A nanopore polypeptide sequencing method based on host-guest interactions

Abstract

This invention discloses a nanopore peptide sequencing method based on host-guest interactions. The method includes the following steps: 1) sequentially hydrolyzing the peptide to be sequenced into monomeric amino acids using a proteolytic enzyme; 2) linking the monomeric amino acids to a load molecule containing the guest molecule structure; 3) performing single-channel recording in a nanopore after the host molecule and guest molecule have bound. This invention provides a de novo sequencing method that utilizes the sequential hydrolysis of peptides by exopeptidases to obtain the peptide sequence information; by linking the load molecule to a single amino acid, the current blocking ratio of the characteristic signals generated after the binding of 20 encoding amino acids to the load molecule is accurately measured. The characteristic current is contributed by a single amino acid, eliminating decoding difficulties; the target peptide dosage is small, and the detection limit is low; the experimental operation is safe and simple, with most reactions carried out in an aqueous phase, without involving toxic reagents, making it green and safe.

Description

Technical Field

[0001] This invention belongs to the field of protein sequencing technology, specifically relating to a nanopore polypeptide sequencing method based on host-guest interactions. Background Technology

[0002] Protein sequencing has wide applications in biology, medicine, and biotechnology. It is crucial for understanding the structure, function, and interactions of proteins in organisms and provides guidance for drug development and clinical diagnosis. Originating in the 1950s, Swedish biochemist Pehr Edman used phenyl isothiocyanate for N-terminal sequencing. This method, known as the Edman degradation method, remains in use today, and protein sequencers based on this principle are among the most mature protein sequencing technologies. However, this method has significant limitations, including high purity requirements, complex reaction processes, long processing times, limited read lengths, and the inability to identify post-translational modifications (PTMs). With the development of modern biotechnology, researchers have turned to mass spectrometry sequencing, inferring sequence information from fragment quality. Mass spectrometry sequencing, with its advantages of short detection time, high accuracy, and strong reproducibility, has become widely accepted and is currently the gold standard in protein sequencing. However, it still suffers from drawbacks such as low sensitivity, limited read lengths, and difficulty in identifying PTMs.

[0003] In recent years, single-molecule sequencing technology has developed rapidly and has been widely applied in the field of protein sequencing. For example, electron tunneling current is used to detect post-translational modifications on peptide chains by detecting different amino acids. Many fluorescence methods, combined with enzymatic digestion and Edman degradation, are also used to locate specific amino acids in peptide chains and compare them with protein libraries to interpret sequence information. In 2022, Brian D. Reed and colleagues proposed a real-time dynamic single-molecule protein sequencing method integrated into semiconductor devices, which can identify specific amino acids in the sequence in real time. However, the above methods rely on identifying and locating specific amino acids and then comparing them with known sequences in a protein library; they cannot directly obtain all sequence information.

[0004] Nanopore single-channel technology is an emerging detection method developed in the mid-1990s based on electrophysiological research. It utilizes nanoscale channels connecting two mutually insulated structures filled with electrolyte. When a bias voltage is applied, electrolyte ions in the solution migrate directionally through the nanopore, generating a current. This current is measured, amplified, and converted by a patch-clamp system before being recorded. When an analyte is present in the detection system, it diffuses or is voltage-driven through the nanopore. During this process, the occupancy of the nanopore by the analyte alters the number of ions passing through the channel, further changing the recorded current and generating characteristic signals with varying current retardation amplitudes and times. Analysis of these signals reveals information including the type, structure, conformational changes, and molecular composition of the substance. In 2021, three research teams proposed constructing DNA-peptide complexes, using molecular motors to pull DNA and move the peptide within nanopores, thus measuring the peptide's current signal. However, because amino acids are small in size, the current information generated by directly reading the polypeptide chain is contributed by multiple amino acids. The large number of encoding amino acids makes accurate reading of amino acids difficult, making it impossible to read sequence information. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a nanopore peptide sequencing method based on host-guest interactions. This method combines host-guest interactions with nanopore single-channel technology to achieve accurate de novo reading of the amino acid sequence of peptides. The invention utilizes the characteristic of exopeptidases to sequentially hydrolyze peptides to obtain sequence information; by linking a single amino acid to a load molecule, it accurately measures the current blocking ratio of the characteristic signals generated after the binding of 20 coding amino acids to the load molecule. Since the characteristic current is contributed by a single amino acid, there is no decoding difficulty, enabling the reading of the amino acid sequence.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A nanopore peptide sequencing method based on host-guest interaction includes the following steps: 1) using a proteolytic enzyme to sequentially hydrolyze the peptide to be sequenced into monomeric amino acids; 2) linking the monomeric amino acids with a load molecule containing a guest molecular domain; 3) after the host molecule and the guest molecule are bound, performing single-channel recording in a nanopore.

