Scanning a single protein, one amino acid at a time

Using nanopore DNA sequencing know-how, researchers from TU Delft and the University of Illinois have managed to scan a single protein. By slowly transferring a linearized protein by way of a tiny nanopore, one amino acid on the time, the researchers had been capable of learn off electrical currents that relate to the knowledge content material of the protein. The researchers revealed their proof-of-concept in Science at the moment. The new single-molecule peptide reader marks a breakthrough in protein identification, and opens the way in which in direction of single-molecule protein sequencing and cataloging the proteins inside a single cell.

Proteins are the workhorses of our cells, but we merely do not know what proteins all of us carry with us. A protein is an extended peptide string fabricated from 20 various kinds of amino acids, similar to a necklace with completely different sorts of beads. From the DNA blueprint, we’re capable of predict of which amino acids a protein consists. However, the ultimate protein can vastly differ from the blueprint, for instance attributable to post-translational modifications. Current strategies to measure proteins are costly, restricted to giant volumes, and so they can not detect many uncommon proteins. With nanopore-based know-how, one is already capable of scan and sequence single DNA molecules. The workforce led by Cees Dekker (TU Delft) has now tailored this system to as an alternative scan a single protein, one amino acid at a time.

“Over the past 30 years, nanopore-based DNA sequencing has been developed from an idea to an actual working device,” Cees Dekker explains. “This has even led to commercial hand-held nanopore sequencers that serve the billion-dollar genomics market. In our paper, we are expanding this nanopore concept to the reading of single proteins. This may have great impact on basic protein research and medical diagnostics.”

Like beads down the drain

The new approach reveals traits of even single amino acids inside a peptide, however how? Lead writer of the paper Henry Brinkerhoff, who pioneered this work as a postdoc in Dekker’s lab, explains, “Imagine the string of amino acids in one peptide molecule as a necklace with different-sized beads. Then, imagine you turn on the tap as you slowly move that necklace down the drain, which in this case is the nanopore. If a big bead is blocking the drain, the water flowing through will only be a trickle; if you have smaller beads in the necklace right at the drain, more water can flow through. With our technique we can measure the amount of water flow (the ion current actually) very precisely.”

Cees Dekker enthusiastically provides, “A cool feature of our technique is that we were able to read a single peptide string again and again. We then average all the reads from that one single molecule, and thus identify the molecule with basically 100% accuracy.”

This ends in a novel read-off which is attribute for a selected protein. When the researchers modified even one single amino acid inside the peptide (“a single bead within the necklace”), they obtained very completely different alerts, indicating the acute sensitivity of the approach. The group, led by Alek Aksimentiev on the University of Illinois, carried out molecular dynamics simulations that confirmed how the ion present alerts relate to the amino acids within the nanopore.

Scanning the barcode for identification

The new approach may be very highly effective for figuring out single proteins and mapping minute adjustments between them—very similar to how a cashier within the grocery store identifies every product by scanning its barcode. It additionally could present a brand new route in direction of full de novo protein sequencing sooner or later. Henry Brinkerhoff clarifies, “Our approach might lay a basis for a single-protein sequencer in the future, but de novo sequencing remains a big challenge. For that, we still need to characterize the signals from a huge number of peptides in order to create a ‘map’ connecting ion current signals to protein sequence. Even so, the ability to discriminate of single-amino-acid substitutions in single molecules is a major advance, and there are many immediate applications for the technology as it is now.”

Glimpsing the ‘dark matter’ of biology

Using the present nanopore peptide reader, researchers can begin analyzing what proteins float round in our cells. After synthesis in cells, proteins nonetheless bear adjustments that have an effect on their perform, known as post-translational modifications. The ensuing hundreds of thousands of protein variants are troublesome to measure, and might be thought-about the “dark matter of biology.” Cees Dekker notes, “To continue the metaphor, after a necklace with its beads is made, it will still be changed: Some red beads get a phosphoryl attached to it, some blue beads a sugar group, etc. These changes are crucial to protein function, and also a marker for diseases such as cancer. We think that our new approach will allow us to detect such changes, and thus shine some light on the proteins that we carry with us.”