Two-dimensional benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE for membrane proteomics.
Despite the importance of membranes in any living system, the global analysis of membrane subproteomes is still a common obstacle. In particular, the widely used 2-DE technique consisting of IEF in the first dimension and SDS-PAGE in the second dimension has some major drawbacks regarding the separation of hydrophobic proteins. Therefore, we applied an alternative electrophoretic technique for separating membrane proteins: two-dimensional BAC/SDS electrophoresis (2-DB) using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride in the first and the anionic detergent SDS in the second dimension. The use of 2-DB resulted in an improved separation of hydrophobic proteins. Thus, extremely hydrophobic proteins such as cytochrome-c oxidase subunit I with a grand average hydrophobicity (GRAVY) index of 0.74 and a total of 12 known transmembrane domains (TMD) or Sec61alpha with a GRAVY index of 0.56 and a total of ten known TMD could be identified by MS/MS analyses of protein spots derived from 2-DB gels.
Zahedi
René-Peiman
RP
Rudolf-Virchow-Center for Experimental Biomedicine, University of Wuerzburg, Germany.
Meisinger
Chris
C
Sickmann
Albert
A
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005233:Fatty Alcohols; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008928:Mitochondria / Q000737:chemistry; D040901:Proteomics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry
D005233:Fatty Alcohols; D008565:Membrane Proteins; D000644:Quaternary Ammonium Compounds; C012817:cetalkonium chloride