Analysis of proteins from membrane-enriched cerebellar preparations by two-dimensional gel electrophoresis and mass spectrometry

✍ Scribed by Giulia Friso; Lilian Wikström

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 183 KB
• Volume: 20
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/(sici)1522-2683(19990101)20:4/5<917::aid-elps917>3.0.co;2-6

✦ Synopsis

Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry is a powerful combination for the separation of complex protein mixtures in biological samples and the subsequent identification of individual polypeptides. We have used this approach to construct a database of proteins of the porcine cerebellum, with emphasis on membrane-bound proteins, as part of our studies on the structure and function of the central nervous system. We compared the ability of different solubilization conditions (using zwitterionic and nonionic detergents; urea and thiourea) to improve the resolution of high molecular weight and hydrophobic proteins, and found the combination of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Tris, thiourea and urea to give the best results in our experiments. As a marker membrane protein, the NR1 subunit of the N-methyl D-aspartate receptor, a 120 kDa hydrophobic protein, was identified using a monoclonal antibody in combination with Western blotting. Sodium chloride treatment of the membrane preparation prior to solubilization caused further enrichment of membrane proteins. Fifty-six spots were identified using matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray mass spectrometry.