Scientists at the Department of Energy’s Pacific Northwest
National Laboratory have obtained the most complete protein coverage of any
organism to date with the study of a radiation-resistant microbe known to
survive extreme environments. This research potentially could open up new
opportunities to harness this microorganism, called Deinococcus radiodurans,
for bioremediation.

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,

Business inquiries on PNNL research and technologies should be directed to
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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.