Nanopore scanner reads individual proteins one amino acid at a time

2021-11-16 07:43:28 By : Mr. Leo Li

The helicase (red) pulls the DNA molecule (black) attached to the peptide (red), causing the molecule to slowly translocate through the nanopore (green). This allows researchers to read the ionic current signal peptides that characterize amino acids because they are temporarily Clogs the pores. [Source: Henry Brinkerhoff, Cees Dekker Lab TU Delft]

In a proof-of-concept study, scientists at the Delft University of Technology in the Netherlands and the University of Illinois successfully reused DNA nanopore sequencing technology to scan individual protein molecules.

When the helicase pulls the DNA-bound peptide chain through the tiny membrane channel, researchers can now decode the changes in the ionic current passing through the nanopore, reading the individual amino acid building blocks of the peptide at a time. This capability is a milestone in protein identification, paving the way for single-molecule protein fingerprinting, protein de novo sequencing, and dynamic cellular proteome analysis.

The results of the study were published in an article in the journal "Science" entitled "Using nanopores to reread a single protein multiple times with single amino acid resolution."

So far, information about the primary sequence of proteins is mainly obtained from DNA sequences. However, neither DNA nor RNA sequences provide information about protein abundance, splicing, or post-synthesis modification. Although proteins are special functional machines in our cells, we still rely on skeletal DNA blueprints to understand proteins.

Methods of analyzing proteins, such as mass spectrometry, cut them into fragments, and identify the proteins from the fragmented spectral features by comparing them with protein databases. Such methods are expensive, are limited to large volumes, and cannot detect low-abundance proteins in cells.

In the past few decades, scientists have used a cost-effective and portable nanopore-based technology to sequence individual DNA molecules that can identify epigenetic markers in long reads. Delft University of Technology professor Dr. Cees Dekker and his team have now used this method to sequence individual protein molecules, one amino acid at a time.

"In the past 30 years, nanopore-based DNA sequencing has evolved from an idea to a practical working device," Decker said. "This has even led to commercial handheld nanopore sequencers serving the multi-billion dollar genomics market. In our paper, we extend this nanopore concept to read individual proteins. This may be a study of basic proteins. And medical diagnosis."

As a postdoctoral researcher in Dekker's laboratory and the first author of the paper, Dr. Henry Brinkerhoff said: "Imagine the amino acid string in a peptide molecule as a necklace with beads of different sizes. Then, Imagine you turn on the faucet and slowly move the necklace to the sewer. In this case, it is a nanopore. If a large bead blocks the drain pipe, the water flowing through will only be a trickle. If the necklace is at the drain pipe With smaller beads, more water can flow through. Using our technology, we can measure water flow (actually ion current) very accurately."

"The challenge of using nanopore technology for peptide sequencing is mainly around the use of controlled translocation of molecules of interest through nanopores to read signals from ionic currents. For protein sequencing, this problem is due to the overall charge distribution and volume of the molecules. It becomes more complicated," said Nicholas Schork, Ph.D., associate director and distinguished professor of quantitative medicine at the Institute of Translational Genomics (TGen) in Phoenix, Arizona. Has been working on nanopore technology for genomics, but has not participated in the current research.

"The study by Brinkerhoff et al. solved the problem of irregular translocation speed by linking a small piece of DNA to the target peptide, including the use of DNA helicase to pull the DNA component of the target molecule through the biological nanopore (MspA) at a controlled rate. ). When the DNA peptide molecule passes through the pore, the change in the ionic current of the DNA part is read, and then the peptide part," Schork said.

This new protein sequencing method has a high degree of specificity and sensitivity. The authors showed that it can detect changes in individual amino acid substitutions from changes in ion current readings.

Dekker added: “A cool feature of our technology is that we can read a single peptide string again and again. Then we average all the readings from a single molecule to identify that molecule with essentially 100% accuracy. ."

In this study, the authors demonstrated that they were able to rewind peptide readings to obtain many independent scans of the same molecule, with an error rate of less than one in a million when identifying individual amino acid variations.

Dr. Aleksei Aksimentiev, professor of physics at the University of Illinois, and his team performed molecular dynamics simulations to show the relationship between ionic current signals and amino acids in nanopores. These simulations indicate that the change in the ionic current signal detected when the peptide string descends the nanopore drain is due to size exclusion and the binding of the peptide or its side chain to the inner wall of the nanopore.

"My laboratory uses high-end computer simulations to determine how the sequence of the polypeptide chain affects the blocking current through the MspA nanopore, which explains the counterintuitive dependence of blocking current on the physical size of amino acids," Aksimentiev said.

The chemical groups attached to the protein after protein synthesis, such as sugar or phosphate groups, will change their natural configuration and add another layer of complexity to determine the true state of the cell proteome. Dekker said: "These changes are critical to protein function and are also markers of diseases such as cancer. We believe that our new method will allow us to detect such changes and thus understand the proteins we carry with us."

De novo protein sequencing directly determines the amino acid sequence of a protein without referring to known sequences or protein databases. In its current form, this technology cannot perform de novo protein sequencing.

"Due to the complexity of this mapping, this method lacks a method to map ionic currents to amino acids, so true de novo protein sequencing is still a challenge. However, the proposed method is suitable for identifying peptide sequence variations, as the authors put their The amino acid sequencing results are shown when compared with a known reference protein containing amino acid substitutions," Schork said.

"The real advantage of this technology is that it can use existing nanopore sequencing hardware (such as the commercial Oxford Nanopore MinION system) by simply changing the protocol for sample preparation and data analysis. This will make research exploring this strategy easier. And to adapt to the necessary bioinformatics workflow improvements to explain the analysis,” Schork added.

Brinkerhoff said: "Our method may lay the foundation for future single protein sequencers, but de novo sequencing is still a huge challenge. For this reason, we still need to characterize the signals from a large number of peptides to create connections between ionic current signals and proteins. The "map" of the sequence. Even so, the ability to distinguish between single amino acid substitutions in a single molecule is a major advancement, and the technology now has many direct applications."

The researchers pointed out that the technology has several limitations that require further attention. For example, positively charged peptides may not pass through the nanopore efficiently. The author points out that this can be solved by modifying the pore so that the peptide can pass through it smoothly regardless of its charge specificity. It is currently possible to scan peptides of approximately 25 amino acids in length by this method, limiting its application to short peptides. Although this is an improvement over peptides less than 10 amino groups long that can be identified using mass spectrometry, the read length can be increased through the fragmentation and shotgun strategies used in traditional protein sequencing.

The author concludes: "Our findings are the first step towards a low-cost method that can perform single-cell proteomics at the ultimate limit of concentration sensitivity, and is widely used in basic biology and clinical practice. Applications."

The improvement and adoption of this new technology is expected to provide real-time, scalable, affordable and easy-to-use molecular sequencers for proteins, thereby changing the daily work of life scientists and healthcare researchers.

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