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New 4D Fractionation Method Detects Ng/ml Proteins, Relevant For Biomarkers

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Researchers at the Wistar Institute have developed a novel method for detecting low-abundance proteins in human plasma and serum by fractionating the proteins in four dimensions before submitting them to a mass spectrometer.

Detecting proteins in the nanogram-per-milliliter concentration range and lower is important because biomarkers for diseases and conditions are most likely to be present in plasma and serum in that range, said David Speicher, the lead developer of the new method.

"The protein biomarkers that are currently in use, such as PSA for prostate cancer and CEA for cancers in general, are present in blood in the low nanogram-per-milliliter level," said Speicher, who is a professor of molecular and cellular oncology at the Wistar Institute in Philadelphia. "If some cancer biomarkers are in low nanogram-per-milliliter level, one might guess that many biomarkers will be present at that concentration level."

Gil Omenn, the chairman of Human Proteome Organization's Plasma Proteome Project, agreed with Speicher. "If proteins are released from cells through disease-mediated apoptosis … shedding of cell surface proteins during cell proliferation, or secretion of over-expressed proteins, their concentration after dilution into four liters of blood or 17 liters of extracellular fluid will surely be quite low," said Omenn, who is also a professor at the University of Michigan.

Speicher's new approach, which is scheduled to be published in an upcoming issue of Proteomics, identifies proteins that differ in concentration by a range of nine orders of magnitude: The highest concentration proteins are present in milligram/ml ranges, while the lowest concentration proteins are present in picogram/ml ranges.

Conventional fractionation approaches, which generally separate proteins in two dimensions, usually identify proteins that differ in concentration by a range of three orders of magnitude. The lowest concentration proteins identified with the conventional approaches are generally in the microgram/ml range.

Few labs have conducted fractionation as extensive as Speicher's because "the more [fractionation] steps, the lower the throughput and the higher the cost," said Omenn. Notable exceptions are Dennis Hochstrasser's laboratory at the University of Geneva; the laboratory of Sam Hanash, who is now at the Fred Hutchinson Cancer Research Center; and the laboratory of Matthias Mann, who is now at the Max Planck Institute.

To achieve ng/ml protein identifications, Speicher and his research group first deplete the top six most abundant proteins using immunoaffinity columns. Researchers then further separate proteins according to their pHs using a technology called MicroSol-Isoelectric Focusing, or MicroSol-IEF. For the third dimension, researchers run fractions out on a one-dimensional electrophoresis gel. Then, for the final dimension, researchers slice up the 1D gel, digest each slice with trypsin, and run the resulting fractions out on a nano-capillary reverse high performance liquid chromatography column.

After going through 4D fractionation, each sample typically results in 150 fractions, Speicher said. The entire process of fractionation, followed by mass spec analysis using an LTQ-MS/MS, takes about 11 to 15 days, he added. That includes three days for the front-end separation, and 8 to 12 days for LCMS/MS.

Though Speicher's technique may seem slow and laborious, it has could prove to be useful for discovering biomarkers, Omenn said. Out of 18 laboratories that participated in a pilot HPPP study in 2003 and 2004 to identify proteins in plasma and serum, Speicher's laboratory — using their 4D technique — identified the most proteins overall, with a significant portion of those proteins in the ng/ml range, and a small number of proteins in the pg/ml range.

Results of the pilot HPPP study were presented last October at the HUPO conference in Beijing, and also published around the same time in a special issue of Molecular and Cellular Proteomics.

Methods such as Speicher's "are unlikely to have sufficient throughput for routine assay of large numbers of clinical or epidemiological specimens, but they can be very useful for discovery," said Omenn. "Once the markers have been discovered and validated, a complementary method, like microarrays or multiplex ELISA, is needed for obtaining [routine assay] throughput."

When asked if he plans to collaborate with any companies to improve and commercialize his method, Speicher said the method does not have much potential of being patented.

"The method doesn't have much potential for IP because we're basically taking a series of established protein separation methods and stringing them together in an efficient manner," he said. "It's really easy to do something different — you just substitute a different separation step."

Omenn said Speicher's method is as fast or faster than a three-dimensional fractionation method developed by Hanash's group. Speicher's technique should not be compared with SELDI, which is also used for biomarker discovery, because SELDI identifies protein profiles and not proteins, Omenn noted.

Speicher said he is working on ways to improve the throughput of his method. "It won't be high throughput, but we can improve the throughput," he said. "I think we can easily reduce the time in half by setting up two columns for HPLC."

In addition to further developing his technique, Speicher is currently using his 4D-fractionation strategy to identify biomarkers for skin, colon, lung, and breast cancers. "We have a substantial number of potential biomarker candidates," Speicher said. "We're in the early discovery phase."

Speicher said that he and his research group are exploring options for collaborating with a commercial company to develop the biomarkers they have discovered into diagnostics. The researchers are working with mice for the discovery phase, but they plan to validate the biomarkers using human samples.

— TSL

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