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. GM1 and GD1a gangliosides serve as trafficking receptors for cholera toxin and the related LTIIb toxin, respectively. LTIIb is not active in human intestinal cells because the LTIIb-GD1a complex does not move retrograde from the plasma membrane into the endoplasmic reticulum. We have tested the idea that structural variation in the GD1a lipid anchor explains the failure of this ganglioside to act as a trafficking receptor. To address this problem, we are using our previously developed method of direct coupling of TLC plates with vibrationally cooled (VC) MALDI-FTMS (2). This allows direct TLC-MALDI-FTMS without adversely affecting the FT high resolution and mass accuracy by the surface irregularity of the TLC plate. Collisional cooling is necessary for stabilization and detection of intact gangliosides (3-5). Previous experiments have descibed ganglioside purification from polarized intestinal epithelial cell line T-84 and monkey kidney Vero cells and functional studies on the mechanism of toxin biology (6). We have described ganglioside separations and instrumental parameters for VC MALDI-FTMS (2). In the current study, the whole HP-TLC plate or the strip cut from TLC plate containing the sample track was attached to the MALDI sample probe and matrix solution was deposited on top of the analyte. The samples were MALDI- desorbed directly off TLC plate surfaces with ~0.5 mm sampling steps. Collisional cooling gas raises the pressure in the ion source up into the 1-10 mbar region during the desorption/ ionization event. Fragmentation was subsequently performed by SORI-CAD and IRMPD techniques (10.6 m CO2 laser, 200-400 ms). GC-MS studies were performed on Finnigan/Thermoquest GCQ ion trap mass spectrometer. Gangliosides were subject to methanolysis, followed by N-acetylation, and addition of TMS reagent. GC peaks were integrated, adjusted according to the detection sensitivity for standards, and plotted as normalized amounts in moles. Fatty acid methyl esters also were analyzed separately after extraction with chloroform following the methanolysis. It was shown that, in addition to a variety of oligosaccharide headgroup compositions, the ceramide structure in gangliosides from both cell lines exhibit substantial heterogeneity. The high separation efficiency of the HP-TLC plate allowed for observation of numerous homologs following each scanning step. This was demonstrated by analysis of the ?pure? commercially available and synthetic gangliosides and whole brain extract. A higher level of ganglioside fucosylation was observed in the T-84 than in the Vero cell line by both FTMS and GC-MS, with both having an extended glycan moiety compared to traditional ganglioside structures. Vibrational cooling resulted in stabilization of the labile sialic acid and fucose glycosidic linkages, and this feature was highly advantageous for the analysis of the heterogenous mixtures. Mass accuracy and resolution were not affected by desorption from the uneven TLC plate surface. The effect of space charge on mass accuracy can be overcome by applying the InCAS technique (7). Compared to SORI-CAD, IRMPD demonstrated more efficient fragmentation of both parent and product ions for the GM1 and T-84 gangliosides. Structural elucidation of the ceramide moiety in terms of length, degree of unsaturation, hydroxylation and branching is required, as these factors are assumed to be responsible for the toxin traffic in an infected cell. The two cell lines also expressed different distributions of fatty acids. Further analysis will focus on characterization of individual long-chain bases and fatty acids. Previous experiments have shown that cleavage within the ceramide is feasible by optimization of SORICAD/IRMPD techniques and/or doping the samples with alkaline salts (8), and this property was now demonstrated by an enhancement of the cerebroside fragmentation with Li salt. 1) Fujinaga, Y., Wolf, A.A., Rodigherio, C., Wheeler, H., Tsai, B., Allen, L., Jobling, M.G., Rapoport, T., Holmes, R.K., and Lencer, W.I. Ganglioisides that Associate with Lipid Rafts Mediate Transport of Cholera and Related Toxins from the Plasma Membrane to Endoplasmic Reticulum Mol Biol Cell 2001, 14, 4783-4793 2) Ivleva V.B., Elkin, Y.N., Budnik B.A., Moyer, S.C., O'Connor, P.B., Costello, C.E. Coupling Thin Layer Chromatography with Vibrational Cooling Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry for the Analysis of Ganglioside Mixtures Anal. Chem. 2004, 76, 6484-6512 3) O'Connor, P.B., Costello, C.E. A High Pressure Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry Ion Source for Thermal Stabilization of Labile Biomolecules Rapid Commun. Mass Spectrom. 2001, 15, 1862-1868 4) O'Connor P.B., Mirgorodskaya, E., Costello, C.E. High Pressure Matrix-Assisted Laser Desorption/Ionization Fourier Transform Mass Spectrometry for Minimization of Ganglioside Fragmentation J. Am. Soc. Mass Spectrom. 2002, 13, 402-407 5) Mirgorodskaya, E., Costello, C.E. TLC/MALDI-FT-ICR MS and SORI-CAD Analysis of Glycosphingolipids Proc. of 47th ASMS Conf. on Mass Spectrom. and Allied Topics 1999 6) Wolf, A.A., Jobling, M.G., Wimer-Mackin, S., Madara, J.L., Holmes, R.K., and Lencer, W.I. Ganglioside Structure Dictates Signal Transduction by Cholera Toxin and Association with Caveolae-like Membrane Domains in Polarized Epithelia J. Cell Biol. 1998, 141, 917-927 7) O'Connor, P. B., Costello, C.E. Internal Calibration on Adjacent Samples (InCAS) with Fourier Transform Mass Spectrometry Anal. Chem. 2000, 72, 5881-5885 8) Mirgorodskaya, E., Costello, C.E. Proc. of 48th ASMS Conf. on Mass Spectrometry and Allied Topics 2000
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