BALTIMORE – Oxford Nanopore Technologies showcased early progress toward nanopore protein sequencing at its London Calling user meeting this week.
Speaking to a live and online audience at the company’s flagship annual event, Oxford Nanopore Chief Technology Officer Clive Brown said the company’s “very active” protein sequencing R&D pipeline has achieved “significant proof of concept that we can generate unique, reproducible profiles of more than one amino acid in a plain background.”
But he also emphasized that the company’s development of protein sequencing technology is still in its early days. Therefore, it remains to be seen if or when the company is able to turn its blueprint into reality.
According to Brown, although charged molecules can travel through a nanopore quite easily, protein nanopore sequencing is difficult because the molecules are often folded, may not be charged, and are often heavily modified with sugars.
While he said that Oxford Nanopore is exploring many different schemes to achieve protein sequencing, he highlighted a DNA-peptide conjugate method, where peptides can be tethered to a double-stranded DNA molecule while translocating through the pore.
“The advantage of that is that we can use the DNA motor that we use for DNA sequencing,” Brown said, adding that theoretically, as the DNA-peptide conjugates travel through the pore, it would generate DNA and peptide signals, though he did not disclose technical details.
Still, he acknowledged that getting reliable peptide signals “has been quite a struggle.” To generate the signal, Oxford Nanopore designed a “flat, bland, small peptide backbone” with one or two amino acids embedded and fed the molecule through the pore, similar to the early signal detection efforts with DNA nanopore sequencing.
Unlike DNA, which is made up of four bases, protein structures can be complex due to the combination of 20 amino acids. However, Brown said the silver lining of this complexity is that “even a not very good sequence can be very unique to a protein.”
He said the company is starting to generate unique profiles for more than one amino acid with “pretty good type profiles” in a "vanilla background." “That's quite good compared to the state of the art,” he added.
But Brown acknowledged that Oxford Nanopore is not the only group working on the DNA-protein conjugation technology. In fact, researchers at Delft University of Technology and the University of Illinois at Urbana-Champaign also developed a peptide-DNA linkage approach for nanopore protein sequencing, which appears to be quite similar to Oxford Nanopore’s method.
The Delft researchers detailed their method in a 2021 Science paper and have filed for a patent covering the approach. An Oxford Nanopore spokesperson declined to comment whether there is any IP overlap between the Delft group and the company.
Touting the early results as “promising,” Brown said that in the long run, Oxford Nanopore hopes to be able to do shotgun sequencing of a complex mixture of proteins from a biological sample. Describing the envisioned workflow, he said the proteins would preferably not be separated from the mixture, chewed up with a protease, typically trypsin, and converted into DNA-peptide conjugates for sequencing.
Compared with current mass spectrometry-based methods, which typically require protein separation and complete trypsinization, nanopore sequencing may have the advantage of working with partially digested proteins, allowing for long or overlapping peptides, he added.
Ultimately, Brown said, the company’s goal is to create a technology that allows researchers to sequence proteins on the existing Oxford Nanopore DNA sequencing platform, eliminating the need for any special hardware.
But for now, “this is really hard stuff,” Brown told the audience. “I don't want to oversell this.”