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HUPO Launching Quantitative Test Sample Study in Late Summer, Advances Human Proteome Project

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Clarification: A HUPO official erroneously said that Invitrogen paid for the full cost of carrying out its protein-standard test sample study completed last year. In fact, others contributed funding to the project, which included labor, personnel, and instrument time.

By Tony Fong

The Human Proteome Organization hopes to launch its second protein test sample study at this year's annual conference in the fall, and is developing an action plan for the next phase of the organization's goal to create a human proteome map, its secretary general told ProteoMonitor this week, furthering along two of HUPO's most ambitious projects in recent years.

The protein test sample is a follow up to a similar study that HUPO began in 2006 assessing researchers' abilities to identify 20 proteins in equimolar amounts in a mixture. Based on that work, HUPO is trying to commercialize the 20-protein mixture to be used by the proteomics community as standard.

In May, HUPO published the results from that effort, in which only seven out of 27 participating labs were able to correctly identify all 20 proteins and one lab identified all 22 tryptic peptides with a mass of 1,250 Daltons [See PM 05/21/09].

In the next study, participants will be asked to detect and quantify proteins in a 20-protein mixture, Pierre Legrain, HUPO's secretary-general and a research director at the French Commissariat à l'Energie Atomique, said. With proteomics — and especially clinical proteomics — increasingly emphasizing the need for quantitative studies, the study addresses an important issue, he added.

"Right now, if you look at the literature, you see many, many papers [in which the authors claim] that they can quantify proteins in complex biological fluids or cellular extracts," he said. "[But] if you look carefully … it appears that probably most of those quantifications were not properly done, so that's why we really want to improve the common way of doing those experiments."

HUPO is still in the process of developing the protocols for the test. In 2006, however, John Bergeron, the former president of HUPO, said that this second study would contain 20 proteins in four different samples at varying concentrations spanning three or four logs. Some would be kept constant at high abundance, some at low abundance, and others mixed in [See PM 07/20/06].

Legrain said this week that once a first draft of the study design is made, it will be sent to a small group of researchers, funding agency officials, and editors of proteomics journals for input and suggestions for changes.

In the meantime, Legrain will also be raising funds for the project. The first protein-standard mixture study was completely funded by Invitrogen, now part of Life Technologies, with the intent that it would commercialize the mixture. However, last year, the company chose not to do so after determining that HUPO's requirement that the mixture be at least 95 percent pure would make it cost-prohibitive to manufacture and sell, a HUPO official told ProteoMonitor in the fall. A new partner to sell that protein mixture has not yet been found.

For the proposed study, Legrain said that he will be approaching both commercial vendors and the public sector for funding. The study is anticipated to cost about $300,000.

"We probably need up to $3,000 to prepare the reagent [for each laboratory] and to make sure that every lab will have exactly the same reagent," he said. "We don't want any single lab to prepare its own reagent or to buy its own reagent."

In May, he hopes to have a one-day meeting with potential participants to discuss the study and start planning for the actual work. The expectation is that between 20 and 40 labs will participate.

The study will use both mass spectrometry and antibody technology as the technology platforms.

Onward, Human Proteome Project

Legrain also reported some progress on HUPO's continuing push to create an international initiative to map the entire human proteome, first proposed in 2008 as a 10-year, $1 billion effort called the Human Proteome Project [See PM 05/09/08].

Next week, a working group will meet in Seattle to start plotting out, in effect, an action plan for HPP.

The goal of the group, composed of about 15 members, including one representative from the European Commission and two from the National Institutes of Health, will be to "see if we can design a research plan for the worldwide Human Proteome Project," Legrain said.

Topics such as bioinformatics, quantitation, or poorly annotated proteins will be defined as areas to be addressed by HPP-based research.

"Basically the goal … is to show that if we make a coordinated Human Proteome Project [at an international level] that we create another value compared to what biologists are right now doing using the classical way of funding through individually based research grants," Legrain said.

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Next week's meeting is just one of several that HUPO will conduct in the next few months as HUPO seeks to create momentum for the project, which has progressed glacially amid a measured reception from funding agencies. Though numerous research projects begun and continued independent of HPP are carrying out some of its goals, the project has existed mostly as a dream.

In addition to the Seattle meeting, this week Legrain met with scientists and research organizations in Montreal and Strausbourg, France, related to making HPP a reality. "I can tell you there will be a lot going on [with HPP] in the coming months," he said.

The goal is to create a first draft of the action plan by the end of the summer in time to be presented at the HUPO meeting in September, and then to start a pilot project in 2011. Among projects being considered for the pilot is one that would look at "redefining biomarkers" so that they have special use for drug makers and diagnostic firms. In short, it would tie a human proteome map to personalized medicine, Legrain said.

HUPO's vision is to take the human proteome "starting from the proteins [that are] coded by all these genes and coded in the human genome" — approximately 20,000 to 25,000 genes — "and we will define the way we want to create value on each of these proteins individually," he said.The approach will be mass spec-based, though it will also incorporate antibody-based technology to verify the proteins.

In addition, work will be done to annotate the proteins in order to gain a better understanding of their biological functions. "The key point now is to define the research program for that," Legrain said.

The idea is that rather than having one centralized project being directed monolithically, researchers from different geographies would be involved in HPP and fulfill some goal of the project, Legrain said. For example, a substantial amount of antibody research has been done in Sweden — in particular by Matthias Uhlen, who is overseeing the Human Protein Atlas — so HPP-based research in that area could continue being directed at antibodies, he said. Meanwhile, researchers in other regions could direct their studies at specific diseases or specific organs.

"At the end, HUPO will try to organize common workshops and common meetings such that knowledge about new proteins will be shared," he said.

HPP will have a gene-centric approach. There had been some debate within the community whether an approach that is tied to the genome would be the most useful, and at the HUPO conference in October, some attendees at a session to discuss HPP said that a sample-focused approach, which would map proteins and their variants in specific tissues, served a clearer and more utilitarian purpose because the results would shed light on disease, rather than produce a list of proteins and their variants [See PM 10/01/2009].

There was also talk of adopting a chromosome-by-chromosome strategy, but that option has been completely "abandoned," Legrain said, because such a method may not yield information that is particularly important from a proteomic perspective.

There remain concerns that a gene-centric approach will provide little biological information, he acknowledged. "The key point is really to see with all the new technology available now in mass specs and also the new algorithms in bioinformatics, how we can implement more biology or more annotation in terms of biological function on all these proteins that will be discovered," he said.

Funding for HPP remains an uncertainty, however. While some agencies have told HUPO that they may fund the pilot project, none have made any hard promises. Legrain downplayed any skepticism that funding agencies may have for the project, though. Rather, he said, agencies such as the EC and NIH want to gauge "whether the community is mature enough to define a credible business plan" that will result in "good deliverables" for a project as large as HPP.

"The fact that the three people that I contacted in the working group from the funding agencies said, 'I am interested, I will be in the working group, I am committed to see it through, and I am ready to work with you to match our expectations in terms of funding agencies and the way that scientists want to deliver their science,' I think that there is not resistance," he said.

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