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Nanopores Distinguish Protein Isoforms

Jeffrey M. Perkel, Ph.D.

Nanopores can sequence DNA, but what else can they do? Find out...

As the world awaits the first shipments of Oxford Nanopore’s tiny MinION sequencers, one of the researchers who founded the company has announced a new proteomics application for nanopore technology.

Writing in Nature Biotechnology, Hagan Bayley, Professor of Chemical Biology at the University of Oxford, demonstrated that nanopores can distinguish different phosphorylated isoforms of thioredoxin (1). Though far from protein sequencing—the analog of what MinION does for DNA— the report represents a first step towards single-molecule protein analysis, Bayley said.

Hagan Bayley

Bayley’s team inserted single alpha-hemolysin protein pores in a membrane with electrodes on either side. In this setup, the only electrical path is through the pore, where the system records the current. To assess the system’s sensitivity to phosphorylation, the team produced several mutants of thioredoxin, all of which contain a single phosphorylation site near the C-terminus, and capped that terminus with a 30-mer DNA oligonucleotide.

Application of a current draws the DNA oligonucleotide into the pore, tugging on the protein and causing it to unfold, like pulling on the end of a ball of string. As the protein passes through the pore, the detection system records the changes in current.

As it turns out, phosphorylated proteins disrupt the baseline current slightly differently than nonphosphorylated proteins. And not just in enriched populations. In one experiment, the team used a protein that could be phosphorylated at either of two positions and then partially phosphorylated it. When they passed 202 of the resulting protein molecules through a pore, the system differentiated each of the 4 possible isoforms.

Other methods also can distinguish protein isoforms, but whereas those methods sample protein populations, nanopores read single molecules, and that, Bayley said, is a key advantage. “You’re analyzing individual molecules. And if you do this over several minutes, you can look at hundreds of molecules, and you can get a reading of the phosphorylation state of each one.”

But the method is far from ready for primetime. This study used purified model proteins with known phosphorylation states. The proteins were not threaded through, but rather began to enter the pore, paused for a short while, and then popped out the other side. The method therefore could not distinguish protein isoforms that differed in the middle of the amino acid sequence.

Eventually, Bayley said, the team would like to address this problem, possibly by using antibodies to cause the protein to pause at specific positions as it passes through the pore. He also would like to apply the approach to native proteins and test its efficacy on larger molecules. If nanopore DNA sequencing is any guide, longer proteins will likely prove harder to work with than shorter ones, he said.

“This is the first paper ever of its kind,” Bayley said, “and such papers generally are quite primitive and crude. Over the coming years we hope to greatly refine this method.”


Rosen, C.B., Rodriguez-Larrea, D., and Bayley, H., “Single-molecule site-specific detection of protein phosphorylation with a nanopore,” Nat Biotechnol, 32:179–81, 2014.