April 23, 2001
New Proteomics Tool Offers a Clear
Look at Cellular Proteins
IDAT Can Detect Proteins at an Unprecedented Resolution
for Research and Clinical Use
(Philadelphia
- PA) Scientists may have identified the genes in the
human genome, but proteomics is the growing field of
research that describes how proteins encoded in those
genes work. 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 Proceedings
of the National Academy of Science, 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 that, 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 at
the University of Pennsylvania Cancer Center.
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