Weston Pimlott

Application of a simple methylation procedure for the analyses of glycosphingolipids

Acid and neutral glycosphingolipids (0.01-1 mumol) were completely methylated in high yields and with little formation of by-products in 10 min at room temperature, using methyl sulphoxide, methyl iodide, and powdered NaOH. Re-methylation of methylated and LiAlH4-reduced gangliosides gave a new derivative that was useful for the analysis of gangliosides by mass spectrometry.

Mass spectrometry of mixtures of intact glycosphingolipids

Publisher Summary This chapter discusses mass spectrometry of mixtures of intact Glycosphingolipids. Glycosphingolipids usually occur in nature in complex mixtures and in small amounts. The number of sugars may range from one to about forty. These substances show distinct cell, tissue, strain, and species specificity in their structures and undergo successive changes during development and on tumor transformation. Of particular biomedical importance are the determinants within the major blood group systems (ABO, Lewis, I, P) and the glycolipids which may act as tumor-associated antigens and as receptors for microbes. Structural studies are, therefore, of major importance for the progress of transplantation and tumor biology and for the understanding of infectious diseases. Such studies require sensitive and rapid methods giving information with high structural specificity and with the capability to work with mixtures. Mass spectrometry is not the only method that matches these demands, but taken overall it is perhaps the most valuable tool in this respect. The electron ionization (EI)/MS distillation technique for the analysis of glycosphingolipid mixtures is the principal subject of this chapter. A few aspects of TLC/MS are also included.

Structure determination of blood group type glycolipids of cat small intestine by mass fragmentography

1. Introduction The finding of a relatively high glycolipid content in human small intestine [ I] and the fact that epithelial cells of intestine offer a system for study of the differ- entiation of eukaryotic cells [2], opens a unique possibility to test the postulated role of cell-surface carbohydrates in differentiation and regulation [3]. We are therefore developing specific preparative and analytical techniques for microscale characterization of complex glycosphingolipids. The present work is an application of a novel technique of mass frag- mentography on a glycolipid mixture from an individ- ual cat small intestine. 2. Experimental One lyophilized small intestine was extracted with chloroform-methanol in a Soxhlet apparatus. The total non-acid glycolipid fraction was prepared by conventional steps like mild alkaline degradation, sili- tic acid and DEAE-cellulose chromatography [4], and a combinatory use of native and acetylated [5] sub- stances. Methylation and reduction was done as in [6]. The mass spectrometer (MS 902, AEI Ltd, England) was connected on line to a data system (Instem Ltd, England). The sample probe temperature was raised linearly l”C/min or S”C/min and scans were taken every 2 min. A detailed description of mass scale cali-

The resolution into molecular species on desorption of glycolipids from thin-layer chromatograms, using combined thin-layer chromatography and fast-atom-bombardment mass spectrometry

Using a specially designed, motorised t.l.c.-f.a.b.-m.s. probe with continuous desorption and scanning over a moving t.l.c. plate, it was shown that glycolipids with identical carbohydrate sequences were well resolved into molecular species with differences in long-chain base and fatty acid. There was no serious diffusion of the glycolipids into the matrix. The technique is demonstrated for sulphatides (one and two sugar residues) isolated from human kidney, GM3 ganglioside isolated from human malignant melanoma, and chemically modified gangliotetraosylceramide from mouse intestine. T.l.c.-f.a.b.m.s. is convenient for sequencing and composition analysis of receptor-active glycolipids, the biological activity of which can be monitored in parallel by overlay on the t.l.c. plate with proteins, viruses, bacteria, or animal cells.

SEQUENCING OF OLIGOSACCHARIDES BY MASS SPECTROMETRY APPLIED ON A 12-SUGAR GLYCOLIPID

Protein or lipid-bound oligosaccharides of the cell surface are at present of great research interest as they are targets for antibodies [I] and toxins [2] and are supposed to take part in intercellular recognition processes [3]. A problem has been the lack of chemical methods for the specific detection of separate saccharide sequences on a microscale. We will show that mass spectrometry is applicable for the microscale sequencing also of large saccharides. Although one would expect less success in this respect for saccharide [4] than for peptide [5], due to a higher mass, more functional groups, different kinds of linkage (position and configuration), branching of the chain and the heterocyclic structure of the saccharide monomers, a careful selection of derivatives and analysis conditions makes the characterization of even as large a molecule as a dodecasaccharide possible. This glycolipid represents the largest biomolecule structurally interpreted directly from mass spectrometry.

The blood group B type-4 heptaglycosylceramide is a minor blood group B structure in human B kidneys in contrast to the corresponding A type-4 compound in A kidneys. Structural and in vitro biosynthetic studies

Blood group A glycolipid antigens have been found based upon at least four different core saccharides (types 1 to 4). The biological significance of this structural polymorphism is not known, although the successful outcome of transplantations of blood group A2 kidneys to blood group O individuals have been partly explained by the low expression of A type-3 and -4 chain glycolipid antigens in A2 kidneys. If graft rejection due to ABO incompatibility is, in any way, correlated to the expression of type-3 and -4 chain blood group glycolipids, it is of interest to identify possible blood group B structures based on these core saccharides. In a non-acid glycosphingolipid fraction isolated from human blood group B kidneys, mass spectrometry, high-temperature gas chromatography-mass spectrometry and probing of thin-layer chromatograms with Gal alpha 1-4Gal-specific Escherichia coli and monoclonal anti-B antibodies provided evidence for minute amounts of a Gal alpha 1-3(Fuc alpha 1-2)Gal beta-HexNAc-Gal alpha 1-4Gal beta-Hex-Ceramide structure consistent with a B type-4 chain heptaglycosylceramide. In contrast, blood group A kidneys have the corresponding A type-4 chain heptaglycosylceramide as the predominant blood group A glycolipid. No, or very low activity of the blood group B gene enzyme on the type-4 chain blood group H hexaglycosylceramide precursor was found by biosynthetic experiments in vitro, which might explain the low expression of type-4 chain blood group B heptaglycosylceramides in human blood group B kidneys.

Identification of a blood group A active hexaglycosylceramide with a type 1 carbohydrate chain in plasma of an A1 Le(a-b-) secretor

A blood group A active hexaglycosylceramide with a type 1 carbohydrate chain was identified in the plasma of an A1 Le(a-b-) secretor. The analysis was done on the total non-acid glycosphingolipid fraction using mass spectrometry, NMR spectroscopy, and anti-A antibody immunostaining on thin-layer chromatograms.