Ready, Set, Sequence!

Back in 1996, a research team from the University of California introduced the concept of nanopore sequencing (1) – originally developed to sequence DNA. 

Now, nanopore based sequencing of DNA and RNA is fully commercialized – leaving one big question unanswered: could the same technology be used to sequence proteins?

Recently, a research group from Nanjing University, China, modified a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore – incorporating a nickel based sensor and machine learning – to investigate the potential of this specific technology in protein sequencing (2). 

Here, we discuss with Shuo Huang, professor at the School of Chemistry and Chemical Engineering in Nanjing University and corresponding author about his latest nanopore-based proteomics research and the future of this potentially disruptive technology… 

What inspired you to explore the possibility of nanopore-based protein sequencing?
 

Nanopore DNA and RNA sequencing has already been achieved, so naturally the next goal is to successfully perform the technique for protein analysis. But distinguishing and determining all the different amino acids simultaneously as well as their rich post translational modifications is the main challenge in nanopore based protein sequencing. 

However, with the high resolution of Mycobacterium smegmatis porin A (MspA) nanopore, this becomes achievable. With the invention of the hetero-octameric MspA nanopore assembly, we managed to resolve a large variety of epigenetically modified nucleoside monophosphates (3) and monosaccharides (4). 

To resolve different amino acids, we need to install a different adapter to the hetero-octameric MspA. The NTA-nickel adapter immediately came into our mind, so we decided to focus our research on developing and optimizing this sensor.

What are some of the challenges involved? Have nanopore sensors been used in protein sequencing before?
 

With the previous invention of the hetero-octameric MspA techniques pioneered by us, the only remaining challenge is to find a suitable reactive adapter for this pore. We were fortunate that the first choice that came into our mind – NTA-nickel – worked the best. We also tried other metal ions as an attempt of optimization – but nickel was the best option. 

A variety of nanopores/nanopore techniques have been previously reported in an attempt to sequence protein. However, to date, there is not a single nanopore technique that has truly sequenced protein solely using nanopore technology, largely due to the limited resolution of nanopore to resolve so many different amino acids. By showing unambiguous discrimination of all 20 proteinogenic amino acids using nanopore technology, the nickel modified MspA nanopore shows great promise in achieving this goal.

Could you explain some key features of your sensor?
 

As noted, perhaps the key feature of this sensor is that it is equipped with an NTA-nickel adapter. The NTA (nitrilotriacetic acid), serves to coordinate a nickel ion in the pore constriction. The immobilized nickel ion then serves to further coordinate with amino acids to achieve nanopore sensing. To the best of our knowledge, prior to this work, there is no previously reported nanopore that contains a sole nickel ion in its lumen. This specially designed configuration appears to be critical to resolving all 20 proteinogenic amino acids along with four post translational modifications (PTM).

What role is played by machine learning?
 

The nickel adapter at the pore constriction serves to extend the dwell time of each amino acid, when captured by the pore. Rich identity information is disclosed with this fully extended event dwell time so that highly characteristic events can be reported. 

With machine learning, we can more easily and more efficiently find even minor nanopore event feature differences. It eventually turned out to be quite efficient to fully discriminate between events generated by different types of amino acids. This is however extremely difficult to do solely by human judgment.

What does the future of nanopore protein sequencing look like? 
 

We are currently developing techniques to show direct sequencing of a short length of peptides or proteins using this MspA-NTA-Ni sensor. We aim to disclose the results very soon in a scientific publication.

The future of nanopore protein sequencing would be extremely portable, low-cost and fast in data production, similar to what has been demonstrated by the nanopore sequencer of DNA/RNA. 

I am optimistic that it might eventually replace mass spectrometry by being much smaller, more economic, faster, while providing more information. Right now, nanopore protein sequencing has not yet been demonstrated – but the unambiguous discrimination of all 20 proteinogenic amino acids suggests a bright future for it!

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