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Novartis Develops Alternative Peptide Separation Method Using Perfluorine

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Scientists at the Genomics Institute of the Novartis Research Foundation have developed a new method for selective and reversible capture of modified peptides. The new method serves as an alternative to conventional biotin-streptavidin methods, which have drawbacks, including cost and capture reversibility.

"People are always looking for multi-dimensional ways of separation," said Eric Peters, the leader of the research team that developed the technique, described in the April 4 issue of Nature Biotechnology. "This is another entirely different type of dimension that people can separate by. It's a unique selectivity."

The new method uses perfluorine as an affinity handle. Any peptide that is attached to the perfluorine can be pulled out by passing it through a fluorous column, or by using a fluorous plate.

Peters said several companies are currently looking at commercializing his fluorous technique, which is in the process of being patented. The technique should be cheaper than biotin-streptavidin techniques because it uses silicon columns rather than the more expensive streptavidin columns, he added.

To demonstrate that the technique works, Peters and his research team used fluorous affinity tags to isolate specific peptide classes, including cysteine-containing peptides and polyubiquitin peptides.

Any number of peptide classes can be pulled out by different types of "warheads" attached to perfluorine affinity handles, Peters explained. The concept is the same for biotin-strepatvidin methods, where biotin is the affinity handle that is pulled out by passing the peptide mixture through a streptavidin column.

"What we wanted to see is if we could come up with something that has all the advantages of streptavidin without its problems," said Peters.

According to Peters, the fluorous technique was developed about two and a half years ago as a side project in the laboratory to arrive at a less-problematic alternative to biotin-streptavidin isolation methods. Peters and his research group got the idea to use fluorous phase combinatory chemistry from Dennis Curran, a professor of chemistry at the University of Pittsburgh who had used flourous tags to help him synthesize high-purity small molecules.

One difficulty in working with biotin-streptavidin methods is that it is difficult to pull peptides off of the streptavidin column once they are bound, and the biotinyl groups often fragment when submitted to a mass spectrometer, Peters pointed out.

With the fluorous method, peptides bound to the fluorous column can be rinsed out with a highly organic solution, then submitted directly to a mass spectrometer for identification, Peters said. The fluorous affinity tag does not interfere with mass-spec analysis, and because fluorine is highly inert, there is no fragmentation of the fluorous groups.

Another advantage of the fluorous affinity tags over biotin-strepativdin methods is that peptides with different functionalities can be separated out based on the number of fluorines attached to them, or the length of their fluorous affinity tag. This was demonstrated in Peters' study by the separation of differentially modified polyubiquitin peptides based on the length of their fluorous tags.

"This unique property opens up other types of separation schemes," said Peters. "You have selectivity in a very simple solid-phase reaction."

Michael Gelb, a professor in the departments of chemistry and biochemistry at the University of Washington, said that Peters' technique is a "clever and novel addition to the proteomic techniques arsenal."

The technique does not present a major advancement, however, given that some of the disadvantages of biotin-streptavidin techniques have already been overcome, Gelb said.

The problem of stripping biotin-labeled peptides off of streptavidin columns can be overcome by using modified forms of biotin or streptavidin, Gelb noted.

"Yes, it is true that biotin-streptavidin is essentially irreversible, but you can use monomeric avidin, which solves the problem," he said. "I think also that Molecular Probes sells a modified biotin that binds irreversibly to streptavidin."

In response to Peters' remarks that biotinylated peptides often fragment during MS/MS analysis, Gelb pointed out that many biotinylating reagents are eventually cleaved off the peptide, so there is no need to be concerned about the biotinyl group fragmenting inside the mass spectrometer.

Gelb also pointed out that the relatively high expense of biotinylation reagents is due to high mark-up by companies that produce it, and that fluorous reagents would probably not be cheap either after being marked up.

"I bet commercially available fluorous reagents will be overpriced as well," he said. "I think [the cost difference] is not important given the tiny amounts of reagents used."

Peters said in the future, his research group would continue to play around with the fluorous separation technique, and to use it in a variety of different fractionation applications.

— TSL

 

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