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Team Led by Oxford Nanopore Founder Uses Nanopores for Phosphoprotein Analysis

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A team led by Oxford Nanopore co-founder Hagan Bayley has demonstrated the ability of a nanopore sensor to distinguish between differentially phosphorylated forms of the protein thioredoxin.

Detailed in a study published this week in Nature Biotechnology, the findings "provide a step toward nanopore proteomics," the authors wrote, adding that "applications of nanopore proteomics could be available in as few as two or three years."

Speaking to ProteoMonitor this week, Bayley cautioned, however, that while nanopore-based proteomic applications might be in the offing several years down the line, actual nanopore-based protein sequencing remains a distant goal.

"We aren't even close to doing that at the moment," he said, noting that the challenge of sequencing DNA, with its four different bases, pales in comparison to nanopore sequencing of the 20 different amino acids plus possible post-translational modifications present in proteins.

"I wouldn't say it's an impossible goal, but it is a bit of a stretch," he said.

More likely, Bayley said, are applications like the phosphorylation assay described in the Nature Biotechnology paper, in which he and his colleagues used a nanopore to distinguish between several known species of phosphorylated thioredoxin.

"You could take a specific protein – you might isolate it from a cell with an antibody, and then look at the different species of that protein, the different modified forms," he said. "That is a much easier target than actually sequencing a protein by nanopore technology."

In the study, the researchers generated unphosphorylated, monophosphorylated, and diphosphorylated versions of thioredoxin, creating a mixture of these proteins which they analyzed by passing them through an α-hemolysin nanopore, detecting the different variants by measuring the changes in conductivity resulting from their movement through the pore.

Of the 202 molecules they observed via nanopore, 67 proteins (33 percent) were diphosphorylated; 123 (61 percent) were monophosphorylated at Serine 112; and one (0.5 percent) was monophosphorylated at Serine 107. They were unable to classify 11 proteins.

An isoelectric focusing-based analysis of the sample found roughly the same composition, estimating that it consisted of 39 percent diphosphorylated thioredoxin and 61 percent monophosphorylated protein.

The researchers are now investigating methods of analyzing proteins extracted and enriched from actual cells, Bayley said. Currently, they are working to transform cells with plasmids for specific target proteins that they will then analyze via nanopore, he said, adding that if this goes well they hope to then attempt analysis of native proteins.

Unfolding and driving target proteins through a nanopore requires certain chemical modifications, which, Bayley said, presents a challenge for analysis of native proteins.

"If we can transform cells with specific plasmids to express specific target proteins then we are pretty sure we can develop methods to fish those proteins out once they are expressed in the cells and modify them so they will go through the nanopores," he said. If, on the other hand, "we have to use, say, a tissue sample, then it is going to be a lot harder to do that modification of the protein to pull it through the pore."

In the case of a protein expressed from a plasmid, the researchers can "make sure it comes out with the amino acid we want at the N-terminus, for example, and then we could modify that amino acid," Bayley explained. "But if it's a natural protein it could have any amino acid at the N-terminus, or it could be acetylated or myristoylated at the N-terminus, which would make [the necessary modification] a lot more difficult to do."

It is only recently that researchers have demonstrated the feasibility of passing proteins through nanopores for analysis. In February 2013, researchers from the University of California, Santa Cruz, published a paper in Nature Biotechnology in which they used the protein unfoldase ClpX to unfold three differentially modified Smt3 proteins and pull them through an α-HL nanopore. A month later, Bayley and his colleagues published a paper in Nature Nanotechnology in which they used oligonucleotide leaders to pull target proteins through nanopores.

In addition to work on detecting proteins by passing them through nanopores, Bayley has also in the past published on nanopore-based protein analyses in which his team functionalized nanopores with aptamers and then used the nanopore to detect binding of target proteins. This approach is more technically straightforward than nanopore protein sequencing, as it relies on an affinity agent, as opposed to the nanopore itself, to detect target proteins. However, it doesn't take advantage of the nanopore's potential ability to detect protein modifications or small differences in amino acids via sequencing.

A similar technique was also demonstrated in 2012 by researchers at the Technical University of Munich, who published a paper in Nature Nanotechnology in which they functionalized a solid state nanopore with recombinant his-tagged proteins to sense target analytes.

This affinity reagent-based approach is also the tack that Oxford Nanopore, where Bayley is a board member, is taking in its efforts to apply nanopores to protein analysis. While the company's GridION and MinION platforms sequence DNA by passing molecules through nanopores, it is working to apply these platforms to protein analysis by linking them to aptamers.

Oxford Nanopore declined to comment on any specific plans for moving into protein analysis, but Zoe McDougall, the company's director of communications, did tell ProteoMonitor that it hoped "to be able to open [the MinION platform] up for a broader range of applications at a later stage."

"Proteins are certainly something of interest to the company," Bayley said, suggesting that "once they have taken care of [the DNA] market, I expect they will be looking for other things that they can analyze using nanopores."

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