[0008] In step 1) of the above method, the proteolytic enzyme is selected from at least one of the following: carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase Y, leucine aminopeptidase, arginine aminopeptidase, cysteine ​​aminopeptidase, glycine aminopeptidase, histidine aminopeptidase, proline aminopeptidase, trypsin, trypsin B, chymotrypsin, and pepsin.

[0009] In step 2) of the above method, the loaded molecule is designed to bind with amino acids and interact with the host molecule, and to generate characteristic signals by passing through nanopores under the drive of an electric field.

[0010] In this invention, the loaded molecule comprises a combination of guest molecule structural domains, connecting functional structural domains, and traction sequences.

[0011] Further, the guest molecular structural domain is selected from at least one of ferrocene and its derivatives, adamantane and its derivatives, phenylalanine and its derivatives, tyrosine and its derivatives, histidine and its derivatives, and methionine and its derivatives. The linking functional structural domain is selected from at least one of azide, acyl halide, alkynyl, allyl, mercapto, amino, succinimide ester, and maleimide ester. The traction sequence is selected from at least one of polynucleotides such as PolyT and PolyA, random nucleotide sequences, charged polypeptides such as PolyR, PolyD, PolyK, and PolyE, or polyelectrolytes.

[0012] According to one embodiment of the present invention, the loaded molecule may be FGCD8, FGGCD8, FGGGCD8, FGGCGGD8, FGK(AZA)D8, an organotransition metal compound-amino acid-polynucleotide complex structure with the sequence ferrocene-GC-TTTTTTTTTTT, etc.

[0013] Specifically, FGCD8 is a polypeptide with an amino acid sequence of FGCDDDDDDDD from the N-terminus to the C-terminus; in this loaded molecule, C provides a linking functional group, and G is used to fix C at different positions and reduce steric hindrance to meet different detection requirements.

[0014] The FGGCD8 is a polypeptide with an amino acid sequence of FGGCDDDDDDDD from the N-terminus to the C-terminus, as shown in SEQ ID No: 1;

[0015] The FGGGCD8 is a polypeptide with an amino acid sequence of FGGGCDDDDDDDD from the N-terminus to the C-terminus, as shown in SEQ ID No: 2;

[0016] The FGGCGGD8 is a polypeptide with an amino acid sequence from the N-terminus to the C-terminus of FGGCGGDDDDDDDD, as shown in SEQ ID No: 3;

[0017] The FGK(AZA)D8 is a polypeptide with an amino acid sequence of FGKDDDDDDDD from the N-terminus to the C-terminus; wherein the K at the 3rd position is an azide-modified lysine, as shown in SEQ ID No: 4.

[0018] The loaded molecules in this invention can be synthesized using solid-phase synthesis technology or purchased from major peptide synthesis companies, such as Qiangyao Biotechnology Co., Ltd.

[0019] Charged peptides in the traction sequence can be synthesized using a peptide synthesizer, polynucleotides can be synthesized using a DNA synthesizer, and polymers can be purchased directly. Linking functional domains on the load molecule can be attached to the traction sequence via coupling reactions such as amide reactions. Guest molecule domains can be attached to the load molecule-traction sequence via coupling reactions such as amide reactions and click chemistry reactions.

