Gangliosides make a wide family of glycosphingolipids ubiquitously expressed in mammalian tissues and particularly abundant in the brain and nervous system. They exhibit a huge diversity due to structural variations in both their oligosaccharidic chain and ceramide moiety, which represent a real analytical challenge. Since their discovery in the 1940s, methods have persistently improved until the emergence of Liquid Chromatography/Mass Spectrometry (LC/MS) which offers a high level of specificity and sensitivity and is suitable with high-throughput profiling studies. We describe here a comprehensive approach relying on various techniques and aiming at fully characterizing gangliosides in biological samples. First, total ganglioside content was determined by a biochemical assay. Second, ganglioside class composition was assessed by high performance thin layer chromatography followed by colorimetric revelation. Then, ceramide types of ganglioside classes were identified and their relative quantification was performed thanks to the development of a powerful and reliable LC/MS method. Finally, ceramides were structurally characterized and minor and less common ganglioside classes were identified using high resolution MS. These methods were applied to the rat retina to provide an exhaustive description of its ganglioside composition, giving the base for a better understanding of the precise roles of gangliosides in this tissue.
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