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Mayer Lab Developing Method for Five-dimensional Protein Analysis Using Nanopores


NEW YORK(GenomeWeb News) – With funding from Oxford Nanopore,University of Michigan researcher Michael Mayer is developing protein-detection approaches using nanopore technology.

In particular, Mayer is developing nanopores with fluid lipid bilayer coatings for the analysis of amyloid aggregates and other protein biomarkers. He is slated to present on aspects of this work next month at Oxford Nanopore's London Calling conference on nanopore sensing.

Mayer first published on fluid lipid bilayer coated-nanopores in a 2011 paper in Nature Nanotechnology, turning to such structures due to difficulties he and his team had encountered in using nanopores to analyze amyloid aggregates.

Implicated in a range of diseases from type II diabetes to Alzheimer's, amyloid aggregates are challenging to research due to their high heterogeneity and a lack of good analytical methods, Mayer said.

"The problem with these [amyloid] samples is that they are incredibly heterogeneous," he said. "There are monomers and dimers all the way up to these huge fibers that can be micrometers long and everything in between."

Each of these different forms of aggregates is associated with a different level of toxicity, Mayer noted, which makes it desirable to characterize not only total levels of amyloid but also the specific sizes of the aggregates present.

"This is one area where we thought nanopores could play a role, and the reason is that fundamentally a nanopore experiment is a single-particle experiment," he said. "So if you could somehow pull amyloids through nanopores one at a time, you might have a shot at characterizing this complicated heterogeneous mixture one by one and getting large statistics by measuring thousands of these particles on an array of nanopores."

One problem the group encountered, though, was that the amlyoid aggregates clogged their nanopores almost immediately. And so they developed the lipid bilayer coating, which prevented clogging and allowed the aggregates to move through the pores.

The coating also allowed them to attached ligands to the pore that they could use to bind analytes of interest. This binding, Mayer said, served to anchor the target molecules, slowing them down as they passed through the pore and enabling the researchers to collect better data.

"The time resolution of these recording systems is limited, and they aren't fast enough to capture freely moving proteins," he said. "They slip through the pore so fast you aren't able to resolve the entire resistance pulse."

By slowing down movement of the proteins through the pore, Mayer and his colleagues were able to collect more detailed information – enough detail that he believes the lipid-coated nanopores could in the future enable detection of unknown proteins in complex mixtures.

His lab has continued to work on amlyoid analysis, but its primary focus has since shifted to efforts to identify proteins via multidimensional analysis using nanopores.

"When we pull individual proteins through these pores, we get five dimensions of information," Mayer said, noting that the researchers are able to measure a protein's volume, charge, shape, dipole moment, and rotation as it passes through the pore.

"My hope is that by measuring those five [characteristics] we will be able to identify proteins," he said.

To date, he and his colleagues have demonstrated the approach in small mixtures of three different protein species. "And there we can do it," Mayer said, adding that if he picked suitable proteins the approach could likely identify specific proteins in mixtures containing up to 10 species.

"We need to be able to go in and distinguish between 100,000 or so proteins, and we are not that far," he said. "But we have demonstrated that this might be feasible."

Key to improving the method is upping the accuracy of the five individual measurements used to characterize the proteins, Mayer said. "The analogy to a gel would be we need to make the bands more sharp. But there is a point where if you get them very precisely, if you can pin it down and say particle this has this volume, this charge, this shape, this dipole moment, and this rotation — then it can only be this protein."

He and his team have not yet looked into identifying protein post-translational modifications, but, he said, it may be possible in principle.

Mayer is inventor on two patents covering his lab's nanopore technology.

He is also not alone in exploring nanopores for protein detection. Contacted by GenomeWeb this week, Oxford Nanopore declined to comment on any specific plans for moving into protein analysis, but a company spokesperson did tell GenomeWeb last year that it hoped "to be able to open [its technology] up for a broader range of applications at a later stage."

The company is working on applying its GridION and MinION platforms to protein analysis by linking them to aptamers, which, in theory, would bind the target analytes with the nanopores then detect the binding event.

This is similar to a technique 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.

Tacking a different tack, 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.

Additionally, in a paper published last year in Nature Biotechnology, Oxford Nanopore co-founder Hagan Bayley (working independently of the company) demonstrated the ability of a nanopore sensor to distinguish between differentially phosphorylated forms of the protein thioredoxin.