[0020] In step 2) of the above method, the connection between the monomeric amino acid and the load molecule containing the guest molecular structure refers to the cross-linking of the amino acid and the load molecule using a bifunctional cross-linking reagent. The bifunctional crosslinking agent is selected from at least one of the following: 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), trans-2,5-dioxapyrrolidone-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane carboxylate (SMCC), di(N-hydroxysuccinimide) ester of 3,3'-dithiodipropionic acid (DSP), maleimide-acetic acid succinimide ester (AMAS), iodoacetic acid-N-succinimide (SIA), bromoacetic acid-N-hydroxysuccinimide ester (SBA), disuccinimide carbonate (DSC), 3-methyl-1-propynyl isocyanate, and 5-azido-2-nitrobenzoic acid-N-succinimide ester.

[0021] In step 3) of the above method, the main molecule is selected from at least one of α-cyclodextrin and its derivatives, β-cyclodextrin and its derivatives, γ-cyclodextrin and its derivatives, other cyclodextrins and their derivatives, cucurbita[5], cucurbita[6], cucurbita[7], cucurbita[8], cucurbita

[10] and various cucurbita derivatives.

[0022] In step 3) of the above method, the nanopore single-channel recording refers to the acquisition of characteristic electrical signals using nanopores.

[0023] Furthermore, the nanopores can be selected from biological nanopores with a diameter of less than 2 nm and solid nanopores with a diameter of less than 3 nm. Specifically, the nanopores can be selected from at least one of α-HL and its mutants, MspA and its mutants, Phi29 and its mutants, Aerolysin and its mutants, CsgG and its mutants, PA63 and its mutants, ClyA and its mutants, FhuA and its mutants, SPP1 and its mutants, PET nanopores, glass nanopores, SiNx nanopores, Al2O3 nanopores, graphene nanopores, hybrid nanopores, carbon nanotubes, and DNA Origami nanopores.

[0024] Furthermore, the characteristic electrical signals were acquired at room temperature (25.0 ± 3.0 °C). The signal sampling frequency was 100 kHz, and the Bessel low-pass filter cutoff frequency was 10 kHz. The characteristic current signals were acquired using single-channel data analysis with Clampfit software, and the data were statistically analyzed using Origin software.

[0025] The present invention provides a more detailed method for nanopore peptide sequencing based on host-guest interactions, comprising the following steps:

[0026] (I) Enzymatic hydrolysis of polypeptides:

[0027] a1: Dissolve the target peptide thoroughly in the enzymatic digestion buffer to a final concentration between 0.5-20 mM; the enzymatic digestion buffer is 100 mM HEPES, pH 7.5;

[0028] a2: Add proteolytic enzyme (a mixture of aminopeptidase or carboxypeptidase) to the solution prepared in a1 to make the final enzyme concentration between 0.1-5.0 U / mL;

[0029] a3: Incubate the a2 mixture at 37°C for 2–60 minutes, then quench with trifluoroacetic acid, and finally separate by ultrafiltration to obtain hydrolyzed amino acids.

[0030] (ii) Coupling of amino acids with supporting molecules:

[0031] b1: Concentrate the separated amino acid solution and remove the solvent;

[0032] b2: The amino acid to be tested, the bifunctional cross-linking reagent, and the organic base are reacted to form an amino acid-bifunctional cross-linking reagent complex; wherein the molar ratio of the amino acid, the bifunctional cross-linking reagent, and the organic base is (1-100):1:(2-10), and the reaction time is 1-4 h; the bifunctional cross-linking reagent is added in the form of DMF solution, and the organic base is added in the form of DMF solution;

[0033] b3: The amino acid-bifunctional cross-linking reagent complex is reacted with the supporting molecule to obtain the amino acid molecule-supported molecule composite probe; wherein, the molar ratio of the amino acid-bifunctional cross-linking reagent complex to the supporting molecule is (1-100):1, and the reaction is carried out at room temperature for 1-8 hours; the supporting molecule is added in the form of an aqueous solution;

[0034] The organic base is selected from at least one of TEA, NMM, DIPEA, Py, DBU, 2,6-Lutidine, Imidazole, or NMI;

[0035] Specifically, the molar ratio of the amino acid, the bifunctional cross-linking agent, and the organic base is 1:1:2.

