Proteomic analysis of the cell envelope fraction of Escherichia coli.
We applied proteomics technologies to analyze a membrane preparation of Escherichia coli, wild type strain and of transformants expressing human cytochrome P450s. The proteins were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. The membrane proteins were solubilized with both mild detergents such as CHAPS and strong detergents, such as sodium and lithium dodecyl sulfate, sodium cholate and sodium deoxycholate. In the E. colimembrane fraction, 394 different gene products were identified. Approximately 28% of them were predicted to be integral membrane proteins, of which 100 proteins have been predicted to carry one transmembrane region, ten proteins to carry two, and two proteins to include three transmembrane domains. The remaining are probably membrane-associated and cytosolic proteins. Cytochrome P450s did not enter the immobilized pH gradient strips but were efficiently analyzed in a two-dimensional, two-detergent system. Use of strong solubilizing agents resulted in the detection of about 20 membrane proteins, which were not detected following extraction with mild detergents and chaotropes. The present database is one of the largest for membrane proteins.
Fountoulakis
M
M
Center for Medical Genomics, F. Hoffmann-La Roche Ltd., Basel, Switzerland. michael.fountoulakis@roche.com
Gasser
R
R
D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000378:metabolism
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D020543:Proteome