title: Sequential GC/MS analysis of sialic acids, monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives.
abstract: A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.
authors.affiliation: CNRS Unité Mixte de Recherche 8576, Laboratoire de Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, Bâtiment C9, 59655 Villeneuve d'Ascq Cedex, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Pons
authors.forename: Colette
authors.initials: C
authors.lastname: Richet
authors.forename: Catherine
authors.initials: C
authors.lastname: Robbe
authors.forename: Annkatrin
authors.initials: A
authors.lastname: Herrmann
authors.forename: Philippe
authors.initials: P
authors.lastname: Timmerman
authors.forename: Guillemette
authors.initials: G
authors.lastname: Huet
authors.forename: Yves
authors.initials: Y
authors.lastname: Leroy
authors.forename: Ingemar
authors.initials: I
authors.lastname: Carlstedt
authors.forename: Calliope
authors.initials: C
authors.lastname: Capon
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Zanetta
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D005466:Fluorocarbons / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D015394:Molecular Structure; D009005:Monosaccharides / Q000032:analysis*; D019158:N-Acetylneuraminic Acid / Q000032:analysis*
chemical_list: D000596:Amino Acids; D005466:Fluorocarbons; D006023:Glycoproteins; D009005:Monosaccharides; C033094:perfluorobutyric acid; D019158:N-Acetylneuraminic Acid