Methods, kits and applications for sequencing polypeptides and proteins

Abstract

The application provides a polypeptide and protein sequencing method, a kit and application. The polypeptide sequencing method comprises the following steps: S1, attaching a target polypeptide to a nanopore; S2, introducing a recognition molecule specifically recognizing a terminal amino acid of the target polypeptide into a nanopore sequencing system; under the action of an electric field, the detection of the terminal amino acid of the target polypeptide is realized by utilizing the electric signal change generated in the binding and dissociation process of the recognition molecule and the target polypeptide. By utilizing the unique electric signal change generated in the binding and dissociation process of the recognition molecule and the terminal amino acid of the target polypeptide, the detection of a single amino acid is realized.

Description

Peptide and protein sequencing methods, kits and applications Technical Field

[0001] The present invention relates to the field of protein sequencing, and in particular to a polypeptide and protein sequencing method, a kit and applications. Background Art

[0002] Proteomics is a core area of ​​life science research following the completion of human genome sequencing. The complexity of the human protein composition far exceeds that of the genome. A deeper understanding of proteins and further understanding of the molecular mechanisms of life and disease requires the support of appropriate protein sequencing technologies.

[0003] Currently, protein sequencing technology is mainly performed through Edman degradation and mass spectrometry. Edman degradation sequencing is an earlier developed protein sequencing technology that uses chemical methods to sequentially degrade amino acids from the N-terminus of the protein, and then uses high-performance liquid chromatography to identify the amino acids. However, the Edman degradation method can only analyze the sequence of a single protein sample and has high requirements for sample purity; it has certain limitations for the sequence analysis of peptides with N-terminal blocking or chemical modifications. Mass spectrometry is currently the most widely used protein sequencing method, but this method has significant limitations in terms of detection limit and dynamic range, and cannot fully meet the needs of real-world applications, especially in the detection of low-abundance protein markers.

[0004] In recent years, nanopore single-molecule sequencing has received widespread attention. Its basic principle is that when the protein to be tested passes through a solid-state nanopore or biological nanopore under the action of an electric field, it causes a change in the current on the resistive membrane. Electrodes are used to capture the curve of the nanopore current, and by analyzing the change in current value, the type and sequence composition of the protein to be tested are determined. The application of nanopore technology in the field of nucleic acid sequencing is becoming increasingly mature. Proteins and nucleic acids are both chain-like biological macromolecules, so further expanding nucleic acid sequencing technology to the field of protein sequencing has become a research hotspot in recent years. However, the amino acids that make up proteins are numerous and have different physical and chemical properties. Relying solely on the theoretical system of nucleic acid sequencing is insufficient to achieve the protein sequencing requirements of single amino acid resolution.

[0005] Ahmad M. et al. proposed a nanopore sensor for protein detection with single-molecule precision. When analytes of different properties pass through the nanopore, they interact with the target molecules on the nanopore to produce unique electrical characteristics, thereby identifying the protein (Ahmad M, Ha JH, Mayse LA, et al. A generalizable nanopore sensor for highly specific protein detection at single-molecule precision. Nature Communications, 2023, 14(1): 1374.).

[0006] The terminal amino acid fluorescence recognition method proposed by Quantum SI refers to the use of a recognition molecule with a fluorescent group that has the ability to recognize a specific N-terminal single amino acid to bind to the protein to be tested, and then use a fluorescence detector to detect and analyze fluorescence intensity, fluorescence lifetime, kinetic information, etc., thereby achieving optical detection at the amino acid single molecule level. However, this method system is relatively complex and has high requirements for optical equipment (Reed BD, Meyer MJ, Abramzon V, et al. Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Science, 2022, 378(6616):186-192.).

[0007] Huang Shuo's laboratory used phi29 DNA polymerase as a motor protein in combination with MspA nanopore protein to achieve relatively stable control of the perforation rate of specific negatively charged polypeptide chains and reading of electrical signals (Yan S, Zhang J, Wang Y, et al. Single molecule ratcheting motion of peptides in a Mycobacterium smegmatis Porin A (MspA) nanopore. Nano letters, 2021, 21(15): 6703-6710.).

[0008] Oxford Nanopore Technologies Limited (ONT) (WO2021 / 111125A1) has disclosed a protein sequencing solution based on oligonucleotide-controlled protein rate control. The design involves first synthesizing an adaptor-peptide-dsDNA tail complex, then partially annealing the single-stranded DNA to form double-stranded DNA, which is then bound to the oligonucleotide control protein and read via a nanopore to generate an electrical signal.

[0009] The Bai Jingwei research group published a protein sequencing scheme based on MTA helicase speed control similar to the technology disclosed in WO2021 / 111125A1. Its design is also to first synthesize a ssDNA-peptide-ssDNA (polyT) complex, bind to the MTA helicase without annealing to form a double strand, and drive the polypeptide fragment through the MspA-M2 nanopore protein through the regular sliding of the MTA helicase on the ssDNA chain to measure the sequencing electrical signal. (Chen Z, Wang Z, Xu Y, et al.Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application. Chemical science, 2021, 12 (47): 15750-15756.).

[0010] The above-mentioned technical methods using fluorescence signals as indicators have the problem of not being universal when measuring polypeptides; and the protein sequencing system using optical three-dimensional signals is relatively complex, making it difficult to achieve single-molecule measurement and placing high demands on optical equipment.

[0011] All of the previously disclosed nanopore-related technical solutions only display effective sequencing current fingerprints containing peptide signals, resulting in relatively low resolution. However, if resolution is to be improved, relying solely on the theoretical framework of nucleic acid sequencing will not be enough to achieve protein sequencing with single amino acid resolution.

[0012] Therefore, there is currently no effective solution to provide a peptide or protein sequencing solution with higher resolution.

[0013] Summary of the Invention

[0014] The main purpose of this application is to provide a polypeptide and protein sequencing method, kit and application to solve the problem that it is difficult to achieve the polypeptide sequencing requirement of single amino acid resolution in the existing technology.

