Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Oak Ridge National Laboratory and Pacific Northwest National Laboratory
8
Center for Molecular and Cellular Systems: High-Throughput Identification and Characterization of Protein Complexes
Michelle Buchanan1, Frank Larimer1, Steven Wiley2, Steven Kennel1, Dale Pelletier1, Brian Hooker2, Gregory Hurst1, Robert Hettich1, Hayes McDonald*1 (mcdonaldwh@ornl.gov), Vladimir Kery2, Mitchel Doktycz1, Jenny Morrell1, Bob Foote1, Denise Schmoyer1,Manesh Shah1, and Bill Cannon2
1Oak Ridge National Laboratory, Oak Ridge, TN and 2Pacific Northwest National Laboratory, Richland, WA
The Center for Molecular and Cellular Systems (CMCS) focuses on Goal 1 of the Genomics:GTL program, which aims to identify and characterize the complete set of protein complexes within a cell to provide a mechanistic basis of biochemical functions. Over the past two years, the CMCS has emphasized developing technologies that can be incorporated into a high throughput “pipeline” for the robust analysis of protein complexes. Several approaches for the isolation and identification of protein complexes from microbial cells were evaluated. Our experience has demonstrated that no single approach will be sufficient to handle the diverse types of complexes present in a cell. Thus, an integrated pipeline has been developed that uses two affinity-based approaches to isolate protein complexes in which tagged proteins are either expressed endogenously or exogenously. The individual technologies have been refined, validated and assembled into a semi-automated pipeline has been in operation for over 30 continuous weeks. A comprehensive laboratory information management system has been developed for sample tracking, process management, and data control. Experiments using over 200 tagged proteins have been conducted using Rhodopseudomonas palustris and Shewanella oneidensis cultures grown under different states. Data from the two types of pull-down approaches have been compared and have been found to provide complementary information. This suggests that both approaches are needed for comprehensive identification of protein complexes.
During the past year research tasks have been designed to improve the analysis pipeline. “Top-down” mass spectrometry has been used to identify modifications of constituent protein in the complexes. Several types of imaging tools have been employed to observe the complexes in live cells, including co-localization assays and fluorescence resonance energy transfer (FRET)-based assays. Additional effort has been placed on identifying new approaches for minimizing sample handling, such as microfluidic devices and automation. All of these research efforts have focused on development and validation of approaches to provide improved confidence of complex identification, increased sample throughput, and enhanced complex characterization.
* Presenting author
