(11a) Multiplexed Analysis Of Microbial Proteomes

A broad range of protein separation and analytical methods are now available for in depth characterization of the protein complement of microbial cells. A multiplexed approach utilizing a variety of electrophoresis, chromatography, and mass spectrometry methods is being used to study the response of bacterial to different growth conditions at the level of total steady state protein content, membrane protein composition, protein-protein interactions, and post-translation modification. As an example of the comprehensive data sets produced using this multiplexed analysis strategy, proteome studies of Shewanella oneidensis, an exquisitely responsive metal-reducing bacterium, are revealing changes in the steady-state abundance of specific proteins as well as post-translational modifications when cells are grown with different terminal electron acceptors. Comparison of Shewanella oneidensis total and membrane protein fractions after separation by two-dimensional gel electrophoresis (2DE) is used to detect changes in steady-state protein levels, and protein identifications are determined by tandem mass spectrometry. Parallel analyses using two-dimensional liquid chromatography coupled to tandem mass spectrometry (2DLC/MS-MS) provides a broader inventory of protein constituents together with relative abundance. The data produced by such multiplexed analysis necessitates the use of a relational database system for data analysis, management, and integration. To date, over 500 Shewanella oneidensis proteins have been identified using 2DE together with tandem mass spectrometry with an additional 500 identified by 2DLC/MS-MS. Serine/threonine phosphorylation of a small subset of these proteins has been identified and preliminary studies suggest that differential phosphorylation of specific Shewanella oneidensis proteins correlates with electron acceptor availability.