Proteome analysis in days

A team of researchers at the US Department of Energy's Pacific Northwest National Laboratory has developed new instrumentation and a unique approach to obtain the most complete protein analysis of any organism to date. Results were published in the Proceedings of the National Academy of Sciences.

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 and the Uniformed Services University of the Health Sciences 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 micro-organisms, including D. radiodurans, and developed ways to predict the set of possible proteins, that 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 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.

Before Smith's team developed the high-throughput method of mass spectrometry, it took scientists two to three years to analyse 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.

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 analyse 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 ionisation, a technique that ultimately would allow almost any protein to be studied using mass spectrometry. The electrospray ionisation 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 channelled their efforts into developing a better spectrometer. Smith's gamble was that this new ionisation 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 signalling 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 ionising radiation; and starvation. They were able to identify many proteins previously only hypothesised 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 were also`` 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 under way - 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.

To build this system, they started with a 9.4 tesla FTICR system manufactured by Bruker Daltonics. Then, they modified about half the system using technology developed at PNNL, including the electrospray ionisation, an ion funnel technology, and DREAMS. The result is a reliable and powerful automated system.

In the near future this will involve studies that centre 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.

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 organisations 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."