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WEST LAFAYETTE, Ind. - A Purdue University researcher can better retrieve specific proteins needed to study how cancer cells form by using a newly developed technique and synthetic nanopolymer.

W. Andy Tao, an assistant professor of biochemistry, said these specific proteins, called phosphoproteins, can be mapped and analyzed so that we can find ways to inhibit the processes that lead to cancer. But first those few proteins must be fished out of a sea of thousands of others.

Tao developed and patented the polymer-based metal-ion affinity capture, or PolyMAC. The synthetic nanopolymer isolates proteins and peptides that have undergone a process called phosphorylation that is highly associated with cancer, and a patented technique allows Tao to retrieve those proteins. Obtaining the information on these proteins is important for studying how to inhibit the processes that lead to cancer.

"You really want to capture these particular proteins, but there are so many different types of proteins around them," said Tao, whose findings were published in the early online version of the journal Molecular & Cellular Proteomics. "The target proteins are a thousand times lower in amount than other proteins. They are difficult to study without the capturing step."

Normal cells grow, divide and eventually die. But cancer cells continue to grow and do not die. Tao said phosphorylation - in which a type of enzyme called a kinase attaches to and catalyzes a protein on a cell - is thought in many cases to be responsible for creating cancer cells.

Tao's nanopolymer is water-soluble and has titanium ions on its surface, which bind with phosphorylated proteins and peptides contained in a solution. The polymer also has a chemical group attached that is reactive and attached to small beads, which allow Tao to retrieve the polymers.

"Once you put the nanopolymer in the solution, you have to retrieve them, so we put a handle on the polymer so we can grab on to it and fish it out of the solution," Tao said.

In laboratory tests, Tao's nanopolymer and retrieval technique isolated about twice as many proteins that had been phosphorylated by an enzyme highly expressed in certain leukemia cells but absent in metastatic breast cancer cells.

Tao is now seeking opportunities to get the polymer and technique into wider use to aid in the development of new cancer drugs.

"This technique is very useful and can be used widely in research for cancer as well as infectious diseases," Tao said.

The National Institutes of Health and the National Science Foundation funded Tao's research. A million NIH grant under the American Reinvestment and Recovery Act paid for a mass spectrometer Tao uses to analyze and map the proteins he recovers using his nanopolymer and retrieval technique.

 

Writer:  Brian Wallheimer, 765-496-2050, bwallhei@purdue.edu

 

Source:  Andy Tao, 765-494-9605, taow@purdue.edu

Ag Communications: (765) 494-2722;
Keith Robinson, robins89@purdue.edu
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ABSTRACT

In-depth Analyses of Kinase-Dependent Tyrosine Phosphoproteomes Based on Metal Ion Functionalized Soluble Nanopolymers

Anton B. Iliuk, Victoria A. Martin, Bethany M. Alicie, Robert L. Geahlen
and Weiguo Andy Tao

The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomic tool, Polymer-based Metal-ion Affinity Capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine (PAMAM) dendrimers multi-functionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared to current strategies based on solid phase micro- and nano-particles, PolyMAC demonstrates outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identify 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allows us to identify novel components in a variety of major signaling networks including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling.