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Novel method for mapping and sequencing of gangliosides by capillary electrophoresis/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Introduction: Efficient separation and structural characterization of gangliosides from biological sources are prerequisites for current ganglioside research strategies tending to elucidate specific function of each particular structure and to use it accordingly, as therapeutic agents in treatment of diseases and/or as specific diagnostic markers. In order to develop a strategy for rapid separation followed by sensitive detection and structural analysis of ganglioside species present in complex mixtures isolated from biological materials, a novel approach combining capillary electrophoresis (CE) and nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS and tandemMS) was implemented using a native mixture of bovine brain gangliosides. Methods and Instrumentation: All CE experiments were performed on a P/ACETM 5000 series instrument (Beckman, Fullerton, CA, USA) using fused silica tubing capillaries with 50mm i.d.x 375 mm o.d and 57 cm in length. The running buffer was 50 mM aqueous ammonium acetate adjusted to pH 10.0 and 12.0 with 32% ammonia. The analyte components arising from the CE separation were collected in different fractions, which were subjected to the negative ion mode nanoESI-QTOF-MS and tandem MS analysis. Mass spectrometry was performed on an orthogonal hybrid quadrupole time-of-flight mass spectrometer (QTOFTM Micromass, Manchester, U.K.). Preliminary Data: Ganglioside mixture has been analyzed by CE in forward polarity. The separation in ammonium acetate/ammonia has been first evidenced by monitoring the UV absorption at 200 nm. Increasing the pH of the running buffer from 10.0 to 12.0, the relative mobilities of components increased, providing a high resolution separation. Although in the case of underivatized gangliosides the best separation accompanied by UV detection has been previously obtained only by using borate or phosphate buffer, gangliosides containing GM1, GM2, GD1 and GT1 could be resolved into separated UV- detectable fractions in ammonium acetate/ammonium hydroxide at pH 12.0. Further characterization of the separated components was achieved in off-line CE/ESI-QTOF-MS and tandem MS experiments. The nanoESI-QTOF-MS analysis performed in the negative ion mode revealed ions related to ganglioside structure in four out of seven CE collected fractions. Instrumental as well as solution parameters were found to play crucial role in the CE separation-MS detection influencing the abundance of product ions produced in tandem MS experiments for mapping and sequencing of single ganglioside molecule in CE separated fractions. Optimization of the experimental parameters provided novel and efficient CE/ESI-MS protocols, as generally required to effect the CE separation followed by mass spectrometric analysis of this type of glycoconjugates.

gangliosides; capillary electrophoresis; ESI-MS; tandem mass spectrometry

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