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HUPO scientists tackle human blood plasma proteome

Collaboration will create master proteomics database; lay groundwork for future studies

University of Michigan Health System

SEATTLE - Although the Human Genome Project made a remarkable contribution to scientific knowledge and biomedical research, it was merely the preamble to proteomics, according to Gilbert S. Omenn, M.D., Ph.D.

"If we really want to understand what genes are doing in cells, we have to focus on proteins - the effector molecules of the cell," says Omenn, a professor of internal medicine and human genetics in the University of Michigan Medical School and School of Public Health.

Omenn directs the Human Plasma Proteome Project (PPP) - one of several initiatives organized by the Human Proteome Organization (HUPO). Established in 2001, HUPO is an international collaboration of academic, government and corporate scientists working in the field of proteomics.

At this year's meeting of the American Association for the Advancement of Science, Omenn will present an update on the Plasma Proteome Project, discuss the challenges of comparing data among laboratories, and provide examples of how scientists are analyzing initial protein data sets from reference specimens developed specifically for the project.

"This is a work in progress," Omenn says. "Nothing like this has ever been attempted in proteomics before. The human proteome contains hundreds of thousands of proteins, which are constantly changing, so we are tracking a moving target. The proteome's complexity goes to the heart of why proteins are so important, because they are responsive to and mediate changes associated with health and disease."

HUPO selected the plasma proteome as a major initiative, because blood is the most accessible human tissue, making it feasible to obtain samples and informed consent from volunteers for research and diagnostic use. In addition, specimen banks containing stored samples of human serum and plasma are available. Since blood bathes all cells and organs in the human body, it contains potential protein biomarkers, which indicate changes associated with specific diseases in various organs of the body.

The long-term goals of the Plasma Proteome Project are to identify all the protein constituents of human blood plasma and serum, find biological sources of protein variation within individuals, and determine the extent of variation across populations and across individuals within populations.

"The complexity of the task is daunting," Omenn says. "Differences between concentrations of the most abundant and least abundant proteins range over at least seven orders of magnitude. Many basic questions involving sample preparation and the relative advantages and limitations of a broad array of technology platforms need to be addressed. Based on the clear consensus of the proteomics community, PPP's initial goal is to establish scientifically valid methods for proteomics analysis of human plasma. Such progress and standardization of procedures will be vital for future studies of important biological, clinical, and public health problems."

In the PPP Pilot Phase, scientists from 13 countries working in 47 laboratories are using various technology platforms to analyze reference specimens of serum and plasma. Technology platforms include 2D gel electrophoresis, 3D liquid phase separation, MALDI-MS, tandem MS-MS, Fourier transform, ion-trap, and SELDI mass spectrometry, microarrays, and combinations of fractionation and analytical technologies.

Following extensive bioinformatics work at the University of Michigan, detailed data about the peptides identified and proteins inferred in PPP reference specimens will be stored in a master database, freely accessible to all, at the European Bioinformatics Institute (EBI) in Cambridge, England. EBI maintains and annotates the International Protein Index (IPI).

Since all laboratories are analyzing the same human reference specimens, Omenn says that comparing results will help scientists answer many key questions, such as:

  • How can we improve the confirmation rate of protein identifications in the same specimens after analysis by different laboratories?

  • Is it better to use serum or plasma with various technologies?

  • If plasma, does the choice of anti-coagulant (EDTA, heparin, citrate) matter?

  • How many freeze-thaw cycles can proteins in plasma tolerate?

  • Is it necessary to remove the most abundant proteins, such as albumin and immunoglobulins, in order to detect lower-abundance proteins, such as cytokines or cellular receptors?

  • What are the advantages and limitations of each technology platform?

  • Which database search engines generate the most reliable protein identifications?

    "It is unusual to be able to organize such an effort, while the technology is still in flux," Omenn says. "But the feedback will be crucial to improving future technology and making assays reproducible."


    To stimulate widespread analysis and utilization, sponsors and scientists participating in the pilot phase of the Plasma Proteome Project have agreed that the results of their work should be openly available and accessible. Participating scientists have agreed to submit their data sets for extensive cross-specimen, cross-laboratory, and cross-technology analyses. Publication of initial data is anticipated in late 2004.

    Samir M. Hanash, M.D, Ph.D., professor of pediatrics and communicable diseases in the University of Michigan Medical School, is president of the Human Proteome Organization.

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