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Katie Cottingham reports from the 16th International Mass Spectrometry Conference—Edinburgh, Scotland Subdivide and conquer Although the human genome only comprises ~35,000 genes, the number of proteins is expected to be in the millions because of alternatively spliced forms and posttranslational modifications. One might wonder—how can a proteomics researcher ever hope to get a handle on so many proteins? One answer, according to Dave Speicher and colleagues at the Wistar Institute, is to divide whole proteomes into more manageable chunks by a new method called batch 2-D array/pixelation. In the first separation step, the researchers separate proteins with a Zoom isoelectric focusing (IEF) device, which they developed. The Zoom IEF device has a row of seven sample chambers that are separated by thin polyacrylamide membranes. These partitions
scientists’ ability to analyze lipids, says Dennis. LIPID MAPS GOVERNMENT AND SOCIETY project leaders also expect that their research will lead to new tools, methods, and technologies for measuring cellular lipids. A hom e forHUPO The Human Proteome Organization (HUPO)—Vida finallyFoubister has a place to call home—Montreal, Canada. “We’re very excited to move from a loosely connected operation to an operation that has headquarters in one location with continuity from year to year, from workshop to workshop, from initiative to initiative,” says Sam Hanash, president of HUPO. HUPO’s new international headquarters will reside in a building that is jointly operated by McGill University and Genome Quebec. According to Hanash, there is a lot to do before the projected opening of the office in early 2004. “We are going to start one step at a time,” he says. “We need to
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are embedded with immobilines, which are acrylamide derivatives with buffering capability. “When an electric field is applied across those chambers, proteins migrate back and forth through those polyacrylamide membranes until they reach a chamber where their pIs are between the pHs of the boundary polyacrylamide/immobiline membranes,” says Speicher. To provide an orthogonal separation based on protein size, each group of proteins is run in separate lanes on a 1D gel for a short distance. The gel is stained, and every lane is cut into 10–20 pieces, or pixels, which are placed into separate tubes for digestion. The pixels are then analyzed by LC/MS/MS. On the basis of past experiments, Speicher estimates that each pixel may contain as many as 30–100 proteins. Although there is some overlap of proteins between the slices, it is much less than the researchers expected with a very short, low-resolution 1-D gel. The batch 2-D array/pixelation technique is similar to a 2-D gel because, Speicher says, “Each point in the array has a known pI range, and it has a known
Batch 2-D protein array
Subdividingtheproteom e.Schematic of the 2-D array/pixelation method. Courtesy David Speicher, Wistar Institute
molecular weight range.” But the similarity ends there. He says, “[The method] can detect far more proteins than 2-D gels alone, and it does not suffer from some limitations of 2-D gels in that it can detect very large proteins and membrane proteins.” Speicher estimates that as many as 8400 proteins could be identified at one time with batch 2-D array/pixelation, which is more than five times the number of proteins that can be analyzed with one 2-D gel. As participants in the Human Proteome Organization’s plasma proteome pro-
ject, the researchers intend to use batch 2-D array/pixelation to analyze human samples. According to Speicher, plasma samples are more complex and more challenging than the cell lysates and extracts that the team initially examined with the method, and they have added a step to deplete the six most abundant proteins that interfere with the detection of low-abundance proteins in plasma. “Even with the six most abundant proteins depleted, the next six or so most abundant proteins now are interfering,” he says. “Those are certainly more of a problem than I
decide how much of the different initiatives that we have will be coordinated out of the central office and how much will be done in regional locations.” Although the organization had considered other locations, such as London, Paris, and Washington, D.C., Hanash cites many reasons for choosing Montreal. The city made HUPO an offer it couldn’t refuse, including free space and partial support for full-time employees. The fact that Montreal is a large city near the U.S. border with a reasonable cost of living also made it attractive.
© 2003 American Chemical Society
had anticipated.” Speicher adds that this is a challenge with any current method.
Hum an Brain Proteom e Project China got the liver, America the blood plasma, and Germany, the “nation of poets and philosophers”, the brain. Now German researchers, once called the “dwarfs in biological research” by James Watson, are to lead the international Human Brain Proteome Project (HBPP), which aims to analyze all the proteins of the human brain. Alzheimer’s and Parkinson’s diseases will be a specific focus. HBPP is a project under the patronage of HUPO, the Human Proteome Organization, which has set itself the goal of discovering all human proteins, beginning with those of the liver, the blood plasma, and now the human brain. “It is a great success for a country in which researchers are still sobered by the realization that they only contributed 2 percent to the decoding of the human genome,” says Joachim Klose of Charité’s Virchow Clinics of Humboldt University, Berlin. “Our first projects on neuroproteomics had already started to receive funding from the German Research Ministry in mid-2001. In America, the planning only started in
™15 million from 2001
until 2004, has announced that brain proteome research will definitely be a fixed part of funding from the German government. —Hanns J. Neubert
LIPID MAPS project launched The prediction of a University of California, San Diego (USCD), professor that metabolomics—the study of metabolites—will be the research focus of the next decade moved a step closer to reality this summer. The National Institute of General Medical Sciences, part of the National Institutes of Health, awarded a five-year, $35 million grant to a UCSD-led ini-
tiative to map all of the lipids in a mouse macrophage cell. “This is the first effort of this magnitude for lipids that’s parallel to the genomics and proteomics assaults,” explains Edward A. Dennis, a professor of chemistry and biochemistry at UCSD and the principal investigator of the Lipid Metabolites And Pathways Strategy (LIPID MAPS) Consortium. Lipids are a subset of metabolites involved in the regulation and control of normal cellular function that includes, for example, the sterols estrogen and testosterone. Abnormalities in these biomolecules have been implicated in diseases ranging from atherosclerosis
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late 2002,” adds Helmut Meyer, head of the Medical Proteome Center at Ruhr University, Bochum (Germany). He and Klose are the coordinators of HBPP, which already involves 125 research teams worldwide. Although single proteins have long been described in the literature, scientists still lack a complete overview, which is not easy to obtain because the proteome is a moving target. The results depend on when a researcher takes a tissue sample—especially in the brain, where the flow of impressions and thoughts result in continuous changes. Meyer and Klose estimate that ~12,000 proteins in the human brain contain key information on serious diseases and on the question of what makes someone a human being. The pilot phase will start in January 2004, and the initial results are to be expected by early summer. Participants in the program will study brain tissues taken from mice to assess the quality of different techniques and to feed proteome databases with reliable data. In addition, biopsy and autopsy tissues of human brains will be compared to evaluate protein stability in postmortem tissue. The German Ministry for Education and Research (known as BMBF), which is funding the project with about
to cancer, diabetes, arthritis, and Alzheimer’s disease. “People think of them negatively because of heart disease, [but] they’re so important,” says Jean Chin, program director of the grant, which will fund more than 30 researchers at 16 universities and 2 companies. The consortium chose to focus on macrophages because “they’ve been well studied in lipid metabolism and they’re also associated with disease transformations,” says Dennis. Furthermore, mouse-derived cells will be used because of the availability of specific gene knockouts. Once all the lipids have been characterized, the consortium plans to look at
quantitative changes in the levels of various species when the cell is exposed to agonists such as endotoxin, a component of many bacterial cell walls. Just as researchers involved in the Human Genome Project didn’t know how many genes they would be sequencing, those involved in the LIPID MAPS Consortium don’t know how many lipid species there will be to characterize. It’s estimated that there are a thousand or more. “There’s a whole bunch of them that have only recently been discovered, and they hope to discover new lipids,” Chin says. Recent advances in HPLC and MS have “revolutionized”
