Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids

We have developed a method involving the formation of heptafluorobutyrate d erivatives of O-methyl-glycosides liberated from glycoproteins and glycolip ids following methanolysis, The stable derivatives of the most common monos accharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, Glc NAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproduci bly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound t o Asn in N-glycans is quantitatively recovered as two peaks. The latter wer e easily distinguished from the other GlcNAc residues of N-glycans, thus al lowing a considerable improvement of the data on structure of N-glycans obt ained from a single carbohydrate analysis, The most common contaminants pre sent in buffers commonly used for the isolation of soluble or membrane-boun d glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degrad ation products of nucleic acids) do not interfere with these determinations . A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significa nt interferences. Since fatty acid methyl eaters and sphingosine derivative s are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 mu g of the initial compound purified by preparative Silicagel TLC, Using elec tron impact ionization mass spectrometry, reporter ions for the different c lasses of O-methyl-glycosides (pentoses, deoxyhexoses, hexoses, hexosamines , uronic acids, sialic acid, and KDN) allow the identification of these com pounds in very complex mixtures. The mass of each compound can be determine d in the chemical ionization mode and detection of positive or negative ion s. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O -methyl glycosides is achieved.