Direct coupling of thin layer chromatography with vibrationally cooled MALDI-MS for the analysis of glycolipids from biological samples

New methods for the minimization of metastable fragmentation of gangliosides by using collisionally cooling matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) were demonstrated; the research involved optimization, improvement, and application of these methods in a routine fashion to glycolipids from biological sources. Direct coupling of thin-layer chromatography (TLC) combined the well-developed separation method with high-resolution FTMS to achieve simple and accurate glycolipid analysis.; Evaluation of the Perkin Elmer prOTOF 2000(TM) instrument was performed with respect to its cooling capabilities and minimization of the metastable decomposition. Coupling of the TLC method with the prOTOF 2000(TM) instrument yielded a simple and fast analysis for gangliosides. The quadrupole time-of-flight orthogonal configuration of the prOTOF mass analyzer allowed for direct desorption of samples from the irregular surface of the TLC plate without impairment of either accuracy or resolution.; The method of direct coupling of TLC plates with vibrationally cooled (VC)-MALDI-FTMS was applied to test the idea that structural variation in the GD1a lipid anchor explains the failure of this ganglioside to act as a trafficking receptor for the LTIIb toxin. The initial hypothesis was that the GD1a ganglioside of human intestinal cells fails to associate with lipid rafts due to structural variation in its ceramide, and this prevents retrograde transport of the LTIIb-GD1a complex from the plasma membrane into the endoplasmic reticulum.; It was shown that the gangliosides from cells contain a variety of oligosaccharide headgroup compositions and the ceramide structures also exhibit substantial heterogeneity. FTMS and GC-MS revealed a higher level of fucosylation in the T-84 cell line than in the Vero line, and showed that both have an extended glycan moiety, as compared to traditional ganglioside structures. No GD1 gangliosides were found in T-84 cells, but this cell line was found to contain glycolipids with both sialic acid and fucose residues, whereas poly-sialylated gangliosides were observed in Vero cells. Numerous ceramide lipoforms were found in the Vero cell line, with a few structures similar to the ones of T-84 glycolipids. Application of various tandem MS techniques demonstrated efficient fragmentation of the neutral and acidic glycosphingolipid standards and these biological samples.