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Olivier Berdeaux, Elodie Masson, Stéphanie Cabaret, Anne Athias, Jean-Paul Pais De Barros, Lionel Bretillon; Analysis of Retinal Gangliosides by Hydrophilic Interaction Liquid Chromatography - Electrospray Ionization Tandem Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3756.
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Gangliosides (GG) are sialic acid-containing glycosphingolipids. A wide variety of GG have been described based on differences in the oligosaccharidic chain, fatty acids and long chain sphingoid bases of the ceramide moiety. The GG profile is specific to the organ, its differentiation and pathophysiological status. An efficient method of separation, identification and quantification is therefore crucial ifthe huge heterogeneity among these compounds is to be understood and changes highlighted. We report here a powerful analytical method based on hydrophilic interaction liquid chromatography-electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS), further tested on rat retina. Indeed, GG are particularly abundant in the central nervous system, including the retina. While their involvement in the development of the retina and their neuroprotective action have been demonstrated, their precise role in the retina and its pathologies is still poorly understood.
GG classes were separated under HILIC conditions. Using a triple quadrupole MS instrument equipped with an ESI source, GM3 and GM2 molecular species were structurally characterized by collision-induced dissociation (CID) of [M-H] - and GD3, GD1a, GD1b, GT1b and GQ1b by CID of [M-XH]X- in negative modes. The semi-quantitative analysis of the GG molecular species was performed in the negative mode by single reaction monitoring (SRM). An N-acetylneuraminic acid fragment at m/z 290 was produced in the collision cell from the GG molecular species and used in SRM for quantification.
This method enables the major and minor GG species to be effectively separated in a highly heterogeneous GG mixture. Furthermore, it provides an accurate characterization of the widely variable ceramide moiety, whose role is not well understood. In rat retina, the main long chain bases are d18:1 and d20:1. Fatty acids are saturated or monounsaturated, mainly 16:0, 18:0, 20:0 and 22:0. The distribution of the different ceramide species varies among the GG classes, even though d18:1/18:0 and d18:1/20:0 (or d20:1/18:0) consistently appear to be the major ones.
Applied to retinal samples, this glycolipidomic approach offers an efficient, sensitive, straightforward, highly accurate and reliable tool to investigate the role of GG in the function and pathologies of the retina.
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