Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Proteomics and Metabolomics
111
Development and Application of New Technologies for Comprehensive and Quantitative High Throughput Microbial Proteomics
Richard D. Smith* (rds@pnl.gov), Mary S. Lipton, James K. Fredrickson, Matthew Monroe, Eric Livesay, Konstantinos Petritis, Joshua Adkins, Gordon A. Anderson, Kim Hixson, Ruihua Fang, Rui Zhao, Ronald J. Moore, and Yufeng Shen
Pacific Northwest National Laboratory, Richland, WA
With recent advances in whole genome sequencing for an increasing number of organisms, biological research is increasingly incorporating higher-level “systems” perspectives and approaches. Biology is transitioning from a largely qualitative descriptive science to a quantitative, ultimately predictive science. Key to supporting advances in microbial and other biological research at the heart of the DOE Genomics: GTL program is the ability to quantitatively measure the array of proteins (i.e., the proteome) in various biological systems under many different conditions. The challenges associated with making useful comprehensive proteomic measurements include identifying and quantifying large sets of proteins that have relative abundances spanning many orders of magnitude, which vary broadly in chemical and physical properties, have transient and low levels of modifications, and are subject to endogenous proteolytic processing. Ultimately, such measurements, and the resulting understandings of biochemical processes are expected to enable development of predictive computational models that could profoundly affect environmental clean-up and energy production by, for example, providing a more solid basis for mitigating the impacts of energy-production-related activities on the environment and human health.
In FY 2001, a project was initiated to develop quantitative and high throughput global proteomic measurement capabilities for microbial systems. The platform is based on a combination of advanced separations and mass spectrometric instrumentation and supporting computational infrastructure. The scope has included the development of an ultra-high pressure nano-scale capillary liquid chromatography platform combined with Fourier transform ion cyclotron resonance mass spectrometry and supporting data analysis and management capabilities. These developments provided the first high throughput mode “24/7” operation of such instrumentation, and resulted in its successful application to a set of microbial systems. The biological applications of this technology and associated activities are the subject of a separate, but interrelated project (J. K. Fredrickson, PI) involving a number of microbial systems (e.g. Shewanella oneidensis MR1, Deinococcus radiodurans R1s) in collaboration with leading experts on each organism. These studies have demonstrated the capability for automated high-confidence protein identifications, broad proteome coverage, and the capability for exploiting stable-isotope (e.g. 15N) labeling methods to obtain high precision relative protein abundance measurements from microbial cultures.
A present emphasis of this project is the need for higher throughput proteomics measurements. A “prototype high throughput production” lab was established in FY 2002 was an early step in this direction. Operations within this lab are distinct from technology development efforts, both in laboratory space and staffing. This step was instituted in recognition of the different staff “mind sets” required for success in these different areas, as well as to allow “periodic upgrades” of the technology platform in a manner that does not significantly impact its production operation. The result has been faster implementation of technology advances and more robust automation of technologies that improve overall effectiveness.
Our efforts currently in progress aim to:
- Significantly increase the overall data quality of global proteome measurements, and provide data that are quantitative and have statistically sound measures of quality.
- Increase overall data production by more than an order of magnitude in conjunction with the improved data quality.
- Provide the informatics tools and infrastructure required to support improved data quality and increased throughput and to efficiently manage, use, and disseminate large quantities of data generated by GTL “users.”
- Develop the foundation for the further extension of proteomics measurements to enable more comprehensive coverage of protein modifications.
This research is supported by the Office of Biological and Environmental Research of the U.S. Department of Energy. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy by Battelle Memorial Institute through Contract No. DE-AC06-76RLO 1830.
* Presenting author
