Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomic analysis of rat liver microsomes: cytochromes P450 and other membrane proteins.
Gel electrophoresis in combination with peptide mass fingerprinting is the method of choice for proteomic profiling of various in vitro and in vivo biological systems. In the investigation reported here we analyzed the protein composition of hepatic microsomes from untreated and phenobarbital treated rats, using one-dimensional (1-DE) and two-dimensional (2-DE) gel electrophoresis, followed by tryptic peptide mapping. To better characterize capabilities of 2-DE 1-DE with regard to microsomal membrane proteins, "ghosts" of microsomal vesicles enriched in membrane proteins were obtained and analyzed. Both 1-DE and 2-DE showed that phenobarbital induces not only cytochromes P450 2B1and 2B2 but such stress related endoplasmic reticulum proteins as protein disulfide isomerase A(3) and A(6) and 78 kDa glucose regulated protein. The analytical performance of 1-DE with regard to endoplasmic reticulum membrane proteins is incomparably greater than that of 2-DE. Twenty-two out of a total of thirty-four known to date microsomal rat membrane proteins were identified by 1-DE in combination with matrix-assisted laser desorption/ionization-mass spectrometry of in-gel digests. At the same time using various types of 2-DE, we were able to identify only three rat microsomal membrane proteins. The data presented in this manuscript clearly demonstrate that 1-DE in combination with peptide mass fingerprinting can be successfully used for cataloging proteins of the endoplasmic reticulum, and that the proteomic analysis of the subcellular organelles containing a considerable number of highly hydrophobic membrane proteins should be performed by combined application of 1-D and 2-D electrophoresis.
Biochemical Research Service Laboratory, University of Kansas, Lawrence, KS 66045-7582, USA.
Nadezhda
N
Galeva
Michail
M
Altermann
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007525:Isoelectric Focusing; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D003577:Cytochrome P-450 Enzyme System