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. Our previously developed method of direct coupling of thin layer chromatography (TLC) with Vibrationally-Cooled (VC) MALDI-FTMS allows simple handling of TLC with FT high resolution which is not affected by the irregular surface of the TLC plate. Use of vibrational cooling is necessary for ganglioside stabilization and detection. We have now shown optimization of this technique leads to high sensitivity for the ions desorbed directly from the TLC plate. Furthermore, accumulation of the ions prior to transferring them into the cell allows application of an internal calibration technique for accurate mass assignment. The ganglioside standards are separated by thin layer chromatography and the positions of the spots are determined prior to MS-analysis. The matrix solution is sprayed onto the TLC plate and the gangliosides are MALDI- desorbed directly from the TLC plate surface. MALDI-FTMS experiments have been carried out on our home-built FT-ICR mass spectrometer with 7T magnet and Nd:YAG (UV) laser, frequency tripled to 355 nm, and Er:YAG (IR) laser (2940 nm). The collision cooling gas raises the pressure in the ion source during the desorption-ionization event. Internal Calibration on Adjacent Samples (InCAS) was performed using peptide standards desorbed from the stainless steel plate as calibrants. The sensitivity the has been obtained for the gangliosides desorbed from TLC is 1 pmole (total amount spotted on a TLC plate prior to separation). The use of InCAS has demonstrated that accuracy is not affected upon desorption of the analyte from uneven TLC plate surface. The accuracy of 0.3-1.5 ppm was obtained for five ganglioside standards. The effect of several parameters on molecule stabilization were studied, such as the type of the cooling gas, gas pulsing coordinating with ionization-desorption event, cooling gas pressure, duration of gas pulse, different laser intensities, focusing and beam profile. Soft IR-desorption of the gangliosides with urea as the matrix, together with cooling in the Vibrationally Cooled source eliminated sialic acid loss. However, the matrix effect on the extent of the ganglioside fragmentation was significant, as was demonstrated by the comparison of desorption using urea as a matrix versus desorption directly from the graphite-coated stainless steel plate. IR-MALDI of the gangliosides directly from the TLC plate was not as efficient as UV-MALDI and the sensitivity was relatively low, apparently because the matrices were poorly incorporated into the bulk of the TLC plate surface, due to their crystallization patterns. Modifications of the ion source that will decrease the pump-down time are now underway, and these should allow use of glycerol as a TLC-compatible IR-matrix that will be able to penetrate into the silica surface of the TLC plate. The developed method is being applied to the samples from biological systems such as whole brain ganglioside mix, Leishmania lipids, and ganglioside rich lipid rafts from neuronal cells. Since the large area XY stage allows facile coupling to many surface techniques, not only TLC, but also polyacrylamide gel electrophoresis (PAGE) and surface plasmon resonance (SPR)- future studies will extend the range of applications to these analytical methods as well.
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