[0015] To achieve the above objectives, according to one aspect of the present invention, a method for sequencing a polypeptide is provided, the method comprising: S1, attaching a target polypeptide to a nanopore; S2, introducing a recognition molecule that specifically recognizes the terminal amino acid of the target polypeptide into the nanopore sequencing system, and detecting the terminal amino acid of the target polypeptide by utilizing the changes in the electrical signal generated during the binding and dissociation process between the recognition molecule and the target polypeptide under the action of an electric field.

[0016] Furthermore, after S2, the sequencing method further includes: S3, introducing an exonuclease into the nanopore sequencing system to remove the terminal amino acid of the target polypeptide; preferably, after S3, the sequencing method further includes: S4, repeating S2 and S3 once or multiple times, thereby completing the sequencing of at least a portion of the amino acids of the target polypeptide.

[0017] Furthermore, after S2, the sequencing method further includes: S3', introducing an exonuclease into the nanopore sequencing system, and further detecting the excised terminal amino acid of the target polypeptide based on the change in the electrical signal generated during the exonuclease excision of the terminal amino acid of the target polypeptide; preferably, after S3', the sequencing method further includes: S4, repeating S2 and S3' once or multiple times, thereby completing the sequencing of at least a portion of the amino acids of the target polypeptide.

[0018] Furthermore, the nanopore is a biological pore or a solid-state pore; preferably, the biological pore is a nanostructured porin; preferably, the porin is selected from any one of the following: α-hemolysin, Mycobacterium smegmatis membrane protein A or channel protein CsgG.

[0019] Furthermore, the solid-state pore is a solid-state nanopore with a diameter of sub-10 nm; preferably, the solid-state pore is selected from any one of the following materials: graphene film, SiNx, SiO2, SiC or Al2O3.

[0020] Furthermore, S1 includes: attaching the C-terminus of the target polypeptide to the nanopore; preferably, S1 includes: selectively modifying the C-terminus of the target polypeptide to form a modified amino acid at the C-terminus; using the modified amino acid at the C-terminus to attach the target polypeptide to the nanopore, more preferably, attaching the target polypeptide to the surface of the nanopore.

[0021] Furthermore, the modified amino acid at the C-terminus is lysine or cysteine.

[0022] Furthermore, attachment includes direct attachment or indirect attachment; preferably, indirect attachment includes any one of the following methods: 1) through a nucleic acid-polypeptide connection method; 2) through a method of connecting a nucleic acid with a nucleic acid on a nanopore based on base complementary pairing; preferably, the surface of the nanopore has a chemical modification, more preferably, the chemical modification is selected from any one of the following: carboxyl modification, amino modification, thiol modification, nucleic acid modification, azide modification, dibenzocyclooctene modification, polyethylene glycol modification, hydroxyl modification or acyl modification; preferably, direct attachment is achieved through any one of the following chemical reactions: amide reaction, click chemistry reaction, thiol phosphoramidite reaction, reaction of amino group with hydroxysuccinimide analogue, disulfide bond polymerization reaction, Diels-Alder reaction or binding reaction of biotin and streptavidin.

[0023] Furthermore, in S2, the recognition molecule that specifically recognizes the terminal amino acid of the target polypeptide is selected from any one or more of the following: an amino acid recognition protein and a nucleic acid aptamer; preferably, the amino acid recognition protein includes any one or more of the following: 1) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of the ClpS family; 2) Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt; 3) human UBR1, human UBR2 or Saccharomyces cerevisiae UBR1 of the UBR family; 4) H. sapiens GID4 or Saccharomyces cerevisiae of the GID4 family. GID4: 5) Drosophila melanogaster BIR2; 6) H. sapiens N-meristoyltransferase NMT1; Preferably, the recognition molecule specifically recognizes the N-terminal amino acid of the target polypeptide.

[0024] Furthermore, the exonuclease is selected from any one or more of the following: yPIP (Y pestisproline iminopeptidase), PhTET (Pyrococcus horikoshii TETaminopeptidase), cVPr (V proteolyticus aminopeptidase), PfuTET, APN (L. pneumophila M1Aminopeptidase) or methionine aminopeptidase (Pfu Methionine Aminopeptidase).

[0025] Furthermore, the electrical signal includes at least one of the following: current block signal strength, current block duration, or interval time between occurrences of current block events.

[0026] Furthermore, between S2 and S3 there is also a step of cleaning to remove the reaction liquid in the reaction system of S2.

[0027] Furthermore, between S2 and S3', a cleaning step is also included to remove the reaction liquid in the reaction system of S2.

[0028] Furthermore, after S2 completes the detection of the first amino acid at the N-terminus of the target polypeptide, the reaction liquid in the nanopore sequencing system is cleared, and S3 is performed. After the exonuclease removes the first amino acid at the N-terminus of the target polypeptide, the second amino acid at the N-terminus of the target polypeptide is exposed; S2 is repeated, and after the detection of the second amino acid at the N-terminus of the target polypeptide is completed, the reaction liquid in the nanopore sequencing system is cleared; then S3 is repeated; and so on, "S2 and S3" are executed n times in a cycle to complete the detection of the nth amino acid at the N-terminus of the polypeptide; where n is a natural number greater than or equal to 2.

[0029] Furthermore, after S2 completes the detection of the first amino acid at the N-terminus of the target polypeptide, the reaction liquid in the nanopore sequencing system is cleared, and S3' is performed. After the exonuclease removes the first amino acid at the N-terminus of the target polypeptide, the second amino acid at the N-terminus of the target polypeptide is exposed; S2 is repeated, and after the detection of the second amino acid at the N-terminus of the target polypeptide is completed, the reaction liquid in the nanopore sequencing system is cleared; then S3' is repeated; and so on, "S2 and S3'" are executed n times in a cycle to complete the detection of the nth amino acid at the N-terminus of the polypeptide; where n is a natural number greater than or equal to 2.

