Gangliosides characterization by high resolution mass spectrometry and ion mobility

Gangliosides (GG) are sialic acid-containing glycosphingolipids particularly abundant in the nervous system. They exhibit a wide variety of structures depending on both the oligosaccharide chain and the ceramide moiety. About 200 GG species have been described so far, but their diversity and biological roles are far from being completely elucidated. An efficient method of identification and quantification is crucial to apprehend this huge heterogeneity. In this study, we focused on the characterization of the ceramide portion of GG, identifying the long chain base (LCB) and the fatty acid. A commercial standard of GD3 was analysed with both a Thermo LTQ-Orbitrap XLTM and a Waters Synapt G2-Si equipped with ion mobility.Whatever the equipment used, fragmentation of GD3 40:1 molecular species revealed the presence of four LCBs (d16:1, d17:1, d17:0 and d18:1) for one ceramide type, suggesting the presence of four GG isomers. This observation was confirmed by the MS3 analyses conducted with the Orbitrap, which allowed isolation and fragmentation of the ion corresponding to the ceramide. The technique of ion mobility offered the possibility to separate the four detected LCBs, after MS/MS fragmentation of GD3 40:1. We were thus able to detect and separate the LCBs, according to their differences of steric hindrance, for each isomer of GG. In this way we deduced the combination LCB/fatty acid (ex: GD3 d16:1/24:0) for each GD3 molecular species. Moreover, this separation presents the advantage to individualize each molecular isobaric species of GG for their ulterior quantification.Ion mobility applied to GG analysis allowed separation and further characterization and quantification of the various ceramide isomers.