Nanopore polypeptide sequencing method based on host-guest interaction

Abstract

The invention discloses a nanopore polypeptide sequencing method based on host-guest interaction. The method comprises the following steps: 1) sequentially hydrolyzing polypeptides to be sequenced into monomer amino acids by using proteolytic enzyme; 2) connecting a monomer amino acid with a load molecule containing a guest molecular structure; and 3) after the host molecules and the guest molecules are combined, performing nanopore single-channel recording. The invention provides a de novo sequencing method. According to the method, sequence information of polypeptide is obtained by utilizing the characteristic that exopeptidase sequentially hydrolyzes the polypeptide; through a method of connecting a load molecule with a single amino acid, the current blocking proportion of a characteristic signal generated after 20 coding amino acids are combined with the load molecule is accurately measured, the characteristic current is contributed by the single amino acid, and the decoding difficulty does not exist; the target polypeptide dosage is small, and the detection limit is low; the experimental operation is safe and simple, most reactions are carried out in a water phase, toxic reagents are not involved, and the method is green and safe.

Description

Technical Field

[0001] The invention belongs to the technical field of protein sequencing, and in particular relates to a nanopore polypeptide sequencing method based on host-guest interaction. Background Art

[0002] Protein sequencing has a wide range of applications in biology, medicine, and biotechnology. It is of great significance for understanding the structure, function, and interaction of proteins in organisms, and is also of guiding significance for drug development and clinical diagnosis and treatment. Protein sequencing originated in the 1950s, when Swedish biochemist Pehr Edman used phenylisothiocyanate for N-terminal sequencing. This method is called Edman degradation and has been used to this day. The protein sequencer developed based on this principle is one of the most mature protein sequencing technologies. However, this method has some obvious limitations, including high purity requirements, complex reaction process, long time consumption, limited read length, and inability to identify post-translationally modified amino acids. With the development of modern biotechnology, researchers have begun to turn to mass spectrometry sequencing to infer sequence information through fragment mass. Mass spectrometry sequencing is widely accepted for its advantages of short detection time, high accuracy, and strong repeatability, and has become the gold standard in the field of protein sequencing. However, it still has the defects of low sensitivity, limited read length, and difficulty in determining post-translational modifications.

[0003] In recent years, single-molecule testing technology has developed rapidly and has been widely used in the field of protein sequencing. For example, electron tunneling current is used to detect post-translational modifications on peptide chains of different amino acids. There are also many fluorescence methods combined with enzymatic hydrolysis and Edman degradation to locate special amino acids in peptide chains and then compare them with protein libraries to interpret sequence information. In 2022, Brian D. Reed and his colleagues proposed a real-time dynamic single-molecule protein sequencing method on an integrated semiconductor device, which can identify special amino acids in the sequence in real time. However, the above method relies on the identification and positioning of special amino acids and then compares them with known sequences in the protein library, and 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. Two mutually insulated formats filled with electrolyte are connected using nanoscale channels. After applying a bias voltage, the electrolyte ions in the solution migrate directionally through the nanopore to generate current, which is measured, amplified, and converted by the patch clamp system and recorded. When the substance to be tested is present in the detection system, the substance passes through the nanopore under diffusion or voltage drive. In this process, the occupation of the nanopore by the substance to be tested will cause the number of examples passing through the pore to change, which will further cause the current recorded by the system to change, generating characteristic signals with different current blockade amplitudes and current blockade times. By analyzing these signals, information including substance type, structure, conformational changes, and molecular composition can be obtained. In 2021, three research teams successively proposed to construct DNA-peptide complexes, using molecular motors to pull DNA to make the polypeptide move in the nanopore, and measured the current signal of the polypeptide. However, due to the small size of amino acids, the current information generated by directly reading the polypeptide chain is contributed by multiple amino acids. The large number of encoded amino acids makes it difficult to accurately read the amino acids, and the reading of sequence information cannot be achieved. Summary of the invention

[0005] Based on the defects of the prior art, the purpose of the present invention is to provide a nanopore polypeptide sequencing method based on host-guest interaction. The method utilizes the host-guest interaction in combination with the nanopore single-channel technology to achieve accurate de novo reading of the amino acid sequence of the polypeptide. The present invention utilizes the characteristics of exopeptidase to sequentially hydrolyze polypeptides to obtain polypeptide sequence information; by means of connecting a single amino acid with a load molecule, the current blocking ratio of the characteristic signal generated by the combination of 20 coded amino acids with the load molecule is accurately measured. The characteristic current is contributed by a single amino acid, there is no decoding difficulty, and the reading of the amino acid sequence can be achieved.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A nanopore polypeptide sequencing method based on host-guest interaction comprises the following steps: 1) using a proteolytic enzyme to sequentially hydrolyze a polypeptide to be sequenced into monomeric amino acids; 2) connecting the monomeric amino acids to a load molecule containing a guest molecule domain; 3) performing nanopore single-channel recording after the host molecule is combined with the guest molecule.