[0030] To achieve the above-mentioned object, according to a second aspect of the present invention, a protein sequencing method is provided, which comprises: obtaining a polypeptide library of the protein, the polypeptide library comprising a target polypeptide from the protein; and sequencing the target polypeptide in the polypeptide library using any of the aforementioned polypeptide sequencing methods, thereby completing the sequencing of the protein.

[0031] Furthermore, the protein is cleaved using an endonuclease to generate a polypeptide library.

[0032] To achieve the above object, according to the third aspect of the present invention, a kit is provided, which includes a recognition molecule for recognizing amino acids and any one or more of the following optional components: an exonuclease, a sequencing buffer, and an endoproteinase.

[0033] Furthermore, the recognition molecule includes any one or more of: amino acid recognition proteins and nucleic acid aptamers; preferably, the amino acid recognition protein includes any one or more of the following: 1) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of the ClpS family; 2) Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt; 3) human UBR1, human UBR2 or Saccharomyces cerevisiae UBR1 of the UBR family; 4) H. sapiens GID4 or Saccharomyces cerevisiae GID4 of the GID4 family; 5) Drosophila melanogaster BIR2; 6) H. sapiens N-meristoyltransferase NMT1; preferably, the exonuclease is selected from any one or more of the following: yPIP (Y pestisproline iminopeptidase), PhTET (Pyrococcus horikoshii TETaminopeptidase), cVPr (V proteolyticus aminopeptidase), PfuTET, APN (L. pneumophila M1Aminopeptidase) or methionine aminopeptidase (Pfu Methionine Aminopeptidase); preferably, the sequencing buffer comprises: 0.2-0.8 M KCI, 5-15 mM HEPES, 0.2-0.8 mM ATP, 0.5-1.5 mM MgCl2, pH 8.

[0034] According to a fourth aspect of the present invention, there is provided use of any of the aforementioned kits in polypeptide sequencing.

[0035] The technical solution of the present invention realizes the detection of a single amino acid by utilizing the unique electrical signal changes (e.g., changes in nanopore blocking current) generated during the recognition and dissociation process between the recognition molecule and the terminal amino acid of the target polypeptide. It should be noted that among the amino acid recognition molecules discovered so far, one recognition molecule can recognize multiple amino acids, but the electrical signal (e.g., blocking current) generated by each amino acid is different, thereby being able to distinguish different amino acids. In practical applications, multiple recognition molecules can be used simultaneously or separately, and using them simultaneously saves more time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0037] FIG1 shows types of nanopores according to the present invention.

[0038] FIG2 shows a schematic diagram of immobilizing a polypeptide on a nanopore according to the present invention.

[0039] FIG3 shows a schematic diagram of kinetic detection of recognition molecules and target polypeptides based on a nanopore sensor according to the present invention.

[0040] FIG4 shows a schematic diagram of a “recognition-excision-recognition” dynamic detection model based on a nanopore sensor according to the present invention.

[0041] FIG5-1 shows a current signal diagram when streptavidin is not introduced according to the present invention.

[0042] FIG5-2 shows a current signal diagram when streptavidin is pushed in according to the present invention.

[0043] FIG6-1 shows a current signal diagram when the recognition molecule ClpS is not introduced according to the present invention.

[0044] FIG6-2 shows a current signal diagram when the recognition molecule ClpS is pushed in according to the present invention.

[0045] FIG7-1 shows a current signal diagram when the recognition molecule UBR1 is not introduced according to the present invention.

[0046] FIG7-2 shows a current signal diagram when the recognition molecule UBR1 is pushed in according to the present invention.

[0047] FIG7-3 shows a current signal diagram when the exonuclease PhTET is pushed in according to the present invention.

[0048] FIG7-4 shows a current signal diagram when the recognition molecule ClpS is pushed in according to the present invention. DETAILED DESCRIPTION

[0049] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0050] As mentioned in the background technology section, existing nanopore-related protein sequencing methods only display effective sequencing current fingerprints containing polypeptide signals and cannot achieve resolution at the single amino acid level, that is, they have the defect of low resolution. To improve this situation, the applicant has proposed an improvement plan.

[0051] The improved scheme of this application is based on the study of the single-molecule dynamics mechanism of nanopore sensing technology to achieve specific recognition of single amino acids. The improved scheme of this application adopts a sequencing system that uses simultaneous recognition and excision to establish a dynamic sequencing model to complete protein sequencing. Based on this improved approach, the inventors conducted a series of verification experiments, and the experimental results show that the improved approach of this application can achieve peptide or protein sequencing at the single-molecule level.

[0052] Based on the above research results, the applicant has proposed an improved solution of the present application. In a typical embodiment, a method for sequencing a polypeptide is provided, comprising: S1, attaching a target polypeptide to a nanopore; S2, introducing a recognition molecule that specifically recognizes the terminal amino acid of the target polypeptide into the nanopore sequencing system, and detecting the terminal amino acid of the target polypeptide by utilizing the changes in the electrical signal generated during the binding and dissociation process between the recognition molecule and the target polypeptide under the action of an electric field.

[0053] The above-mentioned peptide sequencing method realizes the detection of a single amino acid by utilizing the unique electrical signal changes (for example, changes in nanopore blocking current) generated during the recognition and dissociation process between the recognition molecule and the terminal amino acid of the target polypeptide. It should be noted that among the amino acid recognition molecules discovered so far, one recognition molecule can recognize multiple amino acids, but the electrical signal (for example, blocking current) generated by each amino acid is different, so that different amino acids can be distinguished. In practical applications, multiple recognition molecules can be used simultaneously or separately, and using them simultaneously saves more time.

[0054] In some preferred embodiments, the sequencing method further comprises: S3, introducing an exonuclease into the nanopore sequencing system to remove the terminal amino acid of the target polypeptide, so as to initiate the binding of the recognition molecule to the next amino acid at the end of the target polypeptide.