[0036] The molar ratio of the amino acid-bifunctional cross-linking reagent complex to the loaded molecule is 1:1, and the reaction is carried out at room temperature for 1 hour.

[0037] (III) Single-channel recording in nanopores:

[0038] c1: Diphytylphosphatidylcholine (DPhPC) was used to form a synthetic lipid bilayer with a diameter of 100-150 micrometers on a 25-micrometer-thick polytetrafluoroethylene film (Goodfellow, Malvern, PA).

[0039] c2: The nanopores are embedded in the phospholipid bilayer, which divides the sample cell into cis and trans chambers, each containing 1.0 mL of buffer solution; the amino acid molecule-loaded molecule composite probe to be tested is added to the cis chamber, and the host molecule is added to the cis chamber at the same time;

[0040] The above experiments were conducted in 0.8–4.0 M KCl and 10 mM citrate buffer at a pH of 5.0–8.0 and a temperature of 25.0 ± 3.0 °C.

[0041] The mass of the host molecule is more than 10 times greater than the mass of the amino acid-loaded molecule composite probe.

[0042] c3: Apply a fixed bias voltage in the range of +80–250mV to the nanopore single-channel test system and record it. The load molecules and host molecules move into the nanopore under the drive of host-guest interaction and electrophoretic force, generating a characteristic current signal; the signal sampling frequency is 100kHz, and the Bessel low-pass filter cutoff frequency is 10kHz.

[0043] (iv) Data processing and analysis:

[0044] d1: Use the Single-Channel Search function in Clampfit software to scan the recorded data and extract the amplitude and drain time information for each characteristic current.

[0045] d2: Calculate the current hysteresis ratio of the corresponding signal based on the baseline current;

[0046] d3: Import the obtained data into Origin software, plot the signal hysteresis time-current hysteresis ratio graph, and perform frequency analysis on the signal current hysteresis ratio;

[0047] d4: Determine the order in which amino acids are hydrolyzed according to the frequency of the current retardation ratio from high to low, and determine the polypeptide sequence based on the type of protease used.

[0048] The polypeptide described in the method of this invention can be a polypeptide of any length.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] This invention is a de novo sequencing method that utilizes the sequential hydrolysis of peptides by exopeptidases to obtain the sequence information of peptides. By linking a load molecule with a single amino acid, the current blocking ratio of the characteristic signals generated after the binding of 20 encoding amino acids with the load molecule is accurately measured. The characteristic current is contributed by a single amino acid, eliminating decoding difficulties. The amount of target peptide used is small, resulting in a low detection limit. The experimental operation is safe and simple, with most reactions carried out in an aqueous phase, without involving toxic reagents, making it green and safe. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the nanopore peptide sequencing method based on host-guest interactions.

[0052] Figure 2 The characteristic signal current hindrance ratio generated by different encoded amino acids;

[0053] Figure 3 To assess the response of the nanopore detection system to amino acids and loaded molecules, the sample concentrations were 40 pM, 400 pM, 4 nM, 40 nM, and 400 nM, with a detection limit of 4 nM (based on a signal strength greater than 1 per minute).

[0054] Figure 4 The following is the peptide sequencing data analysis process in the example; a is the sample characteristic signal retardation time length-current retardation ratio diagram, b is the frequency distribution of characteristic signal current retardation ratio, and c is the sequence inference process. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The room temperature mentioned in the following examples refers to 25.0 ± 3.0 °C.

