Bo Nilsson

Determination of binding positions in oligosaccharides and glycosphingolipids by fast-atom-bombardment mass spectrometry

Application of f.a.b.-m.s. to the products obtained from glycoconjugates upon periodate oxidation followed by borohydride reduction and methylation gives the positions of binding of the monosaccharide residues on the basis of the sequences of the primary and secondary ions.

Oligosaccharides released from glycolipids by trifluoroacetolysis can be analyzed by gas chromatography-mass spectrometry.

Carbohydrates are quantitatively released by trifluoroacetolysis of neutral glycosphingolipids that contain 4-sphingenine. During the reaction hydroxyl groups are O-trifluoroacetylated and N-acetyl hexosamines are transamidated to form N-trifluoroacetyl derivatives. Selective removal of O-trifluoroacetyl groups followed by permethylation produces derivatives with properties favorable for analysis by combined gas chromatography/mass spectrometry. Electron impact mass spectra of permethylated N-trifluoroacetylated oligosaccharide alditols are characterized by strong primary and secondary fragments in which a positive charge is localized on the N-trifluoroacetyl hexosamine residue. The method enables rapid separation and identification of components in mixtures of carbohydrate chains from glycosphingolipids containing up to six simple sugars.

The carbohydrate of human thrombin: Structural analysis of glycoprotein oligosaccharides by mass spectrometry

The carbohydrate structure of human thrombin has been determined by direct probe mass spectrometry of the oligosaccharides released by trifluoroacetolysis from the asialo glycoprotein. The free oligosaccharides were studied as permethylated and N-trifluoroacetylated oligosaccharide alditols. The structure was confirmed by sequential exoglycosidase digestion of intact thrombin and sugar and methylation analysis of the oligosaccharides by gas-liquid chromatography-mass spectrometry. The results indicate the following structure: (formula; see text) with Fuc present on only about 50% of the oligosaccharides.

[2] Gas chromatography and mass spectrometry of hexosamine-containing oligosaccharide alditols as their permethylated, N-trifluoroacetyl derivatives

Publisher Summary Combined gas-liquid chromatography and mass spectrometry (GLC-MS) can be used to separate and identify permethylated oligosaccharide alditols containing up to four hexose units. Many oligosaccharides found in glycosphingolipids and glycoproteins are difficult to analyze by this method because they contain N-acetylhexosamine residues that prolong retention time on GLC. Transamidation of N-acetylhexosamines by trifluoroacetolysis increases the volatility of hexosamine-containing oligosaccharides so that molecules with at least seven monosaccharide units can be separated by GLC as permethylated alditols. In addition to enhancing volatility, the i-trifluoroacetyl (NTF) group stabilizes ions resulting from primary cleavage of the hexosaminidic linkage during electron impact mass fragmentation. As a result, sugar sequences and linkages often are easier to determine by mass spectrometry when NTF-hexosamine residues are present. Electron capture detection provides a sensitive and selective means of monitoring GLC separation of NTF-oligosaccharides.

A method of purification of partially methylated alditol acetates in the methylation analysis of glycoproteins and glycopeptides

A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (1:1, v/v) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures.

Chondronectin: Physical and chemical properties

Chondronectin, the chondrocyte attachment factor, was purified from chicken serum and characterized as to its physical and chemical properties. From sedimentation equilibrium data it was found to have a native molecular weight of 175,800 +/- 800 and a subunit molecular weight of 55,540 +/- 800 in the presence of guanidinium chloride and cysteine, suggesting a trimeric structure linked by disulfide bonds. As visualized by electron microscopy after rotary shadowing, the protein appears compact and globular. The amino acid and carbohydrate compositions of chondronectin are distinct from fibronectin, the fibroblast attachment factor, and laminin, the epithelial cell attachment factor. The activity of chondronectin in promoting attachment of chondrocytes is stable to digestion by collagenase, elastase, and neuraminidase, but is destroyed by trypsin treatment. The data suggest that chondronectin is structurally and chemically distinct from fibronectin and laminin.

Isolation and characterization of 4-O-[3,4-O-(1-carboxyethylidene)-β-d-galactopyranosyl]erythritol from Klebsiella K33 polysaccharide

The tetrasaccharide-alditol, RL 0.8, from one stage of Smith degradation of Klebsiella K33, was subjected to Smith degradation to yield a disaccharide-alditol. The purified disaccharide-alditol was characterized by sugar analysis, methylation analysis, and mass spectrometry. The following structure was found. (formula see text)