[0055] In this preferred embodiment, the detection of a single amino acid is achieved through the electrical signal generated by the recognition molecule in S2 upon recognition of the amino acid. The exonuclease in S3 then cleaves the recognized amino acid, allowing for the recognition and detection of the next amino acid. The number of repetitions of the "S2 and S3" cycle is determined appropriately based on the length of the polypeptide sequence, thereby enabling the detection of polypeptide fragments.

[0056] As described above, the changes in the electrical signals generated during the binding and dissociation process between the recognition molecule and the target polypeptide have enabled differentiation at the single amino acid level. To further improve the accuracy of the detection results, in other preferred embodiments, the sequencing method further comprises: S3', introducing an exonuclease into the nanopore sequencing system, and detecting the excised terminal amino acid of the target polypeptide based on the changes in the electrical signals generated during the exonuclease excision of the terminal amino acid of the target polypeptide.

[0057] In the preferred embodiment described above, the exonuclease cleaves each amino acid at a different rate, resulting in different changes in the electrical signal (e.g., nanopore blocking current) generated by the enzyme cleaving the terminal amino acid, thereby distinguishing the different amino acids. Therefore, the type of amino acid can be further detected and confirmed based on the electrical signal generated by the recognition molecule.

[0058] The aforementioned S2 detects the terminal amino acid of a polypeptide by generating a unique electrical signal generated by the interaction between the recognition molecule and the terminal amino acid of the polypeptide. Simultaneously, S3' further detects and confirms the type of the terminal amino acid based on the unique electrical signal generated by the exonuclease's efficiency in removing the terminal amino acid from the polypeptide. In other words, by combining the electrical signal generated by the recognition molecule with the electrical signal generated by the exonuclease (i.e., the two signals mutually confirm each other), the accuracy of detecting the terminal amino acid is improved.

[0059] In some preferred embodiments, the above sequencing method further includes repeating S2 and S3 (or S3') once or multiple times to achieve accurate detection of at least a portion of the amino acids of the target polypeptide. The detection of each amino acid preferably undergoes recognition by a recognition molecule and enzymatic cleavage by an exonuclease, while the enzymatic cleavage exposes the next amino acid, and thus undergoes recognition detection by the recognition molecule and enzymatic cleavage by an exonuclease again, which can achieve accurate detection of the next amino acid. Depending on the length of the target polypeptide, the above S2 and S3 (or S3') are repeated, and one amino acid is detected in each repetition round. As needed, and so on, in the process of identification and excision, the changes in electrical signals in different biochemical processes are monitored, thereby achieving the purpose of sequencing the target polypeptide sequence.

[0060] It should be noted that during sequencing, a nanopore is provided in the sample detection pool. As shown in Figure 1, the type of nanopore can be not only a biological pore, such as Figure 1 (a). It can also be a solid-state pore, such as Figure 1 (b). Biological pores are nanostructured protein pores constructed based on biological materials that can be inserted into the membrane, also known as "porins". Types of porins include but are not limited to α-hemolysin (α-Hemolysin), Mycobacterium smegmatis membrane protein A (MspA) or the channel protein CsgG responsible for curli secretion. Solid-state pores are solid-state nanopores constructed based on physical materials and inserted into the membrane. The types of solid-state pores include but are not limited to solid-state nanopores with diameters of sub-10 nm such as graphene films, SiNx, SiO2, SiC or Al2O3.

[0061] Different polypeptides can be attached to the nanopore via specific connection methods. Depending on the material and type of nanopore used in the specific sequencing process, the above-mentioned method of attaching the target polypeptide to the nanopore will also vary. In a preferred embodiment of the present application, the C-terminus of the target polypeptide is attached to the nanopore. Preferably, S1 includes: selectively modifying the C-terminus of the target polypeptide to form a modified amino acid at the C-terminus; and attaching the target polypeptide to the nanopore using the modified amino acid at the C-terminus. In some more preferred embodiments, the polypeptide is attached to the surface of the nanopore (such as the surface of a porin protein).

[0062] The purpose of modification is to facilitate attachment, so any existing modification method that can achieve attachment is applicable to the present application. In some preferred embodiments, the amino acid with a modification at the C-terminus is preferably lysine or cysteine, for example, the modification can be performed on the side chain (SH group) of the cysteine ​​at the C-terminus. . The polypeptide can be attached not only by direct attachment, as shown in Figure 2 (a), but also by indirect attachment of the polypeptide using a nucleic acid-polypeptide connection method, as shown in Figure 2 (b). In addition, indirect attachment also includes attachment by connecting the nucleic acid to the nucleic acid on the nanopore based on base complementary pairing.

[0063] Based on this, it is necessary to perform certain chemical modifications on the surface of the nanopore. The surface-modified nanopore can also interact with polypeptides or nucleic acids to form chemical bonds. Among them, the chemical modifications on the surface of the nanopore include but are not limited to carboxyl modification, amino modification, thiol modification, nucleic acid modification, azide modification, dibenzocyclooctene modification, polyethylene glycol modification, hydroxyl modification or acyl modification. The above-mentioned chemical reactions include but are not limited to amide reaction, click chemistry reaction, thiol phosphoramidite reaction, reaction of amino group with hydroxysuccinimide analogue, disulfide bond polymerization reaction, Diels-Alder reaction or binding reaction of biotin with streptavidin, etc. Any of the above chemical reactions can achieve direct attachment of the target polypeptide.