[0057] The loading molecule FGGCD8 used in the following examples is a polypeptide with an amino acid sequence of FGGCDDDDDDDD from the N-terminus to the C-terminus. It can be purchased from major polypeptide synthesis companies or synthesized by ourselves using solid-phase synthesis technology.

[0058] Example 1

[0059] Experimental sample: The artificially synthesized polypeptide has the sequence of SEQ ID No: 5, and its amino acid sequence is: RRRRRRPVGLRSY.

[0060] The experimental procedure is as follows:

[0061] 1. Dissolve the target peptide in 1 mL of enzyme digestion buffer (100 mM HEPES, pH 7.5) to achieve a final concentration of 10 mM.

[0062] 2. Add buffer solutions of carboxypeptidase A and B to the polypeptide solution to make the final concentration of each enzyme 2.0 U / mL.

[0063] 3. After incubating the polypeptide and enzyme solution at 37°C for 2 hours, trifluoroacetic acid was added for quenching.

[0064] 4. After ultrafiltration of the polypeptide solution using a 3k ultrafiltration tube, the filtrate is vacuum dried until all solvent is removed.

[0065] 5. Add 10 μL of ultrapure water to the dried sample (2 μg) to reconstitute it, then add 10 μL of SPDP (solvent DMF, concentration 4 mol / L) and 10 μL of imidazole (solvent DMF, concentration 8 mol / L), and react for 1 h.

[0066] 6. Dissolve the supported molecule FGGCD8 in ultrapure water to a final concentration of 1M. Add 10μL to the reaction system in step 5, react at room temperature for 1 hour, and then store under cold.

[0067] 7. Use of diaphytylphosphatidylcholine (DPhPC) in 25-micron-thick polytetrafluoroethylene films

[0068] A synthetic lipid bilayer with a diameter of 150 μm was formed on (Goodfellow, Malvern, PA); nanopores (α-HL) were embedded in the phospholipid bilayer, which divided the sample cell into cis and trans chambers, each containing 1.0 mL of buffer solution (composition: 3.0 M KCl, 10 mM citric acid, pH 5.0). 3 μL (sample concentration of 150 μM) of the product obtained in step 6 was added to the cis chamber of the nanopore single-channel test system, and 10 μL of the main molecule cucurbita[7] (concentration of 100 mM) was added to the cis chamber at the same time.

[0069] The conditions for the nanopore single-channel testing system were: 3.0M KCl, 10mM citric acid, pH 5.0, bias voltage +200mV, and data recording for more than 1 hour. The loaded molecules and the host molecules moved into the nanopore under the drive of host-guest interactions and electrophoretic forces, generating characteristic current signals. The characteristic electrical signals were acquired at room temperature (25.0±3.0℃), with a signal sampling frequency of 100kHz and a Bessel low-pass filter cutoff frequency of 10kHz.

[0070] 8. Use the Single-Channel Search function in Clampfit software to scan the recorded data and extract the amplitude and drain time information for each characteristic current.

[0071] 9. Calculate the current hysteresis ratio of the corresponding signal based on the baseline current.

[0072] 10. Import the obtained data into Origin software, plot the signal current hysteresis ratio-hysteresis time length graph, and perform frequency analysis on the signal current hysteresis ratio.

[0073] 11. Determine the order in which amino acids are hydrolyzed according to the frequency of the current retardation ratio from high to low, and determine the polypeptide sequence according to the CN end sequence.

[0074] The characteristic signal current hysteresis ratio generated by different encoded amino acids, for example Figure 2 As shown.

[0075] The response of the nanopore detection system to amino acids (glycine) and supported molecules is as follows: Figure 3 As shown. The final concentrations of the test samples were 40 pM, 400 pM, 4 nM, 40 nM, and 400 nM, respectively, and the detection limit was 4 nM (based on a signal strength greater than 1 ohm per minute).

