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Pushing the limits of phosphoproteomics
August 2010
by Amy Swinderman  |  Email the author
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WEST LAFAYETTE, Ind.—Hardly a decade ago, the field of proteomics was so new and exotic that the American Society For Mass Spectrometry (ASMS) dedicated only one section of its four-day, annual conference to the topic. Remembering the humble beginnings of this burgeoning area of research with a laugh, a Purdue University researcher is fresh off presenting to ASMS his own development of a new retrieval method that makes studying cancer proteins easier.

Dr. W. Andy Tao, an assistant professor of biochemistry at the university, says the new technique can be used to retrieve specific proteins that are needed to study how cancer cells form. The difficulty of this task, Tao says, lay in the isolation of these proteins—called phosphoproteins—from thousands of others.

That's how Tao came to develop and patent the polymer-based, metal-ion affinity capture, or PolyMAC. The technique is described in a paper titled, "In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion functionalized soluble nanopolymers," which Tao and his colleagues presented to the ASMS conference in May in Salt Lake City.

"The belief is that phosphoproteins are responsible for a majority of cancer development—yet, more than 30 percent of cancer research focuses 'omics on kinase inhibitors," Tao says. "While it is certainly a major research focus, phosphoproteins regulate a lot of activities in cancer biology. We had been using P-32 radio isotopes, which have a lot of limitations, and mass spectrometry, which has become a very powerful tool for the study of phosphorylation, but also has its own problems."

Specifically, as stated in Tao's paper, despite the great progress that mass spectrometry-based phosphoproteomics has made in the global analysis of protein phosphorylation and molecular signaling in cells, it is still a considerable challenge due to the typically low
stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. As explained by Tao and his colleagues, an early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity and reproducibility. Almost all of the current isolation methods are based on solid-phase extractions, which, due to the nature of the heterogeneous environment and nonlinear binding dynamics when dealing with extremely low abundant phosphopeptides, can yield inconsistent results from one run to the next, even when using the same protocol.  

"The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research," Tao points out. "Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes."

The PolyMAC technique uses polyamidoamine (PAMAM) dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Tao stresses that compared to current strategies based on solid-phase micro and nanoparticles, PolyMAC demonstrates outstanding reproducibility, exceptional selectivity, fast chelation times and high phosphopeptide recovery from complex mixtures.

Using the PolyMAC method combined with antibody enrichment, Tao's team identified 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 allowed the researchers 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," Tao says. "In my mind, this technique also lends itself to the study of infectious disease, because if we can gain a better, more confident understanding of the pathways involved in controlling pathogenic bacteria, we can certainly better treat these diseases."

Tao's team patented the PolyMAC technique in November 2009 and two commercial entities are interested in licensing the product. The university is also sending samples of the system to other research groups for testing.

"We received a lot of attention" at the ASMS meeting, Tao says, "including multiple groups that asked for the use of PolyMAC reagents and potential collaborations."
The National Institutes of Health (NIH) and the National Science Foundation funded Tao's research. A $1 million NIH grant under the American Reinvestment and Recovery Act paid for a mass spectrometer that Tao uses to analyze and map the proteins using the PolyMAC technique. His collaborators were Anton B. Iliuk, Victoria A. Martin, Bethany M. Alicie and Robert L. Geahlen.
 
 
Code: E081018

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