[0064] The sequencing of the above-mentioned terminal amino acids relies on a class of recognition molecules naturally present in organisms, which can bind to different amino acids at the end of polypeptides. Different recognition molecules are introduced into the sequencing system, and by detecting the electrical signals (such as current blockade signals) generated when different recognition molecules bind to their corresponding polypeptides, a single-molecule kinetic model is established during the binding and dissociation process, thereby achieving the recognition of single amino acid accuracy. The kinetic detection of recognition molecules and target polypeptides based on nanopore sensors is shown in Figure 3. Recognition molecules include but are not limited to any one or more of the following: recognition proteins and nucleic acid aptamers that can specifically recognize terminal amino acids. Specifically, the ClpS family (including Agrobacterium tumefaciens ClpS1 and Agrobacterium tumefaciens ClpS2—two recognition proteins that can recognize phenylalanine, tyrosine, and tryptophan; Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS, Plasmodium falciparum ClpS—can recognize phenylalanine, tyrosine, tryptophan, and leucine), Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt—can recognize aspartate and glutamate; UBR family (human UBR1, human UBR2 or Saccharomyces cerevisiae UBR1)—can recognize lysine, arginine, and histidine; GID4 family (H. sapiens GID4 or Saccharomyces cerevisiae GID4)---can recognize proline, leucine, isoleucine, valine and methionine; Drosophila melanogaster BIR2---can recognize alanine; H. sapiens N-meristoyltransferase NMT1---can recognize glycine.

[0065] Preferably, the recognition molecule recognizes the amino acids at the N-terminus of the target polypeptide one by one.

[0066] Based on the static recognition of individual amino acids described above, an exonuclease is further introduced to cleave the terminal amino acid of a peptide. The exonuclease's cleavage efficiency varies for different amino acids, and changes in the electrical signal still occur during the process. During the recognition-cleavage-recognition process, the types of amino acids identified before and after cleavage are determined based on the differences in the current signal. By observing the changes in amino acid recognition signals during different biochemical processes in the time domain, protein sequencing is completed. The dynamic "recognition-cleavage-recognition" detection model based on a nanopore sensor is shown in Figure 4. Exonucleases include, but are not limited to, yPIP (Y pestisproline iminopeptidase), PhTET (Pyrococcus horikoshii TET aminopeptidase), cVPr (V proteolyticus aminopeptidase), PfuTET, methionine aminopeptidase (Pfu methionine aminopeptidase), which specifically cleaves methionine at the N-terminus of peptides, and APN (L. pneumophila M1 aminopeptidase), which specifically cleaves aspartic acid or glutamic acid at the N-terminus of peptides.

[0067] It should be noted that the above-mentioned electrical signal includes the current block signal intensity, the current block duration, or the interval time between current block events.

[0068] In some preferred embodiments, after S2 and before S3, the sequencing method further includes a cleaning step to remove the reaction solution in the reaction system of S2 to reduce interference with subsequent steps.

[0069] As mentioned above, the sequencing method of the present application can realize the identification and detection of a single amino acid with precision, and a polypeptide sequence of a specific length is the result of repeated identification and detection of a single amino acid. Specifically, after S2 completes the detection of the first amino acid at the N-terminus of the target polypeptide, the reaction liquid in the nanopore sequencing system is cleared, and then S3 (or S3') is performed. The first amino acid at the N-terminus is removed by enzyme cutting. While determining the type of the first amino acid at the N-terminus, the second amino acid at the N-terminus is exposed as the amino acid at the N-terminus, and the identification of S2 and the enzyme cutting of S3 (or S3') are performed again to realize the detection of the second amino acid at the N-terminus, and so on. Repeat this process n times to realize the detection of n amino acids at the N-terminus of the target polypeptide. Here, n represents the number of amino acids in the target polypeptide, which is a natural number, preferably n≥2.

[0070] In a second typical embodiment, a protein sequencing method is provided, which includes: obtaining a polypeptide library of the protein, wherein the polypeptide library contains target polypeptides from the protein; and sequencing the target polypeptides in each polypeptide library using the aforementioned polypeptide sequencing method, thereby completing the sequencing of the protein.

[0071] To ensure more accurate protein sequencing results, the aforementioned peptide libraries can be two or more. Specifically, two or more peptide libraries are generated in parallel by cleaving the protein using two or more endonucleases. An activation reagent is then used to selectively modify the C-terminus of each peptide to form a peptide library for the protein to be tested. By analyzing the peptide information obtained from sequencing the peptide library, the sequence information of the protein to be tested can be obtained.

[0072] The above-mentioned protein sequencing method is to fix the target polypeptide (e.g., C-terminus) on the porin after protein digestion and excision, and then specifically modify it. A recognition molecule that can specifically recognize the terminal (e.g., N-terminal) amino acid is then introduced into the nanopore. During the binding and dissociation process between the target polypeptide on the nanopore surface and the recognition molecule in the flow path, the recognition molecule and the polypeptide interact under the action of the electric field to generate an electrical signal. By comparing the differences in the electrical signals generated by the binding and dissociation of different amino acid molecules with the recognition molecule, the different terminal amino acids of the polypeptide can be detected. At the same time, a polypeptide exonuclease is introduced into the nanopore, and the changes in the electrical signal during the excision process are based on the different exonuclease excision efficiencies of different N-terminal amino acids. During the process of identification and excision, the changes in the electrical signals during different biochemical processes are monitored, thereby achieving the purpose of protein identification and sequencing.

[0073] In a third typical embodiment of the present application, a kit is provided, which includes a recognition molecule that recognizes amino acids and any one or more of the following optional components: an exoproteinase, a reaction buffer, and an endoproteinase.

[0074] In some preferred embodiments, the recognition molecule includes at least one of the following: an amino acid recognition protein and a nucleic acid aptamer. Preferably, the amino acid recognition protein includes any one or more of the following: ClpS family (including Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2----two recognition proteins that can recognize phenylalanine, tyrosine and tryptophan; Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS, Plasmodium falciparum ClpS---can recognize phenylalanine, tyrosine, tryptophan and leucine), Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt---can recognize aspartate and glutamate; UBR family (human UBR1, human UBR2 or Saccharomyces cerevisiae UBR1)---can recognize lysine, arginine and histidine; GID4 family (H. sapiens GID4 or Saccharomyces cerevisiae GID4) recognizes proline, leucine, isoleucine, valine, and methionine; Drosophila melanogaster BIR2 recognizes alanine; and H. sapiens N-meristoyltransferase NMT1 recognizes glycine. Those skilled in the art will appreciate that the names of these amino acid recognition proteins include their source, which is typically indicated in italics.

