NEW METHOD TO IDENTIFY BLOOD PROTEINS MAY SPUR NOVEL DISEASE MARKER DISCOVERIES

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Using conventional technologies, researchers supported by the National Cancer Institute (Science Applications International Corporation-Frederick, Inc. (SAIC)) have developed a new method for identifying proteins found in trace quantities in the blood.* The method offers hope for detecting tiny amounts of these blood-borne molecules that signal the presence of certain diseases, such as cancer, infectious diseases, behavioral disorders, developmental defects, and neurodegenerative diseases. These molecules might be useful biomarkers to aid in earlier detection and treatment of ovarian, breast, and prostate cancer. The National Cancer Institute (NCI) is part of the National Institutes of Health (NIH) and is the government's principal agency for cancer research.

Working at the Laboratory of Proteomics and Analytical Technologies at NCI-Frederick, Md., the researchers crafted a multi-step procedure for separating blood proteins derived from serum, which is the clear, yellowish liquid that separates out from blood after clotting and does not contain any cells. Together, all of these proteins are known as the serum proteome. Prior efforts to identify low- abundance proteins were not as successful mainly because separation steps to reduce amounts of large, high-abundance proteins caused a simultaneous loss of the smaller, low- abundance proteins. Separation and fractionation are needed to produce samples that can be analyzed by mass spectrometry, a high-throughput technique for identifying individual proteins.

To address this problem, the researchers performed a series of fractionations on a tiny volume of serum - about 0.04 teaspoons. The steps included several different methods of separation, called isoelectric focusing and chromatography, that are based on the size, electric charge, and other chemical properties that differ between proteins. These samples were then injected onto a mass spectrometer to acquire the raw sequence data that was subsequently linked to a human proteomic database to identify the observed molecules.

"Our investigation resulted in the identification of 1,444 proteins in serum," said Thomas Conrads, Ph.D., associate director of the Mass Spectrometry Center at NCI-Frederick. A wide range of proteins from different cellular compartments was identified. Another research group using different methods previously had identified 490 serum proteins. "The proteins identified by earlier research overlapped only slightly with those characterized by our group," said Conrads. "This emphasizes the wide scope and complexity of the human serum proteome, which has been estimated to contain more than 10,000 proteins."

Previous studies by other scientists had found evidence of blood-borne molecules related to disease states. This new method offers refinement, which may lead to identification of more sensitive disease markers. "This study shows the extent of the number of proteins that can be characterized within serum using conventional technologies commonly available within proteomic laboratories around the world. The next step will be to apply what has been learned and developed in this study to identify disease-specific biomarkers with greater positive predictive value than those presently being used for diagnostic purposes," added Conrads.

The researchers created a publicly available database of the newly identified human blood proteins. Located online at <http://bpp.nci.nih.gov>, the database will serve as a resource for other investigators studying blood proteins.

For more information about cancer, please go to <http://cancer.gov>.

*The official report of the new method appears in the June 2004 issue of "Clinical Proteomics" (Volume 1, Issue 2; Humana Press).

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