Direct analysis of sialylated or sulfated glycosphingolipids and other polar and neutral lipids using TLC-MS interfaces

Abstract

Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids. Gangliosides and sulfatides (STs) are acidic glycosphingolipids (GSLs) that have one or more sialic acids or sulfate substituents, in addition to neutral sugars, attached to the C-1 hydroxyl group of the ceramide long chain base. TLC is a widely employed and convenient technique for separation and characterization of GSLs. When TLC is directly coupled to MS, it provides both the molecular mass and structural information without further purification. Here, after development of the TLC plates, the structural analyses of acidic GSLs, including gangliosides and STs, were investigated using the liquid extraction surface analysis (LESA™) and CAMAG TLC-MS interfaces coupled to an ESI QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer. Coupling TLC with ESI-MS allowed the acquisition of high resolution mass spectra of the acidic GSLs with high sensitivity and mass accuracy, without the loss of sialic acid residues that frequently occurs during low-pressure MALDI MS. These systems were then applied to the analysis of total lipid extracts from bovine brain. This allowed profiling of many different lipid classes, not only gangliosides and STs, but also SMs, neutral GSLs, and phospholipids. Acidic glycosphingolipids (GSLs) include two subcategories, gangliosides and sulfatides (STs), and are composed of an acidic oligosaccharide head group covalently linked to a ceramide backbone (Fig. 1). In addition to variety in the lengths of the lipid chains, their degrees of unsaturation and double bond position(s), the diverse combination of the oligosaccharide types and carbohydrate branching make GSLs an extremely complex group of compounds and a challenge for structural analysis or profiling. Gangliosides are a group of GSLs that are characterized by the presence of one or more sialic acids in the oligosaccharide moiety. The most abundant sialic acid present in gangliosides in humans is N-acetylneuraminic acid. In humans, N-glycolylneuraminic acid is present at minimal levels, but occurs widely elsewhere at levels that are species-, tissue-, and physiological condition-dependent (1Inoue S. Sato C. Kitajima K. Extensive enrichment of N-glycolylneuraminic acid in extracellular sialoglycoproteins abundantly synthesized and secreted by human cancer cells.Glycobiology. 2010; 20: 752-762Crossref PubMed Scopus (45) Google Scholar). STs make up a class of sulfated glycosylceramides, mostly galactosylceramides having a sulfate group on the third position of the sugar. GSLs that distribute in neuronal and myelin membranes, and in other tissues and organs, play essential roles in numerous physiological and pathophysiological processes (2Degroote S. Wolthoorn J. van Meer G. The cell biology of glycosphingolipids.Semin. Cell Dev. Biol. 2004; 15: 375-387Crossref PubMed