Researchers at the University of Pennsylvania School of Medicine have created the first new technology for the proteomic era, a technique sensitive enough to detect individual proteins and robust enough to screen hundreds or thousands of molecules in mass automation.

The versatile technique, called IDAT, has a variety of potential uses from detecting cancer earlier to sifting through samples of molecules to find new candidates for drug research.

In today's edition of the Proceedings of the National Academy of Sciences, the researchers describe how they used IDAT to identify a protein marker for breast cancer at a resolution up to nine orders of magnitude more powerful than conventional techniques, and explain how the technique can be further refined.

"Nine orders of magnitude is a significant jump. If we were discussing computers, we would be talking about the differences between bytes and gigabytes," said Mark I. Greene, MD, PhD, Professor in the Penn Department of Pathology and Laboratory Medicine. "IDAT has the potential to do for proteomics what PCR did for genomics in the last two decades."

IDAT works by snagging a target protein -- even in a vast mixture of separate molecules -- and broadcasting the presence of the targeted protein with a strong signal.

IDAT stands for Immuno-Detection Amplified by T7 RNA polymerase and it combines the detecting ability of antibodies, highly specific immune system molecules, with the speed of a particular enzyme, T7 RNA polymerase, to act as an alarm system.

The enzyme and the antibody both interact through another molecule called a promoter. When the antibody snags the targeted protein, it triggers the promoter, which, in turn, triggers the enzyme into creating the signal.

Researchers could then screen for the signal, actually a molecule of RNA, in amounts that directly correlate to the amount of protein present, using conventional methods.

"IDAT can detect proteins earlier, faster and with more sensitivity than other methods," said James Eberwine, MD, professor in the Departments of Pharmacology and Psychiatry. "Tumors, for example, often shed particular proteins at an early stage and the sooner you can detect the proteins, the sooner you can treat the cancer."

The researchers also compared IDAT's speed and accuracy in detecting a breast cancer marker with current detection methods. Other technologies, such as immuno-PCR, have been adapted to detect proteins, but they lack the ability to tell how much protein is present in absolute terms.

Not only can IDAT quantify the amount of protein -- it is sensitive enough to pick out even a few copies of a protein out of a highly diluted sample. Moreover, IDAT does not rely on radioactive labeling and is far less time- consuming and cumbersome than existing techniques, all of which would allow for easier use and faster results.

Eberwine and Greene worked with colleagues Hong-Tao Zhang, PhD, Janet Estee Kacharmina and Kevin Miyashiro to develop the IDAT technique and further refine it for broader applications. As reported in the Proceedings article, the researchers have also found a way to create universal detection molecules, so that IDAT could detect an unlimited variety of proteins as well as lipids, sugars and other cellular molecules.

"With such an adaptability, researchers might be able to use an automated IDAT array to identify and count, for example, all the molecules present in a cell at a given moment," said Greene.

For patients, IDAT could enable doctors to routinely screen blood samples for early disease indicators, returning results in a matter of hours instead of the days or weeks it often takes now for the most complicated tasks.

For researchers, IDAT could aid in protein identification for a variety of reasons. For example, to study the existence of certain surface molecules on cells -- or to find ways to block those surface molecules in an effort to treat disease. In addition, IDAT could also determine how a protein functions within a cell.

After a protein is translated from the genome, it often gets further modified -- that is, new parts specific to its role in the body may be added. In the Proceedings article, the researchers describe how they were able to detect variations of the breast cancer protein, before and after it was modified.

Greene and Eberwine believe that this technology has the potential of making proteomics a "rational and solvable problem."

The research that developed IDAT has been funded by the National Institutes on Health and The Leonard and Madlyn Abramson Family Cancer Research Institute. - By Greg Lester

[Contact: Greg Lester ]

24-Apr-2001