Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.