[0076] The detection limit was determined as follows: 10 μL of test sample (FGGC(G)D8) with concentrations of 4 nM, 40 nM, 400 nM, 4 μM, and 40 μM and 10 μL of the host molecule cucurbita[7] (concentration: 100 mM) were added to the nanopore single-channel detection system, and a single-channel recording was performed by applying a voltage of +200 mV for a recording time of more than 1 h. The recording results were analyzed for signal frequency, and the signal frequency (min) in the recording was calculated. -1 Using a signal frequency of 1.52 and 4 nM as the sample detection limit, when the final sample concentration is below 4 nM, the signal frequency will be less than 1 min. -1 It cannot meet the requirements for signal statistics and processing.

[0077] The peptide sequencing data analysis process corresponding to this embodiment is as follows: Figure 4 As shown (the full sequence used here is RRRRRPVGLRSY; since carboxypeptidase cannot cleave V, only this part is identified in the figure). a is the sample characteristic signal retardation time length-current retardation ratio graph, b is the frequency distribution of the characteristic signal current retardation ratio, and c is the sequence inference process.

Claims

1. A nanopore peptide sequencing method based on host-guest interaction, comprising the following steps: 1) using a proteolytic enzyme to sequentially hydrolyze the peptide to be sequenced into monomeric amino acids; 2) linking the monomeric amino acids with a load molecule containing a guest molecule structure; 3) performing single-channel recording in a nanopore after the host molecule and the guest molecule are bound. Specifically, the following steps are included: (a) Enzymatic hydrolysis of polypeptides: a1: Dissolve the polypeptide to be sequenced thoroughly in the enzyme digestion buffer to a final concentration between 0.5 and 20 mM; the enzyme digestion buffer is 100 mM HEPES, pH 7.5; a2: Add proteolytic enzyme to the solution prepared in a1 to make the final enzyme concentration between 0.1-5.0 U / mL; a3: Incubate the a2 mixture at 37 °C for 2–60 minutes, then quench with trifluoroacetic acid, and separate by ultrafiltration to obtain the hydrolyzed amino acids; (ii) Coupling of amino acids with supporting molecules: b1: Concentrate the separated amino acid solution and remove the solvent; b2: The amino acid to be tested, a bifunctional cross-linking reagent, and an organic base are reacted to form an amino acid-bifunctional cross-linking reagent complex; wherein, The molar ratio of the amino acid, the bifunctional cross-linking agent, and the organic base is (1-100):1:(2-10), and the reaction time is 1-4 h; the bifunctional cross-linking agent is added in the form of DMF solution, and the organic base is added in the form of DMF solution. b3: The amino acid-bifunctional cross-linking reagent complex is reacted with the loaded molecule to obtain the amino acid molecule-loaded molecule composite probe to be tested; wherein, the molar ratio of the amino acid-bifunctional cross-linking reagent complex to the loaded molecule is (1-100):1, and the reaction is carried out at room temperature for 1-8 h; the loaded molecule is added in the form of an aqueous solution; The organic base is selected from at least one of TEA, NMM, DIPEA, Py, DBU, imidazole, and NMI; (III) Single-channel recording of nanopores: c1: Diphytylphosphatidylcholine was used to form a synthetic lipid bilayer with a diameter of 100-150 micrometers on a 25-micrometer-thick polytetrafluoroethylene film. c2: The nanopores are embedded in a phospholipid bilayer, which divides the sample cell into cis and trans chambers, each containing 1.0 mL of buffer solution; the amino acid molecule-loaded molecule composite probe to be tested is added to the cis chamber, and the host molecule is added to the cis chamber at the same time; The above experiments were conducted in 0.8–4.0 MKCl, 10 mM citrate buffer at a pH of 5.0–8.0 and a temperature of 25.0 ± 3.0 ℃. The mass of the host molecule is more than 10 times greater than the mass of the amino acid-loaded molecule composite probe. c3: Apply a fixed bias voltage in the range of +80 to 250 mV to the nanopore single-channel testing system and record the signal. The load molecules and host molecules move into the nanopore under the drive of host-guest interaction and electrophoretic force, generating a characteristic current signal. The signal sampling frequency is 100 kHz, and the Bessel low-pass filter cutoff frequency is 10 kHz. (iv) Data processing and analysis: d1: Use the Single-Channel Search function in Clampfit software to scan the recorded data and extract the impedance amplitude and impedance time length information of each characteristic current; d2: Calculate the current hysteresis ratio of the corresponding signal based on the baseline current; d3: Import the obtained data into Origin software, plot the signal hysteresis time-current hysteresis ratio graph, and perform frequency analysis on the signal current hysteresis ratio; d4: Determine the order in which amino acids are hydrolyzed according to the frequency of the current retardation ratio from high to low, and determine the polypeptide sequence based on the type of protease used. In step 2), the loaded molecule includes a combination of guest molecule structure, connecting functional structural domain and traction sequence; The guest molecular structure is selected from at least one of ferrocene and its derivatives, adamantane and its derivatives, phenylalanine and its derivatives, tyrosine and its derivatives, histidine and its derivatives, and methionine and its derivatives. The connecting functional structural domain is selected from at least one of azide, acyl halide, alkynyl, allyl, mercapto, amino, succinimide ester, and maleimide ester; The traction sequence is selected from polyelectrolytes.

