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|W. Andy Tao uses nanopolymers and chemical reactions that cause color changes in a solution to detect activity related to cancer cell formation.|
(Purdue Agricultural Communication photo/Tom Campbell)
Phosphorylation Assay Based on Multifunctionalized Soluble Nanopolymer
Anton Iliuk, Juan S. Martinez, Mark C. Hall, and W. Andy Tao
Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high-throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO, that is multifunctionalized with titanium(IV) ions for specific binding to phosphoproteins and with biotin groups that allow for enzyme-linked spectrometric detection. The sensitivity, specificity, and quantitative nature of pIMAGO for phosphorylation assays were examined with standard phosphoproteins and with purified phosphoproteins from whole cell extracts. As low as 100 pg of phosphoprotein can be measured quantitatively with the pIMAGO chemiluminescence assay. The pIMAGO assay was applied to an in vitro kinase assay, kinase inhibitor screening, and measurement of endogenous phosphorylation events. The technique provides a universal, quantitative method for global phosphorylation analysis with high sensitivity and specificity.
A Purdue University scientist's nanopolymer would make it easier and cheaper for drug developers to test the effectiveness of a widely used class of cancer inhibitors.
W. Andy Tao, an associate professor of biochemistry analytical chemistry and a member of the Purdue Center for Cancer Research team, created the Purdue-patented pIMAGO nanopolymer that can be used to determine whether cancer drugs have been effective against biochemical processes that can lead to cancer cell formation. The nanopolymers would attach themselves to target proteins that would later be detected by a relatively simple laboratory procedure called chemiluminescence.
Tymora Analytical, a company Tao started in the Purdue Research Park, will manufacture the pIMAGO nanopolymers. The 'p' stands for phosphor, and the IMAGO comes from the Greek word for image.
Tao's pIMAGO nanopolymers are coated in titanium ions and would attract and bond with phosphorylated proteins, ones in which a phosphate group has been added to a protein activating an enzyme called kinase. Kinase, when overactive, is known to cause cancer cell formation, and many cancer drugs are aimed at inhibiting kinase activity.
"It is universal. You can detect any kind of phosphorylation in a protein," said Tao, whose findings were reported in the early online version of the journal Analytical Chemistry. "It is also cheaper and would be more widely available."
The nanopolymers would be added to a solution of proteins, a chemical agent to start phosphorylation and a drug to inhibit kinase activity. Phosphorylated proteins would only be present if the drug is ineffective.
Avidin-HRP - the protein Avidin bound with the enzyme horseradish peroxidase - would be added. Avidin would bind with a vitamin B acid called biotin that is also on the nanopolymers' surfaces. A chemical called a substrate, added later, would cause a reaction with HRP, causing the solution to change color.
A lightly colored solution would mean there had been little kinase activity and few phosphorylated proteins and that the drug was effective. A darker solution would signal more kinase activity and a less effective drug.
"This could have a lot of applications in pharmaceuticals for drug discovery," Tao said.
Screening kinase inhibitors using antibodies can be cost-prohibitive for many laboratories because antibodies are in short supply and aren't available for many types of cells. Radioisotope tests are highly regulated and possibly dangerous because of radiation involved.
"We want to develop this as a commercial application to replace radioisotopes and antibodies as a universal method for screening kinase inhibitors," Tao said.
The National Science Foundation and the National Institutes of Health funded the research.
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