Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives

The fine structural motifs of sialic acids, a frequent terminal monosacchar ide of glycans, seem to contain essential biological properties. To identif y such subtle structural differences, a reliable method was developed for t he qualitative and quantitative identification of sialic acids present in d ifferent tissues and fluids. This method involved, after liberation of sial ic acids by mild acid hydrolysis, their methyl esterification using diazome thane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas c hromatography coupled to mass spectrometry in the electron impact mode. Thi s technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glyco lyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (K dn). This method allowed also identifying 8-O-methylated and 8-O-sulfated d erivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through thei r fragmentation patterns. Because of the stability of the heptafluorobutyra te derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small am ounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.