Quantitative Profiling of the Membrane Proteome in a Halophilic Archaeon

• Published in: Molecular & cellular proteomics 2006-09, Vol.5 (9), p.1543-1558
• Main Authors: Bisle, Birgit, Schmidt, Alexander, Scheibe, Burghardt, Klein, Christian, Tebbe, Andreas, Kellermann, Joseph, Siedler, Frank, Pfeiffer, Friedhelm, Lottspeich, Friedrich, Oesterhelt, Dieter
• Format: Article
• Language: English
• Subjects: Cell Membrane - chemistry; Chromatography, Liquid; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Halobacterium salinarum; Halobacterium salinarum - chemistry; Isoelectric Focusing; Membrane Proteins - chemistry; Membrane Proteins - isolation & purification; Proteome - analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M600106-MCP200

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.