Glycosphingolipid profiling in cells and tissues using fluorescent LC/MS

Abstract

Glycosphingolipids (GSLs) are a class of glycolipids characterized by two major components; an oligosaccharide chain and a ceramide lipid chain bearing a fatty acid and a sphingosine (Sph) base. Gangliosides belong to the family of GSLs whose distinguishing feature is the presence of one or more sialic acid residues in their glycan. These biomolecules play significant roles in a number of cellular processes and have been associated with the pathology of diseases such as Alzheimer’s, Huntington’s and type 2 diabetes. There is a need for robust methods to analyze GSLs in various biological samples accurately. A significant amount of research has been conducted to identify the analytical tools for this purpose. Unfortunately, there remain limited techniques to identify and quantify GSLs precisely, due to the structural complexity of these molecules. This thesis has attempted to address this problem, through development of two complementary methods using enzymes to modify GSLs. The endoglycoceramidase (EGCase) enzyme cleaves the ceramide moiety, while the sphingolipid ceramide N-deacylase (SCDase) enzyme cleaves the fatty acid chain in the ceramide moiety to generate a lyso-GSL (l-GSL). Both degradation products could be labeled with a fluorophore and analyzed by LC-MS using internal standards to quantify GSL components from a variety of biological samples. The primary strength of our methods is quantification based on fluorescence. Additionally, these two methods are complementary providing information about both the oligosaccharide component of GSLs and the Sph base component in l-GSLs. The latter are seldom analyzed, despite studies that have shown differential expression of different Sph chains in tissues.