Premium
Analysis of proteins from membrane‐enriched cerebellar preparations by two‐dimensional gel electrophoresis and mass spectrometry
Author(s) -
Friso Giulia,
Wikström Lilian
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:4/5<917::aid-elps917>3.0.co;2-6
Subject(s) - chemistry , chromatography , mass spectrometry , chaps , membrane protein , matrix assisted laser desorption/ionization , gel electrophoresis , protein mass spectrometry , tandem mass spectrometry , urea , polyacrylamide gel electrophoresis , top down proteomics , membrane , integral membrane protein , thiourea , biochemistry , desorption , organic chemistry , adsorption , enzyme
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.