Breakthrough mass spectrometry technology
Reduces the time needed to analyze a proteome from years to days—and does it better
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A scientist at the Pacific Northwest National Laboratory saw his 15-year "big gamble" pay off in record-breaking results in proteome analysis using a new high-throughput method of mass spectrometry.
The new instrumentation, a high-throughput technology that uses very high-pressure capillary liquid chromatography (LC) combined with a unique form of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was conceived by Battelle Fellow and Chief Scientist Dick Smith. This breakthrough technology enabled Smith's research team, collaborating with Deinococcus radiodurans experts from Louisiana State University, Baton Rouge, and the Uniformed Services University of the Health Sciences, Bethesda, to identify more than 61% of the predicted proteome (more than 1900 of the almost 3200 proteins predicted) of D. radiodurans, a radiation-resistant bacterium. These results represent the broadest coverage of any organism to date.
A "proteome" is the collection of proteins that make up a cell (or organism) under a specific set of conditions at a specific time. 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.
We've been able to see more proteins, especially those proteins that exist in small quantities," said Mary Lipton, senior scientist and lead author of the recent study published in the August 20, 2002, Proceedings of the National Academy of Sciences (PNAS). "Because our coverage is unprecedented, we're now able to provide biologists with protein-level information they never had access to before.
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Before Smith's team developed the high-throughput method of mass spectrometry, it took scientists two to three years to analyze a proteome with much less accuracy and depth than the recently completed analysis of D. radiodurans. With the high-throughput instrumentation and systems, Smith's team can now complete five to six such analyses of the proteins of a proteome in a day with sensitivities 100 times greater than other methods.
Paradigm Shift
The dramatic improvements in the high-throughput instrumentation and system over the old methods are a direct result of Smith's taking an entirely different research path to his innovative design.
"I have been aiming at doing this for about 20 years," said Smith, who is the principal investigator and leader of a multidisciplinary team of researchers supported through the Office of Science's user facility at PNNL—the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL). "Years ago we made a lot of choices that were big gambles when we designed and selected the kinds of instruments we would develop for the EMSL."
Smith based his vision on using mass spectrometry to make biological measurements, when the conventional method had been (and still is in most laboratories) separations using two-dimensional gels to analyze the proteome. The two-dimensional gels are much less expensive than the present instrumentation, but suffer from limitations in protein coverage and the ability to detect low-level proteins. While the conventional approach can detect many of the more abundant proteins, an extremely time-consuming second stage of analysis called tandem mass spectrometry (MS/MS) is required for the identification of each protein.
When Smith began his research, mass spectrometry had never been used for making the kinds of biological measurements made today because there was no way to transport protein ions into a very high vacuum required for mass spectrometry. The ions were too large.
Smith's team solved this problem in 1985 by using electrospray ionization, a technique that ultimately would allow almost any protein to be studied using mass spectrometry. The electrospray ionization interface coupled separations (capillary electrophoresis in their initial work) with mass spectrometry to open the door for ultra-sensitive studies of biological mixtures. With the mass spectrometer interface problem solved, the team channeled their efforts into developing a better spectrometer. Smith's gamble was that this new ionization method combined with further development of the then fledgling FTICR technology would then allow biological systems to be studied in unprecedented detail.
"The conventional approach requires that you do maybe 10 to 50 times as many analyses to get the information you want," said Smith, "but with the high quality of the FTICR, this extra step (tandem mass spectrometry) can be eliminated. Thus, we can not only study the proteome for an organism much faster, but also with a much smaller protein sample—often a very important consideration in biological measurements." Added to this, Smith's new approach enables researchers to see most or all of the proteome—something that has been missing until now, regardless of the effort applied.
In addition to speeding up the process, Smith and team have recently invented a process called DREAMS (Dynamic Range Enhancement Applied to Mass Spectrometry) as a way to detect low-level proteins much more effectively. Low-level proteins can be important for cell signaling in key cellular functions and other important biological processes. DREAMS functions as a part of the FTICR instrumentation and allows researchers to extend the range between the most abundant and least abundant protein that will be detectable, thus allowing the mass spectrometer to look more deeply into the proteome.
"What we have now is like the Model T of this technology—obviously it has a long way to go before we can call it a Jaguar. But the Model T was a paradigm shift for modern travel in its day," said Smith.
"Now, it's a different era—we can study many different organisms and make many, many more measurements of the proteomes of those organisms than we ever could have before, and that is where you really learn interesting things," said Smith.
For example, to identify proteins involved in various functions like DNA repair, Lipton and team 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.