[0075] In the above kit, the exonuclease is selected from any one or more of the following: the exonuclease is selected from any one or more of the following: yPIP (Y pestisproline iminopeptidase), PhTET (Pyrococcushorikoshii TETaminopeptidase), cVPr (V proteolyticus aminopeptidase), PfuTET, APN (L.pneumophila M1Aminopeptidase) or methionine aminopeptidase (Pfu Methionine Aminopeptidase).

[0076] In a fourth exemplary embodiment of the present application, the use of the above kit in peptide sequencing is provided. Peptide sequencing using the components in the above kit can achieve detection at the level of single amino acids with high detection accuracy.

[0077] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0078] Example 1: Binding of streptavidin and biotin based on nanopore sensor

[0079] In this embodiment, streptavidin is used as an example of a recognition molecule to detect the binding signal between streptavidin and biotin.

[0080] The experimental steps are as follows:

[0081] 1) In this example, a biopore was used as an example. A planar 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids) phospholipid bilayer membrane was used to divide the electrolytic cell into two chambers: the cis chamber and the trans chamber. A pair of Ag / AgCl electrodes was placed in each chamber. The nanopore protein CsgG was added to the bilayer membrane. A voltage of 180 mV was applied to promote the insertion of the pore into the membrane, forming a single nanopore channel. After the single nanopore protein was inserted into the membrane, sequencing buffer (0.5 M KCl, 10 mM HEPES, 0.5 mM ATP, 1 mM MgCl2, pH 8) was introduced to remove excess pore protein.

[0082] 2) Dissolve diphenylcyclooctyne-PEG4-hydrogenated succinimide ester (Sigma, 764019-1MG) in working solution A with a final concentration of 1 mM and set aside.

[0083] 3) Push working solution A into the cis chamber prepared in step 1) and incubate at room temperature for 30 min. Push sequencing buffer into the chamber to remove excess diphenylcyclooctyne-PEG4-hydrogenated succinimide ester.

[0084] 4) Design and order a biotin- and azide-modified nucleic acid sequence A. Sequence A is as follows: 5'-GCCGTGGTCTTCTCGCCGGATGGTCAGTGG-3' (SEQ ID NO: 1) (Shanghai Biotech), with the 3' end modified with azide and the 5' end modified with biotin. Dissolve the modified DNA in double-distilled water to a final concentration of 1 μM in working solution B.

[0085] 5) Add working solution B to the cis chamber after the reaction in step 3) and incubate overnight at room temperature. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 1 (shown in Figure 5-1).

[0086] 6) Prepare 1 μM streptavidin solution (Thermo, 434302) as working solution C, vortex thoroughly and mix thoroughly.

[0087] 7) Add working solution C to the cis chamber after the reaction in step 5) and incubate at room temperature for 30 minutes. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 2 (shown in Figure 5-2).

[0088] Experimental results:

[0089] Comparing the current signal graphs in Figures 5-1 and 5-2, we see a clear current blockade signal after the insertion of streptavidin into the nanopore. This strong current blockade signal is due to the inherent biological properties of streptavidin and biotin, which bind tightly. This experimental result is consistent with biological properties and demonstrates that when a molecule specifically binds to the N-terminus, a current blockade signal is generated. This principle can be exploited to detect amino acids in peptides using nanopore sensors.

[0090] Example 2: Binding of recognition molecules and peptides based on nanopore sensors

[0091] In this example, the recognition molecule ClpS is used as an example to detect the binding signal between the recognition molecule Agrobacterium tumefaciens ClpS1 and the polypeptide.

[0092] The experimental steps are as follows:

[0093] 1) This example uses a biopore as an example. A planar 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids) phospholipid bilayer membrane was used to divide the electrolytic cell into two chambers: the cis chamber and the trans chamber. A pair of Ag / AgCl electrodes was placed in each chamber. The nanopore protein CsgG was added to the bilayer membrane. An 180 mV voltage was applied to promote the insertion of the pore into the membrane, forming a single nanopore channel. Once the single nanopore was inserted into the membrane, sequencing buffer (0.5 M KCl, 10 mM HEPES, 0.5 mM ATP, 1 mM MgCl2, pH 8) was introduced to remove excess pore protein.

[0094] 2) Dissolve diphenylcyclooctyne-PEG4-hydrogenated succinimide ester (Sigma, 764019-1MG) in working solution A with a final concentration of 1 mM and set aside.

[0095] 3) Push working solution A into the cis chamber prepared in step 1) and incubate at room temperature for 30 min. Push sequencing buffer into the chamber to remove excess diphenylcyclooctyne-PEG4-hydrogenated succinimide ester.

[0096] 4) A C-terminally azide-modified peptide was designed and ordered. The peptide sequence information was FRSKGEELFT-Azide (SEQ ID NO: 2) (GenScript). The peptide was prepared into a working solution B with a final concentration of 1 μM for later use.

[0097] 5) Add working solution B to the cis chamber after the reaction in step 3) and incubate overnight at room temperature. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 1 (shown in Figure 6-1).

[0098] 6) Prepare 1 μM ClpS1 recognition molecule working solution C and vortex thoroughly to mix.

[0099] 7) Add working solution C to the cis chamber after the reaction in step 5) and incubate at room temperature for 30 minutes. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 2 (shown in Figure 6-2).

[0100] Experimental results:

[0101] Comparing the current signal graphs in Figures 6-1 and 6-2 , a clear current blockade signal was observed after the recognition molecule ClpS1 was introduced into the nanopore. This signal is significantly different from the binding signals of streptavidin and biotin, indicating that this signal indicates recognition between ClpS1 and the aforementioned polypeptide (SEQ ID NO: 2).

[0102] Example 3: Signal changes in recognition-enzyme cleavage-re-recognition based on nanopore sensors

[0103] In this example, the peptide RYPDDDK-azide (SEQ ID NO: 3) was used as an example to detect changes in binding signals between human UBR1 and ClpS1 (same as in Example 1) and the peptide before and after cleavage of amino acid R by exonuclease PhTET.

