Scanning a single protein, one amino acid at a time

Nov 04, 2021

(Nanowerk News) Using nanopore DNA sequencing expertise, researchers from TU Delft and the University of Illinois have managed to scan a single protein: by slowly shifting 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 data content material of the protein. The researchers revealed their proof-of-concept in Science (“Multiple re-reads of single proteins at single-amino-acid resolution using nanopores”). 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 cataloguing the proteins inside a single cell. Artist impression of the peptide reader, the place a helicase (crimson) pulls up a DNA molecule (yellow) to which a peptide (purple) is hooked up – yielding a sluggish translocation of the molecule by way of the nanopore (inexperienced) which permits to learn off the ion present indicators (orange spotlight) that characterize the amino acids of the peptide as they quickly block the pore. (Image: Cees Dekker Lab TU Delft / SciXel) Proteins are the workhorses of our cells, but we merely don’t know what proteins all of us carry with us. A protein is an extended peptide string fabricated from 20 several types of amino acids, similar to a necklace with totally 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 tremendously differ from the blueprint, for instance as a result of post-translational modifications. Current strategies to measure proteins are costly, restricted to giant volumes, they usually can’t detect many uncommon proteins. With nanopore-based expertise, one is already capable of scan and sequence single DNA molecules. The crew led by Cees Dekker (TU Delft) now tailored this system to as a substitute 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 method 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 throughout the peptide (‘a single bead within the necklace’), they obtained very totally different indicators, indicating the intense sensitivity of the method. The group led by Alek Aksimentiev on the University of Illinois carried out molecular dynamics simulations that confirmed how the ion present indicators relate to the amino acids within the nanopore.

Scanning the barcode for identification

The new method could 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 method would possibly lay a foundation for a single-protein sequencer sooner or later, however de novo sequencing stays a giant problem. For that, we nonetheless must characterize the indicators from an enormous variety of peptides with a view to create a ‘map’ connecting ion present indicators to protein sequence. Even so, the power to discriminate of single-amino-acid substitutions in single molecules is a significant advance, and there are a lot of instant functions for the expertise as it’s 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 endure adjustments that have an effect on their perform, known as post-translational modifications. The ensuing tens of millions of protein variants are troublesome to measure, and could possibly be thought of the ‘dark matter of biology’. Cees Dekker: “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.”