Analyzing glycoproteins separated by two-dimensional gel electrophoresis
| Autor: | Daniel Jardine, Nicolle H. Packer, Margaret A. Lawson, Jean-Charles Sanchez, Andrew A. Gooley |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
PNGase F
Glycosylation alpha-2-HS-Glycoprotein Molecular Sequence Data Clinical Biochemistry Oligosaccharides Biochemistry Mass Spectrometry Amidohydrolases Analytical Chemistry chemistry.chemical_compound Animals Humans Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Protein Isoforms Electrophoresis Gel Two-Dimensional Glycophorins Polyacrylamide gel electrophoresis Chromatography High Pressure Liquid Glycoproteins chemistry.chemical_classification Gel electrophoresis Two-dimensional gel electrophoresis Chromatography Membranes Artificial Blood Proteins Gel electrophoresis of proteins Chromatography Ion Exchange Fetuin Carbohydrate Sequence chemistry alpha 1-Antitrypsin Cattle Electrophoresis Polyacrylamide Gel Polyvinyls alpha-Fetoproteins Glycoprotein |
| Zdroj: | Europe PubMed Central |
| ISSN: | 1522-2683 0173-0835 |
| Popis: | Two-dimensional (2-D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as 'trains' of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the 'rules' which govern the migration of glycoproteins in 2-D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N-linked oligosaccharides) or chemically (beta-elimination of O-linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic removal of the N-linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O-linked oligosaccharides. These approaches have been proved on 1-D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2-D PAGE preparative narrow range (pH 4.5-5.5) blots of human plasma: alpha2-HS glycoprotein (human fetuin) and alpha1-antitrypsin (alpha1-protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity (diversity of structures) of the glycosylation contribute to the separation of protein isoforms in 2-D PAGE. |
| Databáze: | OpenAIRE |
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