[0104] The experimental steps are as follows:

[0105] 1) This example uses a biopore as an example. A planar 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids) phospholipid bilayer membrane was used to divide the electrolytic cell into two chambers: the cis chamber and the trans chamber. A pair of Ag / AgCl electrodes was placed in each chamber. The nanopore protein CsgG was added to the bilayer membrane. An 180 mV voltage was applied to promote the insertion of the pore into the membrane, forming a single nanopore channel. Once the single nanopore was inserted into the membrane, sequencing buffer (0.5 M KCl, 10 mM HEPES, 0.5 mM ATP, 1 mM MgCl2, pH 8) was introduced to remove excess pore protein.

[0106] 2) Dissolve diphenylcyclooctyne-PEG4-hydrogenated succinimide ester (Sigma, 764019-1MG) in working solution A with a final concentration of 1 mM and set aside.

[0107] 3) Push working solution A into the cis chamber prepared in step 1) and incubate at room temperature for 30 min. Push sequencing buffer into the chamber to remove excess diphenylcyclooctyne-PEG4-hydrogenated succinimide ester.

[0108] 4) A C-terminally azide-modified peptide was designed and ordered. The peptide sequence information was RYPDDDK-Azide (SEQ ID NO: 3) (GenScript). The peptide was prepared into a working solution B with a final concentration of 1 μM for later use.

[0109] 5) Add working solution B to the cis chamber after the reaction in step 3) and incubate overnight at room temperature. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 1 (shown in Figure 7-1).

[0110] 6) Prepare 1 μM UBR1 recognition molecule working solution C and vortex thoroughly to mix.

[0111] 7) Add working solution C to the cis chamber after the reaction in step 5) and incubate at room temperature for 30 minutes. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 2 (shown in Figure 7-2).

[0112] 8) Prepare 100 nM PhTET exonuclease working solution D and mix thoroughly.

[0113] 9) Pour working solution D into the cis chamber after the reaction in step 7) and incubate at room temperature for 1 hour. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 3 (shown in Figure 7-3).

[0114] 10) Prepare 1 μM ClpS1 recognition molecule working solution E and vortex thoroughly to mix.

[0115] 11) Pour working solution E into the cis chamber after the reaction in step 9) and incubate at room temperature for 30 minutes. Finally, apply 180 mV and record the nanopore current data at a frequency of 5 kHz. This current data is Result 4 (shown in Figure 7-4).

[0116] Experimental results:

[0117] Comparing the current signal graphs in Figures 7-2 and 7-4 reveals distinct current blockade signals before and after the exonuclease PhTET is introduced into the nanopore, i.e., before and after the cleavage reaction occurs. These signals are generated by the binding of different recognition molecules to different peptides, and can be interpreted as information changes during the peptide (SEQ ID NO: 3) process of recognition, cleavage, and re-recognition.

[0118] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: The present invention demonstrates for the first time the study of single-molecule dynamics mechanisms based on nanopore sensing technology to achieve specific recognition of single amino acids. This system utilizes a simultaneous recognition and excision sequencing system to establish a dynamic sequencing model to complete protein sequencing. The present invention proposes that after protein digestion and excision, the peptide is attached to the porin through specific modification. A recognition molecule that recognizes the N-terminal amino acid is then introduced into the system for kinetic detection of the recognition molecule and the peptide. Simultaneously, an exonuclease is used to sequentially excise the N-terminal amino acid, and the current signal changes during different biochemical processes are monitored to achieve protein sequencing. Compared to existing technologies, the present invention has the following advantages: 1) The present invention enables sequential detection of N-terminal amino acids with high sequencing accuracy. 3) The present invention enables protein sequencing at the single-amino acid level. 4) The present invention avoids signal reading issues caused by pore blockage and other issues.

[0119] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for sequencing a polypeptide, characterized in that, The sequencing method includes: S1, attaching the target polypeptide to a nanopore; S2, introducing a recognition molecule that specifically recognizes the terminal amino acid of the target polypeptide into the nanopore sequencing system. Under the action of an electric field, using the change in the electrical signal generated during the binding and dissociation process between the recognition molecule and the target polypeptide, the detection of the terminal amino acid of the target polypeptide is achieved.

2. The sequencing method according to claim 1, wherein After the S2, the sequencing method further includes: S3, introducing an exonuclease into the nanopore sequencing system to excise the terminal amino acid of the target polypeptide; Preferably, after the S3, the sequencing method further includes: S4, repeating the S2 and the S3 one or more times, so as to complete the sequencing of at least a part of the amino acids of the target polypeptide.

3. The sequencing method according to claim 1, characterized in that, After the S2, the sequencing method further includes: S3’, introducing an exonuclease into the nanopore sequencing system, and further detecting the excised terminal amino acid of the target polypeptide according to the change in the electrical signal generated during the process of the exonuclease excising the terminal amino acid of the target polypeptide; Preferably, after the S3’, the sequencing method further includes: S4, repeating the S2 and the S3’ one or more times, so as to complete the sequencing of at least a part of the amino acids of the target polypeptide.

4. The sequencing method according to any one of claims 1-3, characterized in that, The nanopore is a biological pore or a solid-state pore; Preferably, the biological pore is a nanoporous porin; Preferably, the porin is selected from any one of the following: α-hemolysin, Mycobacterium smegmatis membrane protein A, or channel protein CsgG.

5. The sequencing method according to claim 4, wherein The solid-state pore is a solid-state nanopore with a diameter in the sub-10 nm scale; Preferably, the solid-state pore is a solid-state pore made of any one of the following materials: graphene film, SiNx, SiO2, SiC, or Al2O3.

6. The sequencing method according to claim 1, wherein The S1 includes: attaching the C-terminus of the target polypeptide to the nanopore; Preferably, the S1 includes: Selectively modifying the C-terminus of the target polypeptide to form an amino acid with a modified C-terminus; Using the amino acid with a modified C-terminus to attach the target polypeptide to the nanopore, More preferably, attaching the target polypeptide to the surface of the nanopore.

