Forget DNA Sequencing, Protein Sequencing is Here!

Every so often I have the distinct pleasure of hearing of a new technology that I’d never imagined before. This last week, a study in Nature Biotechnology described using nanopore to perform protein sequencing. Thinking of the difficulty and complexity of sequencing just 4 nucleotides, it’s hard to conceive of the exponential difficulty to sequence 20 amino acids.

The technology is still in a nascent period working on the ability to differentiate 20 amino acids- some with very similar sizes and charges. However, as has been the case with many technologies, the speed of advancement can increase rapidly. The possibilities of this technology are very exciting.

Figure 1. Aerolysin structure from CyroEM. This pore comes from Aeromonas hydrophila, a Gram-negative bacterium associated with diarrheal diseases and deep wound infections.

Nanopores have been used mostly for sequencing DNA. Pacific Biosciences have created a platform that reads long sequences of DNA by synthesizing DNA from a single strand moving through a pore. Another company, Oxford Nanopore has made several devices that use nanopore technology to perform sequencing in portable devices. The key to most of these technologies involve functionalizing the pores to have properties desirable for specific purposes.

For protein sequencing, amino acids need to move through the pores slowly enough to have their charges accurately measured. To accomplish this goal, the French start-up, DreamPore, used a bacterial derived cytolytic pore called aerolysin. Aerolysin was found to slow down the passage of amino acids as it has a single molecule trap, which binds to single amino acids with carrier cations. This method detects 13 of the20 amino acids. Additional chemical modifications, instrumentation advances, and nanopore imaging allowed two additional amino acids to be detected. The last few were too similar in charge to be differentiated. However, this study provides a path forward where it is conceivable to sequence each of the 20 amino acids.

In the future, detecting post-translational modifications such as glycosylation and phosphorylation will be an important advance. It will be fun to follow the progress of this technology to see how it might be applied clinically.

References

  1. Iacovache I, De Carlo S, Diaz Nuria et al. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process. Nature Communications 2016. 7:12062.
  2. Ouldali H, Sarthak K, Ensslen T et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nature Biotechnology 2020; 38: 176-181

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

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