Proteins are one of the fundamental substances for building living organisms, playing important roles in all life activities such as synthesis, catalysis, and signal sensing. Developing a reliable and efficient protein sequencing technology is crucial for understanding life processes and revealing disease mechanisms. As the constituent fragments of proteins, peptides become the "key" to opening the door of proteins.

　　Nanopore sequencing technology is an emerging single-molecule sequencing technique in recent years, among which peptide nanopore sequencing still faces many challenges. Recently, a research team from the School of Chemistry and Chemical Engineering at Nanjing University published a paper in the internationally renowned journal "Nano Express". They used nanopore displacement sequencing technology to construct a peptide DNA chimeric chain, similar to pulling a rope to fetch water from a well. By binding DNA sequencing enzymes to DNA, the movement of DNA drives the ratchet movement of peptide molecules in the nanopore, thus achieving nanopore sequencing of peptides and breaking the technical bottleneck.

　　Unable to perform sequence amplification, limiting the sensitivity of protein sequencing

　　Protein is an important substance that makes up all cells and tissues in the human body. It can be said that without protein, there would be no life. The function of proteins is determined by their sequence, and small changes in the sequence may cause proteins to lose their biological activity or trigger diseases.

　　If protein sequencing can be performed, it can determine whether there are sequence changes in the protein and determine whether it will have an impact on human health.

　　Unlike nucleic acid testing, the current difficulty in protein sequencing lies in the lack of effective sequence amplification methods and stagnant technological development, mainly relying on mass spectrometry and Edman degradation methods for sequencing, "said Huang Shuo, the corresponding author of the paper and a professor at the School of Chemistry and Chemical Engineering, Nanjing University.

　　Simply put, mass spectrometry is the process of using biological enzymes to break down proteins into many peptide fragments, which are then detected by mass spectrometry instruments to determine the information of the protein fragments, and ultimately reassembled into protein sequences. The Edman degradation method involves chemically cleaving an amino acid from the N-terminus of a protein for identification, followed by a second round of amino acid cleavage and identification. After multiple cycles, the sequence of the amino acid is determined.

　　But in Huang Shuo's view, both methods have limitations. Compared to DNA and RNA sequencing, there are more types of amino acids that make up proteins, and mass spectrometry is more difficult to detect than nucleic acid sequencing. For the Edman degradation method, it can only analyze the sequence of a single protein sample. If the purity of the degraded sample is not enough and multiple protein samples are mixed, there will be many types of amino acids cut, making it impossible to determine which amino acid comes from which protein and determine the protein sequence. In addition, some peptide segments have closed ends, so degradation method cannot be used. "Huang Shuo introduced.

　　More importantly, due to the inability to achieve sequence amplification, the sensitivity of protein sequencing is low, which means that some protein samples with low abundance in nature cannot be detected. Huang Shuo believes that using existing technology, it is difficult to directly sequence proteins in complex environments. For example, protein samples in body fluids, soil, and water bodies need to be purified and enriched to meet the sample standards for sequencing before they can be sequenced in a mass spectrometer. Meanwhile, protein chemical modifications are abundant, which may also interfere with protein sequencing.

　　By utilizing the reaction between DNA and enzymes, peptide chains can be pulled to move controllably in nanopores to complete sequencing

　　Can we find a more microscopic sequencing method that can sequence proteins with just one molecule? Since 2015, nanopore sequencing technology has entered the research field of Huang Shuo's research group.

　　Nanopore sequencing technology is an emerging single-molecule sequencing technique in recent years, which has achieved success in DNA and RNA sequencing. It allows single stranded nucleic acids to pass through nanopores and obtain the base information of the nucleic acid strand through detectors inside the nanopores.

　　If protein sequencing can be performed at the single-molecule level, it will provide extremely high sensitivity for detecting low abundance proteins and single-cell proteomics. Inspired by this, Huang Shuo's research group began to study the use of nanopore sequencing technology for single-molecule sequencing of proteins or peptides.

　　However, peptide nanopore sequencing still faces many challenges, one of which is how to achieve controllable ratchet movement of peptides in nanopores, which is mainly limited by the current lack of peptide sequencing enzymes that match peptide chains and have strong affinity.

　　Ratchet motion refers to the uniform and directional movement of a biopolymer that adheres to the inner wall of a nanopore in the same direction, similar to moving on the inner wall of a gear. This requires finding an enzyme to control the speed of the peptide's movement, thereby controlling its speed through the nanopore for precise sequencing

　　Previously, Huang Shuo's research group attempted to sequence non natural nucleic acids, but struggled to find suitable enzymes to match the ratchet motion. Later, they had a sudden idea and used the displacement effect of nanopores to sequence, and pioneered nanopore displacement sequencing technology.

　　The recognition site of nucleic acid nanopores and the reaction site of enzymes have a fixed misalignment of about 14-15 bases, which forms a sequencing window. This window, which is not limited by enzyme reactions, can be used to achieve peptide sequencing, "said Huang Shuo.

　　Can nanopore displacement sequencing technology be applied in the field of peptide sequencing? Recently, Huang Shuo's research group discovered feasibility during technical validation. In this study, they embedded peptides and DNA into a chain, with the DNA part of the chimeric chain being the "traction chain". During the detection process, DNA sequencing enzymes bind to DNA and, by pulling the DNA part, manipulate the controllable ratchet movement of the entire chain in the nanopore, achieving direct reading of peptides from the nanopore.

　　Huang Shuo gave an analogy, "This is like using a rope to lift a bucket in a well. If the well is a nanopore, the rope is DNA, and the bucket is a peptide. The DNA sequencing enzyme at the wellhead pulls DNA to move inside the nanopore, and DNA is then fused with the peptide. The movement of DNA directly pulls the movement of the peptide, thus achieving nanopore sequencing of peptides

　　Huang Shuo introduced that after verification, the nanopore sequencing signals of peptides exhibit high consistency and sequence correlation, and the current changes caused by single amino acid substitutions can also be clearly detected. By coupling the N-terminus or C-terminus of the polypeptide with the DNA drive chain, the reading of amino acid sequence information at both ends of the polypeptide is achieved.

　　This means that protein single-molecules can be sequenced, providing a new tool for high-throughput protein sequencing and protein reconstruction in the future, "said Huang Shuo. With the help of protein sequence research, protein functions can be predicted, thereby helping to understand and interpret related life activities. (Golden Phoenix)