Mapping the membrane proteome ofCorynebacterium glutamicum

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Abstract
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.

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