸性糖脂的检测是 可能只在转移膜上。 观察到的灵敏度 这远远超过了LSIMS实验的报道，即使 用于MALDI-TOF MS分析的实验条件仍在改进中。 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，稍过量 能量被赋予分子;对于所有被检查的化合物， 包括唾液酸化物质（神经节苷脂），稳定的分子离子可以 被发现。 脱附物的源后衰变飞行时间分析 离子提供碎片化以允许结构细节的分配。 碎片化遵循我们先前报告的模式 标准MALDI样品制备。

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.