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

Abstract

Results are reported for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of native glycosphingolipids (GSLs) after development on thin layer chromatographic plates and after heat transfer of the GSLs from the plates to several types of polymer membranes. The spectral quality is better for membrane-bound analytes, in terms of sensitivity, mass resolution and background interference. The sensitivity 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 compensated by delayed extraction. The best results were obtained with a polyvinylidene difluoride (PVDF) P membrane, with irradiation from a nitrogen laser. Although the Nafion membrane could not be used for molecular weight profiling, its acidic character led to sample hydrolysis at the glycosidic linkages, thus yielding a series of fragments that could be used to determine the 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 characterized in a 1:1 combination of 2, 5-dihydroxybenzoic acid and 2-amino-5-nitropyridine. GSLs that exhibited binding to antibodies in an overlay assay on the TLC plate were transferred to membranes and analyzed by MALDI-TOFMS without interference from the antibody or the salts and buffers used during the binding and visualization steps. Taking advantage of the insights into sample 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 an external ion source.

High pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for minimization of ganglioside fragmentation

“…Methods for separation of gangliosides by thin layer chromatography (TLC) are well established [14,[43][44][45], and previous work in our own and in other laboratories has shown that TLC can be easily coupled to MALDI-TOF MS [31,46,47], with some compromise in instrument performance due to the irregular surface [31]. The ability to desorb gangliosides without fragmentation, as shown here, allows coupling TLC separations to MALDI-FTMS while retaining spectral quality.…”

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry

“…The lateral spread of the analyte and its penetration into the silica surface during the spot migration contributed to the apparent "low" sensitivity. Because of its deeper penetration into the surface, an infrared laser (Er:YAG, 2.94 M) has shown a sensitivity increase, compared with UV-MALDI, for desorption of glycolipid ions from a TLC plate [30], but the instrument available at that time (1998) did not have orthogonal injection, and the massive resulting plume degraded the spectral quality. One must keep in mind, however, that for a typical TLC experiment that uses color reagents for detection, the samples often are spotted in the high picomole-nanomole range.…”

“…The direct coupling described previously has been implemented on axial MALDI-TOF mass spectrometers, [30][31][32][33], showing the feasibility of this method. However, axial MALDI-TOF instruments require a flat surface for sample desorption to achieve optimum resolving power and mass accuracy results.…”

High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes

“…In another study, GSLs developed on a TLC plate were transferred to a PVDF membrane then coupled to MALDI MS; this was called the TLC-Blot-MALDI MS method [35][36][37]. There have been only a few recent reports on TLC-Blot-MALDI MS, because the additional work, i.e., the transfer from TLC plates to PVDF membranes, requires, and the orthogonal configuration of the instrument allows for direct desorption of samples from the irregular surface of the TLC plate without impairment of either mass accuracy or resolution [38].…”