Scanning a single protein, one amino acid at a time

Date:
November 4, 2021
Source:
Delft University of Technology
Summary:
Using nanopore DNA sequencing technology, researchers have
managed to scan a single protein: by slowly moving a linearized
protein through a tiny nanopore, one amino acid at the time, the
researchers were able to read off electric currents that relate to
the information content of the protein. The new single-molecule
peptide reader marks a breakthrough in protein identification,
and opens the way towards single-molecule protein sequencing and
cataloguing the proteins inside a single cell.



FULL STORY ========================================================================== Using nanopore DNA sequencing technology, researchers from TU Delft
and the University of Illinois have managed to scan a single protein:
by slowly moving a linearized protein through a tiny nanopore, one
amino acid at the time, the researchers were able to read off electric
currents that relate to the information content of the protein. The
researchers published their proof-of- concept in Science today. The
new single-molecule peptide reader marks a breakthrough in protein identification, and opens the way towards single- molecule protein
sequencing and cataloguing the proteins inside a single cell.


========================================================================== Proteins are the workhorses of our cells, yet we simply don't know
what proteins we all carry with us. A protein is a long peptide string
made of 20 different types of amino acids, comparable to a necklace
with different kinds of beads. From the DNA blueprint, we are able to
predict of which amino acids a protein consists. However, the final
protein can greatly differ from the blueprint, for example due to post-translational modifications. Current methods to measure proteins
are expensive, limited to large volumes, and they cannot detect many
rare proteins. With nanopore-based technology, one is already able to
scan and sequence single DNA molecules. The team led by Cees Dekker
(TU Delft) now adapted this technique to instead 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 technique reveals characteristics of even single
amino acids within a peptide, but how? Lead author 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 adds: "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 results
in a unique read-off which is characteristic for a specific protein. When
the researchers changed even one single amino acid within the peptide
('a single bead within the necklace'), they obtained very different
signals, indicating the extreme sensitivity of the technique. The group
led by Alek Aksimentiev at the University of Illinois performed molecular dynamics simulations that showed how the ion current signals relate to
the amino acids in the nanopore.

Scanning the barcode for identification The new technique is very powerful
for identifying single proteins and mapping minute changes between them
-- much like how a cashier in the supermarket identifies each product
by scanning its barcode. It also may provide a new route towards full
de novo protein sequencing in the future. Henry Brinkerhoff clarifies:
"Our approach might lay a basis for a single-protein sequencer in the
future, butde 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 current nanopore peptide reader,
researchers can start analyzing what proteins float around in our
cells. After synthesis in cells, proteins still undergo changes that
affect their function, called post-translational modifications. The
resulting millions of protein variants are difficult to measure,
and could be considered 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." ========================================================================== Story Source: Materials provided by Delft_University_of_Technology. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Henry Brinkerhoff, Albert S. W. Kang, Jingqian Liu, Aleksei
Aksimentiev,
Cees Dekker. Multiple rereads of single proteins at single-amino
acid resolution using nanopores. Science, 2021; DOI:
10.1126/science.abl4381 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/11/211104140836.htm

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