A study published in the Aug. 20 issue of the Proceedings of the National Academy of Sciences observed a 61 percent coverage of the microbe's possible predicted set of proteins, or its proteome. This is the most complete proteome reporting to date of any organism. (The proteome is the collection of proteins expressed by a cell under a specific set of conditions at a specific time.) PNNL scientists identified more than 1,900 proteins in D. radiodurans.
Studying the amount of each protein present at any time has become more important as scientists attempt to learn which proteins are involved in important cellular functions. DOE's Microbial Genome Program, an element of the Genomes to Life Program, provided the genomic information for various microorganisms, including D. radiodurans, and developed ways to predict the set of possible proteins, which hold the key to why and how these microbes carry out different functions.
D. radiodurans is of interest because of its potential to degrade radioactive materials, its ability to withstand high levels of radiation and its impressive DNA repair capabilities. The Guinness Book of World Records once called it the world's toughest bacterium.
"We've been able to see more of the proteins, especially those proteins that exist in small quantities," said Mary Lipton, PNNL senior research scientist and lead author of the PNAS paper. "Because our coverage is unprecedented, we're now able to provide biologists with protein-level information they never had access to before."
To identify proteins involved in various functions, PNNL researchers exposed D. radiodurans to several stresses and environments: heat shock; cold shock; exposure to chemicals that damage DNA such as trichloroethylene; exposure to ionizing radiation; and starvation. They were able to identify many proteins previously only hypothesized to exist on the basis of DNA information and also proteins that seemed to have little function. New proteins that became active only during a specific condition also were identified, as were proteins that appeared to exist all the time.
To achieve this unprecedented coverage, researchers used a new high-throughput mass spectrometer based on Fourier-transform ion cyclotron resonance developed at PNNL. This instrumentation allows scientists to identify thousands of proteins within hours. The system relies on a two-step process that first uses tandem mass spectrometry to identify biomarkers for each protein.
"We've not only identified the proteins, we have validated our results by using two mass spectrometry techniques," said Richard D. Smith, PNNL principal investigator.
"Once we've identified the protein biomarkers, then we never have to repeat the identification step, thereby speeding up our experiments. As a result we not only have a much more complete view of the proteome than existed previously, but we also can follow changes to it much faster."
The experiments were conducted in the William R. Wiley Environmental Molecular Sciences Laboratory, a DOE scientific user facility supported by the Office of Biological and Environmental Research and located at PNNL.
Other authors involved in the research came from Louisiana State University and the Uniformed Services University of the Health Sciences in Bethesda, Md.
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Pacific Northwest National Laboratory is a DOE research facility and delivers breakthrough science and technology in the areas of environment, energy, health, fundamental sciences and national security. Battelle, based in Columbus, Ohio, has operated the laboratory for DOE since 1965.