[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 load molecule is designed to achieve the purpose of binding with the amino acid and interacting with the main molecule, and passing through the nanopore under the drive of the electric field to generate a characteristic signal.

[0010] In the present invention, the load molecule comprises a combination of a guest molecule domain, a connecting functional domain and a pulling sequence.

[0011] Further, the guest molecule 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 connecting functional domain is selected from at least one of azide, acyl halide, alkynyl, allyl, sulfhydryl, 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, PolyE, or polyelectrolytes.

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

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

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

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

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

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

[0018] The load molecule in the present invention can be synthesized using solid phase synthesis technology, or purchased from major peptide synthesis companies, such as Qiangyao Biotechnology Co., Ltd.

[0019] The charged polypeptide in the traction sequence can be synthesized by a peptide synthesizer, the polynucleotide can be synthesized by a DNA synthesizer, and the polymer can be purchased directly. The connecting functional domain on the load molecule can be connected to the traction sequence through a coupling reaction such as an amide reaction. The guest molecule domain can be connected to the load molecule-traction sequence through a coupling reaction such as an amide reaction and a click chemistry reaction.

[0020] In step 2) of the above method, connecting the monomeric amino acid and the load molecule comprising the guest molecule structure refers to cross-linking the amino acid and the load molecule using a bifunctional cross-linking reagent. Wherein, the bifunctional cross-linking reagent is selected from at least one of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), trans-2,5-dioxapyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl) cyclohexanecarboxylate (SMCC), 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide) ester (DSP), maleimidoacetic acid succinimide ester (AMAS), iodoacetic acid-N-succinimide (SIA), bromoacetic acid N-hydroxysuccinimide ester (SBA), disuccinimidyl 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, cucurbituril [5], cucurbituril [6], cucurbituril [7], cucurbituril [8], cucurbituril

[10] and various cucurbituril derivatives.

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

[0023] Furthermore, the nanopore can be selected from biological nanopores with a diameter of less than 2 nm and solid-state nanopores with a diameter of less than 3 nm. Specifically, the nanopore 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 signal was collected at room temperature of 25.0±3.0°C. The signal sampling frequency was 100kHz, and the Bessel low-pass filter cutoff frequency was 10kHz. The characteristic current signal was analyzed by single-channel data analysis using Clampfit software to collect the characteristic signal, and the data was statistically analyzed and analyzed using origin software.

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

[0026] (I) Enzymatic hydrolysis of polypeptides:

[0027] a1: fully dissolve the target polypeptide in an enzymatic hydrolysis buffer to a final concentration of 0.5-20 mM; the enzymatic hydrolysis buffer is 100 mM HEPES, pH 7.5;

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

[0029] a3: Incubate the mixed solution of a2 at 37°C for 2–60 minutes, then add trifluoroacetic acid to quench, and separate the hydrolyzed amino acids by ultrafiltration.

[0030] (II) Coupling of amino acids and load molecules:

[0031] b1: concentrating the separated amino acid solution and removing the solvent;

[0032] b2: reacting the amino acid to be tested, the bifunctional cross-linking reagent and the organic base 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 hours; the bifunctional cross-linking reagent is added in the form of a DMF solution, and the organic base is added in the form of a DMF solution;

[0033] b3: reacting the amino acid-bifunctional cross-linking reagent complex with a load molecule to obtain a test amino acid molecule-load molecule composite probe; wherein the molar ratio of the amino acid-bifunctional cross-linking reagent complex to the load molecule is (1-100):1, and the reaction is carried out at room temperature for 1-8 hours; the load 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 (2,6-dimethylpyridine), Imidazole (imidazole) or NMI;

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

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

[0037] (III) Nanopore single channel recording:

[0038] c1: diphytylphosphatidylcholine (DPhPC) was used to form a synthetic lipid bilayer with a diameter of 100-150 μm on a 25 μm thick polytetrafluoroethylene membrane (Goodfellow, Malvern, PA);

[0039] c2: embedding the nanopore into a phospholipid bilayer, the phospholipid bilayer divides the sample pool into two chambers, cis and trans, both chambers contain 1.0 mL of buffer solution; adding the amino acid molecule-load molecule composite probe to be tested into the cis chamber, and adding the main molecule into the cis chamber at the same time;