Making the measurement just once is not enough, Smith said. "It's necessary to make hundreds of measurements for an organism and to see how it responds to different changes. Essentially, anything that happens to you is reflected by a change in the proteins in your body, and changes to your proteome. By making sets of such measurements we can learn about the role of each protein part of the proteome. If applied to the human proteome, which is a much bigger problem than the microbes currently being studied, such measurements can provide a molecular level understanding of diseases and a basis for much better and faster drug development, for example."
Next Steps
"We have had a really significant breakthrough," said Smith, who is the first to point out that the analyses they have made of the proteome of D. radiodurans is just the first step in a long-term goal.
The next step is already underway—Smith's team has developed a prototype high-throughput version of the instrumentation that is automated and more robust. Scientists have previously considered FTICR to be difficult to use, said Smith. In response, the PNNL team developed a user-friendlier, automated version of the FTICR.
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To build this system, they started with a 9.4 tesla FTICR system manufactured by Massachusetts-based Bruker Daltonics Inc. Then, they modified about half the system using technology developed at PNNL, including the electrospray ionization, an ion funnel technology, and DREAMS. The result is a reliable and powerful automated system.
"The payoffs are potentially mind-boggling," said Smith. "We don't want to just understand D. radiodurans, but we would like to apply this technology to a range of important biological problems."
In the near future this will involve studies that center on microbes of interest to the DOE. It also has the potential to be applied to understanding human response to drugs—the detailed molecular changes that occur, for example, in cancer progression—and essentially almost every area of biomedical and health-related research. Proteome studies can also reveal the important proteins involved with a specific disease and the roles they play. Pharmaceutical laboratories developing new drugs may be able to design drugs to target these specific proteins.
"This is an area where the national laboratories really have a role to play," said Smith. The Office of Science national laboratories and user facilities, especially the EMSL, will play a major role in making this new technology available to the broader scientific community.
"Right now the technology is just too expensive for others to develop, and the data production rate and its management is too large and too complex for most organizations to manage and use effectively," Smith said. "Additionally, the technology is really still in its infancy, and as powerful as it is already, it will benefit from a series of advances we plan to implement over the next few years."—by Marye Hefty and Sallie Ortiz
Media contact: Staci Maloof, PNNL Media Relations Specialist, (509) 372-6313, staci.maloof@pnl.gov
Technical contact: Dick Smith, Chief Scientist, PNNL Fundamental Sciences Directorate, (509) 376-0723, dick.smith@pnl.gov
Related Web Links
"Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags," Mary S. Lipton, Ljiljana Paša-Tolic, Gordon A. Anderson, David J. Anderson, Deanna L. Auberry, John R. Battista, Michael J. Daly, Jim Fredrickson, Kim K. Hixson, Heather Kostandarithes, Christopher Masselon, Lye Meng Markillie, Ronald J. Moore, Margaret F. Romine, Yufeng Shen, Eric stritmatter, Nikola Tolic, Harold R. Udseth, Amudhan Venkateswaran, Kwong-Kwok Wong, Rui Zhao, and Richard D. Smith, Proceedings of the National Academy of Sciences (PNAS), August 20, 2002. [Abstract] [Full article]
"An Accurate Mass Tag Strategy for Quantitative and High Throughput Proteome Measurements," Richard D. Smith, Gordon A. Anderson, Mary S. Lipton, Ljiljana Paša-Tolic, Yufeng Shen, T.P. Conrads, T.D. Veenstra, and Harold R. Udseth. Proteomics. 2(5): 513-523 May 2002. [Abstract][Full article - PDF 1343 KB]
Identifying the Proteome of Deinococcus radiondurans
"Paving the Way for Proteomics," Breakthroughs, Summer 2002
Microbial Cell Project Archive
The Natural and Accelerated Bioremediation Research (NABIR) Program
"World's Toughest Bacterium Has a Taste for Waste," Rosalind Schrempf, Energy Science News, August 1998.
Funding: This research is supported by the U.S. Department of Energy's Office of Science, Office of Biological and Environmental Research through its NABIR Program, and through its Microbial Cell Program.
The William R. Wiley Environmental Molecular Sciences Laboratory(EMSL), the DOE Office of Science's newest national scientific user facility, is located at Pacific Northwest National Laboratory in Richland, Washington. The EMSL is operated by PNNL for the DOE Office of Biological and Environmental Research.
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.
Authors: Marye G. Hefty is a professor of technical writing at the Oregon Institute of Technology. She practices the craft of science writing during summer breaks for the Pacific Northwest National Laboratory in Richland, Washington. For more science news, see PNNL's News & Information
Sallie J. Ortiz is a science writer for the Pacific Northwest National Laboratory and the managing editor of the DOE Office of Science's award-winning quarterly online science newsletter, Energy Science News, and the features editor of DOE Science News, the weekly science news of the Office of Science website.