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Oxford Nanopore Co-Founder Bayley Working to Apply Nanopore Technology to Protein Analysis

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A research team led by Oxford Nanopore Technologies co-founder Hagan Bayley has devised a method for unfolding proteins and passing them through nanopores.

The technique, which the researchers detailed in a paper published this month in Nature Nanotechnology, uses oligonucleotide leaders to pull target proteins through nanopores, and is a step towards using such molecules for protein sequencing and detection, Bayley, a professor of chemical biology at Oxford University, told ProteoMonitor.

When voltage is applied across a membrane containing a nanopore, molecules passing through it or binding to an agent outside it cause changes in ionic conductivity that can be measured, enabling their sequencing or detection. Thus far, the technology has been applied primarily to the study of nucleic acids, with a number of commercial firms, including Oxford Nanopore, developing it for high-throughput DNA sequencing.

In theory, the method is applicable to proteins, as well, and with nanopore-based nucleic acid sequencing relatively far along in its development, proteins could see increasing research interest, Bayley suggested.

"Much of the work that had to be done on [nanopore sequencing] of DNA, at least in the academic world, is close to finished," he said. "So I think people have become interested in looking at different things, and one of them is proteins."

The Oxford team's study is the second in recent months investigating methods of unfolding and driving proteins through nanopores. In February, 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 (PM 2/8/2013).

Broadly speaking, researchers have adopted two main approaches to using nanopores for protein detection. In one, they have functionalized nanopores with affinity agents like antibodies or aptamers for specific proteins and then used the nanopore to detect binding of the target proteins.

Such a technique was used last year 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 (PM 3/16/2012).

Bayley and colleagues at Oxford Nanopore have also investigated such a method, publishing in January of last year a study in the Journal of the American Chemical Society using nanopores linked to aptamers for protein detection.

The long-term goal of such an effort, Bayley said, would be to create the "equivalent of an antibody chip with which you could measure the concentrations of many different proteins" in a sample using arrays of nanopores as the detection readout.

The second main approach to nanopore-based protein analysis – the one exemplified by the recent papers from Bayley and the UCSC team – consists of passing target proteins through the nanopore and measuring the changes in conductivity that occur as they move through the pore. This method would enable the detection and identification of proteins by determining their specific amino acid sequences, much as nanopore-based nucleic acid sequencing works by associating changes in conductivity with particular nucleobases moving through the pore.

This second approach offers an advantage over the affinity agent-based method in that it could allow researchers to identify protein post-translational modifications and splice variants.

For instance, Bayley said, "you could just run your protein through [the nanopore] and be able to say that it's phosphorylated on positions 47, 63, and 219 on a single molecule" – information that would be more difficult, if not impossible to obtain, using approaches like antibody-based detection or bottom-up mass spec.

The ability to perform nanopore-based protein sequencing on a collection of different proteins taken from, for instance, a cell extract, remains distant, Bayley said, noting that the large number of amino acids involved makes it significantly more challenging than nanopore sequencing of DNA.

"In DNA sequencing there are only four bases, and that has been difficult enough a problem for Oxford Nanopore," he said. "A protein has 20 amino acids, even if you don't think about the modifications of those amino acids. So it's a much more difficult problem."

A simpler challenge — and one that the technology might currently be able to address — is identifying modifications and variants within a particular known protein population, Bayley said.

For instance, he said, if a researcher were to use an antibody to isolate a specific protein from a sample, they could then use nanopore sequencing to identify different isoforms within that isolated protein population.

Such an approach could prove particularly attractive given the growing interest in top-down proteomics and the potential importance of protein isoforms in work including disease biomarker research.

"I think one of the surprises of the human genome was how few protein-coding genes there actually are," Bayley said. "But the variety of proteins is made enormous by alternative splicing and modifications like phosphorylation and glycosylation and so on."

He added that his group is actively working on using nanopore sequencing to identify protein variants and modifications.

The plan, Bayley said, is to first "take a protein and modify it ourselves and look to see how small a modification we can detect."

"Then once we've done that we'll start pulling proteins out of cells and looking at them," he added.

Bayley said he couldn't speak to the level of interest in protein sequencing at Oxford Nanopore, where he is currently a board member. However, he said, "the company is interested in and has always been interested in analytes other than DNA."

Oxford Nanopore declined to comment, but it does note on its website that its GridIon technology can be used for protein analysis and highlights in particular its work with Bayley on using aptamer-linked nanopores for protein detection.

"When I founded Oxford Nanopore, it was as a company to make a sensing platform," Bayley said. "The vision of stochastic sensing with nanopores ranged from small molecules to DNA to peptides, proteins, sugars, and so on. The technology wasn't envisioned specifically for DNA, so all the hardware and software that has been developed for DNA can be used for other applications."

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