[0040] The above experiments were performed in 0.8–4.0 M KCl, 10 mM citrate buffer at pH 5.0–8.0 and 25.0 ± 3.0 °C;

[0041] The mass of the main molecule is more than 10 times the mass of the amino acid molecule-load molecule composite probe to be detected;

[0042] c3: A fixed bias voltage in the range of +80–250 mV is applied to the nanopore single-channel test system and recorded. Driven by the host-guest interaction and electrophoretic force, the load molecules and the main molecules move toward the nanopore, generating a characteristic current signal; the signal sampling frequency is 100 kHz, and the Bessel low-pass filter cutoff frequency is 10 kHz;

[0043] (IV) Data processing and analysis:

[0044] d1: Use the Single-Channel Search function in the Clampfit software to scan the recorded data and extract the information of each characteristic current block amplitude (Amplitude) and block duration (Dwell Time);

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

[0046] d3: Import the obtained data into Origin software, draw a signal block time length-current block ratio diagram, and perform frequency analysis on the signal current block ratio;

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

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

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

[0050] The present invention is a method for de novo sequencing, which utilizes the characteristic of exopeptidase to sequentially hydrolyze polypeptides to obtain polypeptide sequence information; through the method of connecting a load molecule with a single amino acid, the current blockage ratio of characteristic signals generated after 20 coding amino acids are combined with the load molecule is accurately measured, and the characteristic current is contributed by a single amino acid, so there is no decoding difficulty; the target polypeptide is used in a small amount and the detection limit is low; the experimental operation is safe and simple, most reactions are carried out in an aqueous phase, no toxic reagents are involved, and the method is green and safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the process of nanopore peptide sequencing based on host-guest interaction;

[0052] Figure 2 The characteristic signal current blockade ratio produced for different encoded amino acids;

[0053] Figure 3 The response of the nanopore detection system to amino acids and load molecules, the test sample concentrations were 40pM, 400pM, 4nM, 40nM, 400nM, and the detection limit was 4nM (measured as a signal greater than 1 per minute);

[0054] Figure 4 It is the polypeptide sequencing data analysis process in the embodiment; a is the sample characteristic signal blocking time length-current blocking ratio diagram, b is the characteristic signal current blocking ratio frequency distribution, and c is the sequence inference process. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels, and the room temperature described in the following examples refers to 25.0±3.0°C.

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

[0058] Embodiment 1,

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

[0060] The experimental steps are as follows:

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

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

[0063] 3. Incubate the peptide and enzyme solution at 37°C for 2 h and then add trifluoroacetic acid to quench.

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

[0065] 5. Add 10 μL of ultrapure water to the above dried sample (2 μg) to re-dissolve it, 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 hour.

[0066] 6. Dissolve the load molecule FGGCD8 in ultrapure water to a final concentration of 1 M. Take 10 μL and add it to the reaction system in step 5. After reacting at room temperature for 1 hour, store in a refrigerator.

[0067] 7. Use diphytylphosphatidylcholine (DPhPC) for 25 μm thick polytetrafluoroethylene film

[0068] (Goodfellow, Malvern, PA) to form a synthetic lipid bilayer with a diameter of 150 μm; the nanopore (α-HL) was embedded in the phospholipid bilayer, and the phospholipid bilayer divided the sample pool into two chambers, cis and trans, both of which contained 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 cucurbituril [7] (concentration of 100 mM) was added to the cis chamber;

[0069] The conditions of the nanopore single-channel test system are: 3.0M KCl, 10mM citric acid, pH 5.0, bias voltage +200mV, and data recording for more than 1h; the load molecules and the main molecules move toward the nanopore driven by the host-guest interaction and the electrophoretic force, generating a characteristic current signal; the characteristic electrical signal is collected at room temperature 25.0±3.0℃, the signal sampling frequency is 100kHz, and the Bessel low-pass filter cutoff frequency is 10kHz.

[0070] 8. Use the Single-Channel Search function in the Clampfit software to scan the recorded data and extract the information of each characteristic current block amplitude (Amplitude) and block duration (Dwell Time).

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

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

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

[0074] The characteristic signal current blockade ratios produced by different encoded amino acids are e.g. Figure 2 shown.

[0075] The response of the nanopore detection system to amino acids (glycine) and load molecules is shown in Figure 2. Figure 3 The final concentrations of the test samples were 40pM, 400pM, 4nM, 40nM, and 400nM, respectively, and the detection limit was 4nM (measured as a signal greater than 1 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 main molecule cucurbituril [7] (concentration: 100 mM) were added to the nanopore single-channel detection system, and a voltage of +200 mV was applied for single-channel recording for more than 1 h. The recording results were subjected to signal frequency analysis, and the signal frequency (min) in the recording was calculated. -1 ), with a signal frequency of 1.52 at 4 nM as the sample detection limit, when the final sample concentration is lower than 4 nM, the signal frequency will be less than 1 min -1 , cannot satisfy signal statistics and processing.

