Direct matrix-assisted laser desorption/ionization mass spectrometric analysis of glycosphingolipids on thin layer chromatographic plates and transfer membranes

Results are reported fur analysis by matrix-assisted laser desorption/ioniz ation time-of-flight mass spectrometry (MALDI-TOFMS) of native glycosphingo lipids (GSLs) after development on thin layer chromatographic plates and af ter heat transfer of the GSLs from the plates to several types of polymer m embranes. The spectral quality is better for membrane-bound analytes, in te rms of sensitivity, mass resolution and background interference. The sensit ivity gain compared with liquid secondary ion mass spectrometry (LSIMS) of GSLs on thin layer plates is 1-2 orders of magnitude (detection limits of 5 -50 pmol vs. 1-10 nmol). Resolution and mass accuracy (0.1%) are limited by the irregular membrane surfaces and this effect cannot be entirely compens ated by delayed extraction. The best results were obtained with a polyvinyl idene difluoride (PVDF) P membrane,with irradiation from a nitrogen laser. Although the Nafion(R) membrane could not be used for molecular weight prof iling, its acidic character led to sample hydrolysis at the glycosidic link ages, thus yielding a series of fragments that could be used to determine t he sequence of carbohydrate residues. Structural information could also be obtained by post-source decay (PSD) experiments on mass-selected precursor ions. Samples containing both neutral and acidic components were characteri zed in a 1:1 combination of 2,5-dihydroxybenzoic acid and 2-amino-5-nitropy ridine. GSLs that exhibited binding to antibodies in an overlay assay on th e TLC plate were transferred to membranes and analyzed by MALDI-TOFMS witho ut interference from the antibody or the salts and buffers used during the binding and visualization steps. Taking advantage of the insights into samp le preparation gained from these studies, future research will extend this approach to analysis by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) with a n external ion source. Copyright (C) 1999 John Wiley & Sons, Ltd.