Mapping the membrane proteome of Corynebacterium glutamicum.
In order to avoid the specific problems with intrinsic membrane proteins in proteome analysis, a new procedure was developed which is superior to the classical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method in terms of intrinsic membrane proteins. For analysis of the membrane proteome from Corynebacterium glutamicum, we replaced the first separation dimension, i.e., the isoelectric focusing step, by anion-exchange chromatography, followed by sodium dodecyl sulfate (SDS)-PAGE in the second separation dimension. Enrichment of the membrane intrinsic subproteome was achieved by washing with 2.5 M NaBr which removed more than 35% of the membrane-associated soluble proteins. For the extraction and solubilization of membrane proteins, the detergent amidosulfobetaine 14 (ASB-14) was most efficient in a detailed screening procedure and proved also suitable for chromatography. 356 gel bands were spotted, and out of 170 different identified proteins, 50 were membrane-integral. Membrane proteins with one up to 13 transmembrane helices were found. Careful analysis revealed that this new procedure covers proteins from a wide pI range (3.7-10.6) and a wide mass range of 10-120 kDa. About 50% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, protein translocation, and proteolysis while for the others a function is not yet known, indicating the potential of the developed method for elucidation of membrane proteomes in general.
Schluesener
Daniela
D
Lehrstuhl für Biochemie der Pflanzen, Ruhr Universität Bochum, Bochum, Germany.
Fischer
Frank
F
Kruip
Jochen
J
Rögner
Matthias
M
Poetsch
Ansgar
A
D001426:Bacterial Proteins / Q000032:analysis*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D048230:Corynebacterium glutamicum / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D003902:Detergents; D008565:Membrane Proteins; D020543:Proteome