NEW YORK (GenomeWeb) – University of Groningen researchers have demonstrated the ability of nanopores to identify peptide and protein biomarkers in simple mixtures and to distinguish between polypeptides differing by as little as a single amino acid.
Published this week in Nature Communications, the study established the suitability of Fragaceatoxin C (FraC) nanopores for protein analysis and offered a novel approach to driving target proteins into nanopores for detection, said Giovanni Maglia, professor of science and engineering at the University of Groningen and senior author on the paper.
Nanopores have been successfully applied to DNA analysis, with, most notably, Oxford Nanopore using the technology in its sequencing platforms. Researchers are likewise pursuing nanopores for protein measurements, though this has proven a more difficult challenge due in large part to the greater complexity of proteins compared to DNA.
The fact that DNA consists of four bases limits the complexity of the signal generated by such molecules as they pass through a nanopore, Maglia noted. Proteins, on the other hand, consist of combinations of 20 amino acids, making for "a really quite complex signal," he said.
Researchers are working on a variety of approaches to tackle this complexity. For instance, in May a team at the University of California, San Diego published a machine learning-based approach to nanopore protein identification that they said indicated large-scale proteomic profiling via nanopores could be possible.
In 2016, University of Fribourg researchers funded in part by Oxford Nanopore published work exploring how increasing the number of parameters they extracted from nanopore data improved protein analysis. (Maglia's co-author and Groningen colleague Carsten Wloka receives funding from Oxford Nanopore, and Maglia said the company has provided his lab with a nanopore device to work with.)
In addition to the complexity of the signals they generate, proteins are challenging analytes for nanopores because of the difficulty of passing them through a nanopore in a consistent way. As the authors noted, with DNA, the electric field within the nanopore unfolds and stretches the molecule as it passes through the pore. Proteins, however, do not have a uniform charge distribution, meaning that different portions of the molecule interact in different ways with the electric field.
This presents the problem of "how we can actually get the protein into the pore," Maglia noted.
The researchers found that by engineering the electro-osmotic flow — the flow of liquid induced by the electric field — within the nanopore, they were able to draw proteins into the pore regardless of their charge composition.
"That was the first part of protein sequencing that we were able to tackle," he said, noting that the study demonstrates "that there is no fundamental barrier to de novo sequencing of proteins with nanopores."
"Before it wasn't really obvious how you could drag a polypeptide across a nanopore at a constant potential, because you couldn't use the electric field for that," he added. "But now we have found we can use this other [electro-osmotic] force to do that job."
Now, Maglia said, researchers need to establish a method, enzymatic or otherwise, for moving unfolded polypeptides through nanopores at a controlled speed.
"With DNA sequencing you have an enzyme that feeds the DNA across the nanopore and the current reads it base by base," he said. "Now we need to find a system that can unfold and feed polypeptides across the nanopore."
The researchers used FraC nanopores for the work, which, Maglia said, differ structurally from nanopores commonly used for DNA analysis and in previous protein work.
He said that he and his colleagues became interested in the molecule when they saw its crystal structure, which shows it to have a conical shape with a large opening on one side that narrows to a smaller opening at the other end.
This makes it potentially suitable for analyzing folded proteins (using the wider opening) as well as sequencing unfolded proteins (by passing them through the narrow end of the pore), Maglia said.
Using the FraC nanopores, the researchers were able to identify a number of different folded proteins and peptides. Investigating a mixture of β2-microglobulin, EGF, and endothelin 1, they found they were able to consistently distinguish between these three analytes. They were likewise able to distinguish between endothelin 1 and 2, which, Maglia said, are near-isomers, differing in just one out of 21 amino acids.
While, as he noted, technical challenges remain for de novo sequencing of unfolded peptides, targeted analysis of folded proteins using nanopores is more plausible in the short term, Maglia said. And, indeed, the Nature Communications study as well as previous work from other groups like the Fribourg team has demonstrated the capability of nanopores to identify proteins in simple mixtures.
"If, say, you have a way to pull down a group of proteins that you want to study, with, say antibodies, or with a rough kind of separation method that eliminates 95 percent or so of proteins that you don't want, then after this [enrichment], you can imagine that you could analyze this sample and you would see a pattern of different [current] blockades [different] proteins can give you," he said. "And then if you have this pattern, you can compare it with a standard that you analyzed before, and you can probably identify the components that you want to analyze. So that could be possible tomorrow, if you wanted to do it."
Such an approach wouldn't currently be competitive with existing protein analysis technologies like mass spectrometry, Maglia noted, but he said nanopore technology could eventually find a niche for protein analysis due to its portability, low-cost, and ability to detect single molecules in small sample sizes.
"Mass spectrometry is great, and I don't think, at least in the near future, you can compete with mass spectrometry," he said. "However, it has limitations in that a good machine costs a million [dollars] and they are very big and require vacuums to work."
With existing nanopore-based DNA sequencers, "you can send it to space, take it to the jungle, put it in your pocket, and they work," he said. Similarly, with nanopore-based protein analysis, "if you develop a system that can concentrate or carry out a rough purification of [your target] protein, then you should be able to actually measure it on the spot without really any special and expensive technology."