7. The sequencing method according to claim 6, wherein The amino acid with a modified C-terminus is lysine or cysteine.

8. The sequencing method according to claim 6, wherein The attachment includes direct attachment or indirect attachment; Preferably, the indirect attachment includes any one of the following methods: 1) by the nucleic acid-polypeptide ligation method; 2) by the method of connecting nucleic acid on the nanopore with nucleic acid based on base complementary pairing; Preferably, the surface of the nanopore has a chemical modification, More preferably, the chemical modification is selected from any one of the following: carboxyl modification, amino modification, thiol modification, nucleic acid modification, azide modification, dibenzocyclooctene modification, polyethylene glycol modification, hydroxyl modification, or acyl modification; Preferably, the direct attachment is achieved by any one of the following chemical reactions: amide reaction, click chemical reaction, thiophosphoramidite reaction, reaction between amino group and hydroxysuccinimide analog, disulfide polymerization reaction, Diels-Alder reaction, or the binding reaction between biotin and streptavidin.

9. The sequencing method according to any one of claims 1 to 8, characterized in that, In S2, the recognition molecule that specifically recognizes the terminal amino acid of the target polypeptide is selected from any one or more of the following: amino acid recognition protein and nucleic acid aptamer; Preferably, the amino acid recognition protein includes any one or more of the following: 1) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of the ClpS family; 2) Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt; 3) human UBR1, human UBR2 or Saccha-romyces cerevisiae UBR1 of the UBR family; 4) H.sapiens GID4 or Saccharomyces cerevisiae GID4 of the GID4 family: 5) Drosophila melanogaster BIR2; 6) H.sapiens N-meristoyltransferase NMT1; Preferably, the recognition molecule specifically recognizes the N-terminal amino acid of the target polypeptide.

10. The sequencing method according to claim 2 or 3, characterized in that, The exopeptidase is selected from any one or more of the following: yPIP (Y pestis proline iminopeptidase), PhTET (Pyrococcus horikoshii TET aminopeptidase), cVPr (V proteolyticus aminopeptidase), PfuTET, APN (L.pneumophila M1 Aminopeptidase) or methionine aminopeptidase (Pfu Methionine Aminopeptidase).

11. The sequencing method according to any one of claims 1-3, characterized in that, The electrical signal includes at least one of the following: current block signal intensity, current block duration or the interval time when the current block event occurs.

12. The sequencing method according to claim 2, wherein A step of washing to remove the reaction solution in the reaction system of S2 is further included between S2 and S3.

13. The sequencing method according to claim 3, wherein A step of washing to remove the reaction solution in the reaction system of S2 is further included between S2 and S3'.

14. The sequencing method according to claim 2, wherein After the detection of the first amino acid at the N-terminus of the target polypeptide is completed in S2, the reaction solution in the nanopore sequencing system is removed, and S3 is performed, after the exonuclease removes the first amino acid at the N-terminus of the target polypeptide, the second amino acid at the N-terminus of the target polypeptide is exposed; Repeating S2, after completing the detection of the second amino acid at the N-terminus of the target polypeptide, clearing the reaction solution in the nanopore sequencing system; and then repeating S3; By analogy, "the S2 and the S3" are executed in a loop n times to complete the detection of the nth amino acid at the N-terminus of the polypeptide; wherein n is a natural number greater than or equal to 2.

15. The sequencing method according to claim 3, characterized in that: After the detection of the first amino acid at the N-terminus of the target polypeptide is completed in S2, the reaction solution in the nanopore sequencing system is removed, and S3' is performed, and after the exonuclease removes the first amino acid at the N-terminus of the target polypeptide, the second amino acid at the N-terminus of the target polypeptide is exposed; Repeating S2, after completing the detection of the second amino acid at the N-terminus of the target polypeptide, clearing the reaction solution in the nanopore sequencing system; and then repeating S3'; By analogy, "the S2 and the S3'" are executed in a loop n times to complete the detection of the nth amino acid at the N-terminus of the polypeptide; wherein n is a natural number greater than or equal to 2.

16. A method for sequencing a protein, characterized in that, The sequencing method comprises: Obtaining a polypeptide library of a protein, wherein the polypeptide library comprises a target polypeptide derived from the protein; The target polypeptide in the polypeptide library is sequenced using the polypeptide sequencing method described in any one of claims 1 to 15, thereby completing the sequencing of the protein.

17. The sequencing method according to claim 16, characterized in that: The protein is cleaved by using an endonuclease to generate the polypeptide library.

18. A kit, characterized in that, The kit comprises a recognition molecule for recognizing amino acids and any one or more of the following optional components: protein exonuclease, sequencing buffer and protein endonuclease.

19. The kit according to claim 18, characterized in that, The recognition molecules include any one or more of: amino acid recognition proteins and nucleic acid aptamers; Preferably, the amino acid recognition protein comprises any one or more of the following: 1) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of ClpS family; 2) Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt; 3) human UBR1, human UBR2 of the UBR family, or UBR1 of Saccharomyces cerevisiae; 4) H. sapiens GID4 or Saccharomyces cerevisiae GID4 of the GID4 family: 5) Drosophila melanogaster BIR2; 6) H. sapiens N-meristoyltransferase NMT1; Preferably, the exopeptidase is selected from any one or more of the following: yPIP (Yersinia pestis proline iminopeptidase), PhTET (Pyrococcus horikoshii TET aminopeptidase), cVPr (Vibrio proteolyticus aminopeptidase), PfuTET, APN (Legionella pneumophila M1 aminopeptidase), or methionine aminopeptidase (Pfu Methionine Aminopeptidase); Preferably, the sequencing buffer comprises: 0.2 - 0.8 M KCI, 5 - 15 mM HEPES, 0.2 - 0.8 mM ATP, 0.5 - 1.5 mM MgCl2, pH 8.

20. Use of the kit according to claim 18 or 19 in polypeptide sequencing.

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