Detection of N-glycans on small amounts of glycoproteins in tissue samples and sodium dodecyl sulfate-polyacrylamide gels |
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Authors: | Yoshimura Takeshi Yamada Gen Narumi Mai Koike Takako Ishii Akihiro Sela Ilan Mitrani-Rosenbaum Stella Ikenaka Kazuhiro |
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Institution: | Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan. |
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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. |
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