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Purdue Team Says PolyMAC Improves Phosphopeptide Enrichment for Mass Spec Analysis

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This story originally ran on July 8.

By Adam Bonislawski

Researchers at Purdue University have developed a method for isolating phosphorylated proteins from complex samples for analysis by mass spectrometry and plan to work with an as-yet-undetermined commercial partner to bring it to market relatively quickly.

The technique, described in a paper published in the June issue of Molecular & Cellular Proteomics, offers significantly higher levels of reproducibility, selectivity, and sensitivity compared to conventional phosphopeptide isolation approaches, Andy Tao, assistant professor of analytical chemistry and chemical biology at Purdue University and one of the paper's authors, told ProteoMonitor.

Traditionally, isolation of phosphopeptides for mass spec analysis has been done via solid-phase extraction-based methods using IMAC or metal oxides. These techniques suffer from a lack of reproducibility, however.

To address this problem, the researchers devised a method called polymer-based metal-ion affinity capture, or PolyMAC. PolyMAC uses water-soluble dendrimers covered with phosphopeptide-binding titanium molecules. Because these dendrimers are water soluble, the phosphopeptide binding can occur in a homogeneous aqueous solution – eliminating the heterogeneous binding conditions that limit the reproducibility of solid-phase isolation techniques. Once the phosphopeptides have bound to the titanium, the complexes can be pulled out of solution via a second functional group on the dendrimer that binds to agarose beads.

Using PolyMAC, the researchers achieved roughly 80 percent reproducibility across assays compared to only 50 percent for conventional solid-phase titanium dioxide nanobead methods, Tao said.

In addition to allowing isolation to proceed in a homogeneous aqueous solution, the PolyMAC approach offers better reproducibility due to the ease of standardizing the polymers used in the process, Tao said.

With the polymer approach, "the preparation can be standardized much more easily than the [titanium oxide] nanobeads," he said. "We have synthesis that can be characterized by spectroscopy methods, and we can use NMR and IR to make sure you have a controlled amount of functional groups on the polymer. So you have much better quality control in terms of synthesis to make sure that batch-to-batch reproducibility is good."

The researchers also found that the new method offers improved selectivity and sensitivity over previous methods.

For example, using 100 micrograms of lysate, the team demonstrated that PolyMAC has a selectivity of over 96 percent. This compares to an average of 70 percent for solid phase isolation techniques, Tao said. The technique's sensitivity, meantime, was roughly double that of conventional solid-phase approaches. The researchers were able to identify more than 1,000 phosphopeptides via mass spec analysis after isolation using PolyMAC, compared to fewer than 500 phosphopeptides after isolation using conventional methods.

One potential downside of the method is that in the study PolyMAC demonstrated selectivity for singly phosphorylated peptides over peptides with multiple phosphorylation sites, meaning that peptides with more than one phosphate group often aren't isolated by the technique. This is a problem in conventional titanium dioxide methods as well, Tao noted, due to the strong bond between titanium and phosphopeptides.

"Titanium tends to bond mainly to single-phosphate peptides," he said. "Some of the literature has reported that because titanium tends to bond to phosphate groups strongly, if you have two phosphate groups it bonds so strongly that you can't recover it into solution and analyze it by mass spec."

Typically researchers have gotten around this issue by using IMAC and titanium dioxide separation methods in sequence. In the case of PolyMAC, the dendrimer used in the approach can be functionalized with a variety of metal ions, including Fe(III), meaning that a similar strategy could be employed by using dendrimers first functionalized with iron – which shows a preferential enrichment of multiple phosphopeptides – and then with titanium.

The ability to functionalize the PolyMAC dendrimer with a variety of different metal ions suggests the technique could be used to isolate various classes of proteins.

"You can certainly use different metals to enrich metal-bonding proteins. That's one major initiative that we are working on," Tao said.

Currently his lab is working to develop a dendrimer covered in nickel-cobalt that could be used to capture cobalt-binding his-tagged proteins. More generally, the approach could be used to isolate a wide variety of metal-binding proteins, Tao said.

"You can capture endogenous metal-binding proteins. Some enzymes are catalyzed by certain metals, which means they have metal-binding sites. So you could use this method to capture these proteins," he said.

The technique also has potential uses in cancer drug research, Tao added.

"A large number of cancer drugs are palladium based, so potentially we can develop another version of this reagent to mimic these palladium-based drugs if they are binding to palladium-binding proteins," he said.

The researchers have patented the PolyMAC technology and have received interest in commercializing it from two undisclosed companies, Tao said. He said he has held off making a decision so far because he's trying to determine the best route for bringing the method to market.

"Currently I have [interest from] one big company and one small company," he said. "The reason I'm waiting is I want to find a company that can make this a widely used product available to the proteomics and biology community."

While the larger company has the resources to enable broad distribution of the technology, the smaller one might focus more of its energy on it, Tao suggested. He expects that once he does decide, PolyMAC will reach the market fairly quickly.

"The product is actually well developed," he said. "We have all the quality control steps to make sure the synthesis is correct and reproducible from batch to batch. So from a commercial point of view this product is almost ready. I expect it's going to be pretty soon."

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