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

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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.