PNNL expands blood serum protein library

In a significant scientific advance, researchers at the Department of Energy’s Pacific Northwest National Laboratory have identified or confirmed 490 proteins in human blood serum — nearly doubling the number of known serum proteins, according to a paper accepted for publication in the December issue of Molecular and Cellular Proteomics.

“We have performed the most extensive identification of proteins in serum to date,” said Joel Pounds, corresponding author and a PNNL staff scientist. “We studied blood serum because it holds clues to all the major processes in our bodies. We need to know what proteins exist in that serum to know how they might be used to predict disease susceptibility, monitor disease progression or diagnose disease.”

These clues include proteins that “leak” from dead and dying cells, and proteins secreted into blood or released from tumors. Identifying these proteins allows scientists to conduct additional studies to define each protein’s functional role in cells and the body.

The scientific community has studied plasma, the parent component to serum, for more than a hundred years. Recent studies have primarily utilized a technique called two-dimensional gel electrophoresis to study proteins found in plasma, yet this method is limited in its ability to identify proteins that exist in small amounts, known as low-abundance proteins, and is labor intensive. The identification of low-abundance proteins is important as many of these proteins often function as “messengers” that inform cells to turn signaling pathways on or off — such functions are central to cell death or disease development.

“After a long period of slow progress, research on the plasma proteome has begun a period of explosive growth attributable to new multidimensional fractionation methods,” said N. Leigh Anderson, founder and chief executive officer of The Plasma Proteome Institute (www.plasmaproteome.org). “PNNL’s work is an important early demonstration of the power of these methods, and suggests that hundreds, if not thousands, of new candidate markers will be found.”

Studying the proteome of blood serum was a natural fit for scientists at PNNL, which has a strong proteomics capability. A proteome is the collection of proteins expressed by a cell under a specific set of conditions at a certain time. Through its Biomolecular Systems Initiative, the laboratory is supporting multidisciplinary research in systems biology. Scientists have developed unique technologies that allow for more thorough analysis of proteins and have studied the proteome of ovarian cancer as well as other disease states.

Pounds and his team, which included lead author and post-doctoral researcher Joshua Adkins, used chromatography and mass spectrometry instead of the more traditional 2-D gel electrophoresis to identify proteins, including low-abundance proteins not previously identified in serum and proteins with an unknown function. Their overall analysis was conducted on a single human blood serum sample from a healthy anonymous female donor.

The majority of serum protein consists of a few, very abundant proteins. One of the current challenges in the field is that the presence of abundant proteins obscures the measurement of many low-abundance proteins, and that removal of these abundant proteins may result in the simultaneous removal of low-abundance proteins. Here, Pounds and his team kept those abundant proteins, but simplified the mass spectrometry by fractionating the peptides according to charge state.

Once fractionated to allow for the analysis of lower abundance proteins, the samples were analyzed using a mass spectrometer that had been programmed to concentrate on specific ranges of peptide size during several analyses, thereby providing a more complete analysis of the proteome. The researchers employed powerful mass spectrometers housed in the William R. Wiley Environmental Molecular Sciences Laboratory, a DOE national user facility located at PNNL.

The sample preparation and analysis approach allowed PNNL scientists to expand the range of proteins that could be identified. For example, prostate-specific antigen (PSA) was identified in the sample using this approach. The reference value for PSA is in extremely low abundance in women, along the order of less than 1 picogram per milliliter. Detecting its presence provided a control to learn how well PNNL’s approach identified low-abundance proteins.

“With this study, we have taken a large step toward defining the protein composition of serum,” Pounds said. “But many more steps and technological improvements are needed to move beyond these 490 proteins to the thousands of proteins that may be present in blood serum.”

Molecular and Cellular Proteomics is a new journal distributed by the American Society for Biochemistry and Molecular Biology. Pounds’ paper is available online at http://www.mcponline.org/cgi/reprint/M200066-MCP200v1.pdf. A recent paper authored by Anderson and appearing in this journal also is available online at http://www.mcponline.org/cgi/reprint/R200007-MCP200v1.pdf.

Business inquiries on PNNL research and technologies should be directed to 1-888-375-PNNL or e-mail: inquiry@pnl.gov. PNNL’s Biomolecular Systems Initiative is online at http://www.biomolecular.org.

Pacific Northwest National Laboratory is a DOE research facility and delivers breakthrough science and technology in the areas of environment, energy, health, fundamental sciences and national security. Battelle, based in Columbus, Ohio, has operated the laboratory for DOE since 1965.