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Description :
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Visualizing Interaction Networks. Graphical maps display protein interaction data in an accessible form. These visualizations summarize data from multiple experiments and also allow quick determinations of proteins that might be core constituents of a particular protein complex and those that might play roles in bridging interactions among different complexes. The figure above, generated using Cytoscape (www.cytoscape.org), summarizes affinity purification data from Shewanella
oneidensis. Nodes (yellow or red circles) represent proteins identified from the integrated pipeline at the Center for Molecular and Cellular Systems, using both endogenous and exogenous protocols (see sidebar text). Probe proteins for affinity purifications are shown as red circles. Edges (black lines connecting nodes) are drawn between probe proteins and any other proteins confidently identified from a particular affinity-isolation experiment. Capturing and Characterizing Protein Complexes in the Workhorses of the Cell Comprehensively analyzing the molecular complexes that perform life’s most essential functions presents many challenges due to their large number, biochemical variations, and dynamic nature. Some, such as ribosomes and other components of the cell’s basic biosynthetic machinery, are present under most, if not all, growth conditions and are relatively stable. Other proteins and their complexes are expressed only under particular conditions and on an as-needed basis. Isolating and characterizing the range of molecular complexes present in microbial organisms require the development and validation of robust and complementary techniques. GTL researchers at the Center for Molecular and Cellular Systems [a joint project of Oak Ridge National Laboratory (ORNL) and Pacific Northwest National Laboratory (PNNL)] have developed an integrated analysis pipeline that combines two complementary isolation approaches with mass spectrometry (MS) and computational tools for identifying protein complexes. This analysis pipeline uses molecular biology tools for expression of affinity-labeled proteins, highly controlled cell growth, affinity-based isolation of the complexes, and analysis of constituent proteins by MS. In addition, a bioinformatics infrastructure supports the entire pipeline, following samples “from cradle to grave” using a laboratory information management system integrated with data analysis and storage. This pipeline has been in continuous operation for over a year, focusing on two microbes relevant to DOE energy and environmental missions, Rhodopseudomonas palustris and Shewanella oneidensis. Extensive data are available for these organisms, including completed genome sequences. To isolate the complexes, the center employs two complementary affinity-based approaches in which tagged proteins are expressed either endogenously (in Rhodopseudomonas or Shewanella cells) or exogenously (in Escherichia coli or another surrogate cell) under specific experimental conditions. Combined liquid chromatography tandem mass spectrometry (LC MS/MS) is used to identify the isolated complexes. Once a protein complex is identified, additional analytical tools are used to validate the complex. For example, imaging tools are employed to confirm the interactions of protein pairs in live cells using proteins expressed with fluorescent tags. At ORNL, high-performance Fourier transform ion cyclotron (FTICR) MS has been added to the analysis pipeline. This “top-down” approach analyzes the intact protein, relying on the high mass resolving power of FTICR MS to identify the full range of truncations and modifications present on the protein. The “bottom-up” conventional LC MS/MS method analyzes protein fragments and relies on databases to identify the original protein but cannot identify the full range of protein modifications. Thus, integrating the two types of MS provides detailed insights into the full identity of protein complex constituents. Using these integrated methods to study 70S ribosomes from R. palustris, investigators obtained 42 intact protein identifications by the top-down approach, and 53 of 54 orthologs to E. coli ribosomal proteins were identified via bottom-up analysis. Scientists were able to assign post-translational modifications to specific amino acid positions and distinguish between isoforms. The combined MS data also allowed validation of gene annotations for three unusual ribosomal proteins (S2, L9, and L25) that were predicted to possess extended C-termini.1 The low-complexity, highly repetitive sequences common to eukaryotes had not previously been identified experimentally at the protein level in prokaryotes.2 These early results underscore the need for multiple technologies to identify and characterize the thousands of protein complexes GTL studies will require each year and to eliminate the many bottlenecks that remain. [Michelle Buchanan, ORNL, and Steven Wiley, PNNL]
References
1. F. W. Larimer et al., “Complete
Genome Sequence of the Metabolically Versatile Photosynthetic Bacterium
Rhodopseudomonas
palustris,” Nat. Biotechnol. 22, 55–61 (2004).
2. M. B. Strader et al., “Characterization of the 70S Ribosome from
Rhodopseudomonas
palustris Using an Integrated ‘Top-Down’ and ‘Bottom-Up’
Mass Spectrometric Approach,” J. Proteome Res. 3, 965–78 (2004). |
