Sample preparation

Our analytic workflow begins with an organic extraction (Protocol 1) that quantitatively recovers all of the glycosphingolipids while precipitating all of the glycoproteins. Using our protocol, we can simultaneously profile glycosphingolipids, and if needed, N-linked, O-linked glycans, Glucosamino glycans (GAGs), Free oligosaccharides (FOS), Lipid linked oligosaccharides (LLO)  as well as proteins (for protein identification) can be profiled from the same starting material. From the lipid extract (mixture of sphingolipids, glycerolipids and other types of lipids), sphingolipids are enriched by saponification (Protocol 2). The reaction mixture are then desalted by passing through tC18-sep-pak (Protocol 3). Free lipids are removed from the sphingolipid fraction by washing the dried material out with Hexane (Protocol 4). The GSL fraction thus obtained are permethylated (Protocol 5) and profiled by mass spectrometry (Protocol 6).