Analysis of Sulfoglycolipids by Mass Spectrometry

Abstract

Glycosphingolipid (GSL) expression in developing tissues is spatially and temporally regulated. Post-synthetic glycan modifications such as sulfation, phosphorylation, and O-acetylation are associated with specific cell types within complex tissues and with specific developmental stages. Sulfated GSLs (sulfo-GSLs) have been shown to bind a variety of cell surface and extracellular matrix proteins and to also play an important role as adhesion molecules, acting as ligands for P- and L-selectin. In addition, sulfo-GSL expression correlates with cancer progression and facilitates the phagocytic clearance of cancer cells by macrophages. In general, glycoconjugate sulfation impacts a wide variety of intercellular interactions and signaling pathways. Glycomic approaches for analyzing GSLs in biological samples frequently utilize enzymatic release of oligosaccharide chains from their ceramide lipid moieties. Ceramides are structurally heterogeneous, composed of one of several sphingosine bases in amide linkage to a variety of fatty acids. Thus, enzymatic release of the GSL glycan has the useful property of simplifying the complexity of the sample. Unfortunately, this approach suffers from the limited specificity of the enzyme most commonly used for glycan release, an endoglycosylceramidase, EGCase. EGCase releases the glycans of GSLs with complex modifications, such as sulfation, sialylation, or unique monosaccharide sequences, with decreased efficiency. Therefore, in order to explore the full complements of GSLs including glycan and ceramide moieties, a sensitive, robust, and selective methodology for glycolipidomics is required. This chapter describes a procedure for comprehensive glycolipidomics that is also applicable to the characterization of sulfoglycoconjugates.