2. The nanopore polypeptide sequencing method according to claim 1, characterized in that: In step 1), the proteolytic enzyme is selected from at least one of the following: carboxypeptidase A, carboxypeptidase B, carboxypeptidase C, carboxypeptidase Y, leucine aminopeptidase, arginine aminopeptidase, cysteine ​​aminopeptidase, glycine aminopeptidase, histidine aminopeptidase, proline aminopeptidase, trypsin, trypsin B, chymotrypsin, and pepsin.

3. The nanopore polypeptide sequencing method according to claim 1, characterized in that: The traction sequence is selected from at least one of polynucleotides and charged polypeptides.

4. The nanopore polypeptide sequencing method according to claim 1, characterized in that: In step 2), the connection between the monomeric amino acid and the load molecule containing the guest molecule structure refers to cross-linking the amino acid and the load molecule using a bifunctional cross-linking agent. The bifunctional crosslinking agent is selected from at least one of the following: 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), trans-2,5-dioxapyrrolidone-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane carboxylate (SMCC), di(N-hydroxysuccinimide) ester of 3,3'-dithiodipropionic acid (DSP), maleimide-acetic acid succinimide ester (AMAS), iodoacetic acid-N-succinimide (SIA), bromoacetic acid-N-hydroxysuccinimide ester (SBA), disuccinimide carbonate (DSC), 3-methyl-1-propynyl isocyanate, and 5-azido-2-nitrobenzoic acid-N-succinimide ester.

5. The nanopore polypeptide sequencing method according to claim 1, characterized in that: In step 3), the main molecule is selected from at least one of cyclodextrin and its derivatives, cucurbituril[5], cucurbituril[6], cucurbituril[7], cucurbituril[8], cucurbituril[10] and other cucurbituril derivatives.

6. The nanopore polypeptide sequencing method according to claim 1, characterized in that: In step 3), the nanopore single-channel recording refers to the acquisition of characteristic electrical signals using nanopores.

7. The nanopore polypeptide sequencing method according to claim 6, characterized in that: The nanopores are selected from biological nanopores with a diameter of less than 2 nm and solid nanopores with a diameter of less than 3 nm. Specifically, the nanopores are selected from at least one of α-HL and its mutants, MspA and its mutants, Phi29 and its mutants, Aerolysin and its mutants, CsgG and its mutants, PA63 and its mutants, ClyA and its mutants, FhuA and its mutants, SPP1 and its mutants, PET nanopores, glass nanopores, SiNx nanopores, Al2O3 nanopores, graphene nanopores, hybrid nanopores, carbon nanotubes, and DNA Origami nanopores.

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