title: Detection of N-glycans on small amounts of glycoproteins in tissue samples and sodium dodecyl sulfate-polyacrylamide gels.
abstract: N-linked glycans harbored on glycoproteins profoundly affect the character of proteins by altering their structure or capacity to bind to other molecules. Specific knowledge of the role of N-glycans in these changes is limited due to difficulties in identifying precise carbohydrate structures on a given glycoprotein, which arises from the large amounts of glycoprotein required for N-glycan structural determination. Here, we refined a simple method to purify and detect trace amounts of N-glycans. During the N-glycan purification step, most contaminants were removed by two kinds of columns: a graphite carbon column and a cellulose column. N-Glycans were identified with a three-dimensional high-performance liquid chromatography (HPLC) system. Using our method, a global analysis of N-glycans from human muscle biopsy samples and mouse brain sections was possible. By combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with our method, we refined analytical procedures for N-glycans from SDS-PAGE gels using hydrazinolysis to achieve a high N-glycan recovery rate. N-Glycans on as little as 1 μg of the target protein transferrin or immunoglobulin G (IgG) were easily detected. These methods allowed us to efficiently determine glycoprotein N-glycans at picomole (pmol) levels.
authors.affiliation: Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Gen
authors.initials: G
authors.lastname: Yamada
authors.forename: Mai
authors.initials: M
authors.lastname: Narumi
authors.forename: Takako
authors.initials: T
authors.lastname: Koike
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ishii
authors.forename: Ilan
authors.initials: I
authors.lastname: Sela
authors.forename: Stella
authors.initials: S
authors.lastname: Mitrani-Rosenbaum
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ikenaka
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D006023:Glycoproteins; D006834:Hydrazines; D011134:Polysaccharides; D012967:Sodium Dodecyl Sulfate