Analysis of proteins from membrane-enriched cerebellar preparations by two-dimensional gel electrophoresis and mass spectrometry.
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.
Friso
G
G
Department of Cellular and Molecular Pharmacology, Astra Pain Control AB, Huddinge, Sweden. giulia.friso@pain.se.astra.com
Wikström
L
L
D000818:Animals; D002531:Cerebellum / Q000737:chemistry*; D005591:Chemical Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D012965:Sodium Chloride; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine
D008565:Membrane Proteins; D012965:Sodium Chloride