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Fields & Applications Proteomics

Pores for Peptide Profiling

A team of scientists based at the University of Groningen, the Netherlands, have produced – and patented – the smallest biological nanopores ever. They’ve also demonstrated their potential for measuring the mass of single peptides (1).

Giovanni Maglia previously reported that cell membrane nanopores from the sea anemone Actinia fragacea could be used to study DNA, proteins, and peptides; a change in electrical current across the pore as a molecule enters or passes through it results in the analytical signal. However, these pores were too large for studying small peptides – anything below 1.6 kDa moved through the pores too rapidly. Maglia realized that they needed to make much smaller pores.

Maglia teamed up with colleagues Gang Huang and Arnout Voet to reduce the pore size by adjusting the interactions between the nanopores and the lipids in the membranes. Three types of nanopores were created with diameters from 1.6 nm down to 0.84 nm – the smallest biological nanopore reported.

But were peptides able to pass through such pores? Yes and no. The negative charge on the pores pulled water through, carrying peptides with it; however, the team had to adjust the pH to 3.8 so that negatively-charged peptides would not be repelled by the negatively-charged pores. With this modification, the medium-sized pores were able to successfully distinguish between angiotensin peptides, some of which differed by only one amino acid, giving a resolution of 44 Da.

The nanopore system is portable, and can be made cheaply using off-the-shelf technology – plus, peptides of different sizes or post-translational modifications can be measured directly by combining multiple pore sizes in one device. However, for the system to be really useful in proteomics research, the resolution needs major improvement admitted Maglia – and he has several ideas to that end, including modifying the nanopores with synthetic amino acids.

Distinguishing known analytes is useful, but what about identifying unknown peptides? Promisingly, there was a linear relationship between the volume of the analyte in the nanopore and the electrical signal. Enough promise to put a low-cost, single-molecule protein sequencer on your wish list?

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  1. G Huang et al., “FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution”, Nat Commun, 10 [Epub ahead of print] (2019). DOI: 10.1038/s41467-019-08761-6
About the Author
Ryan De Vooght-Johnson

After graduating from the University of Warwick with a masters in instrumental and analytical methods for biological, pharmaceutical, and environmental chemistry, I worked in the laboratory in various analytical development roles. I was then lucky to find my calling in academic publishing and science writing. I’ve been a commissioning editor and launch editor in a biomedical publisher and since 2014, I’ve been working as a freelance science writer and editor.

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