[0077] The polypeptide sequencing data analysis process corresponding to this embodiment is as follows Figure 4 As shown (the full sequence used here is RRRRRPVGLRSY, because carboxypeptidase cannot cut V, so only this point is recognized in the figure.). a is the sample characteristic signal blocking time length-current blocking ratio diagram, b is the characteristic signal current blocking ratio frequency distribution, and c is the sequence inference process.

Claims

1. A nanopore polypeptide sequencing method based on host-guest interaction, comprising the following steps: 1) using a proteolytic enzyme to sequentially hydrolyze the polypeptide to be sequenced into monomeric amino acids; 2) connecting the monomeric amino acids to a load molecule containing a guest molecule structure; 3) after the host molecule is combined with the guest molecule, performing nanopore single-channel recording.

2. The nanopore polypeptide sequencing method according to claim 1, characterized in that: In the 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 or 2, characterized in that: In the step 2), the load molecule includes a combination of a guest molecule domain, a connecting functional domain and a pulling sequence.

4. The nanopore polypeptide sequencing method according to claim 3, characterized in that: The guest molecule domain is at least one selected from ferrocene and its derivatives, adamantane and its derivatives, phenylalanine and its derivatives, tyrosine and its derivatives, histidine and its derivatives, methionine and its derivatives; Or, the linking functional domain is selected from at least one of an azide group, an acyl halide, an alkynyl group, an allyl group, a thiol group, an amino group, a succinimide ester, and a maleimide ester; Alternatively, the pulling sequence is selected from at least one of a polynucleotide, a random nucleotide sequence, a charged polypeptide, and a polyelectrolyte.

5. The nanopore polypeptide sequencing method according to any one of claims 1 to 4, characterized in that: In the step 2), the connection between the monomeric amino acid and the load molecule comprising the guest molecule structure refers to cross-linking the amino acid and the load molecule using a bifunctional cross-linking reagent; Preferably, the bifunctional cross-linking reagent is selected from at least one of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), trans-2,5-dioxapyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxylate (SMCC), 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide) ester (DSP), maleimidoacetic acid succinimide ester (AMAS), iodoacetic acid-N-succinimide (SIA), bromoacetic acid N-hydroxysuccinimide ester (SBA), disuccinimidyl carbonate (DSC), 3-methyl-1-propynyl isocyanate, and 5-azido-2-nitrobenzoic acid-N-succinimide ester.

6. The nanopore polypeptide sequencing method according to any one of claims 1 to 5, characterized in that: In step 3), 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, cucurbituril [5], cucurbituril [6], cucurbituril [7], cucurbituril [8], cucurbituril [10] and other types of cucurbituril derivatives.

7. The nanopore polypeptide sequencing method according to any one of claims 1 to 6, characterized in that: In the step 3), the nanopore single-channel recording refers to the collection of characteristic electrical signals using a nanopore.

8. The nanopore polypeptide sequencing method according to claim 7, characterized in that: The nanopore is selected from biological nanopores with a diameter of less than 2 nm and solid-state nanopores with a diameter of less than 3 nm. Specifically, the nanopore is 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.

9. The nanopore polypeptide sequencing method according to claim 7, characterized in that: The characteristic electrical signal was collected at room temperature of 25.0±3.0°C; the signal sampling frequency was 100kHz, and the Bessel low-pass filter cutoff frequency was 10kHz; the characteristic current signal was analyzed by single-channel data analysis using Clampfit software to collect the characteristic signal, and the data was statistically analyzed and analyzed using origin software.

10. The nanopore polypeptide sequencing method according to any one of claims 1 to 9, characterized in that: The nanopore polypeptide sequencing method based on host-guest interaction specifically includes the following steps: (I) Enzymatic hydrolysis of polypeptides: a1: fully dissolve the target polypeptide in an enzymatic hydrolysis buffer to a final concentration of 0.5-20 mM; the enzymatic hydrolysis buffer is 100 mM HEPES, pH 7.5; a2: Add proteolytic enzyme to the solution prepared in a1 to make the final concentration of the enzyme 0.1-5.0U / mL; a3: Incubate the mixed solution of a2 at 37°C for 2–60 minutes, then add trifluoroacetic acid to quench, and separate the hydrolyzed amino acids by ultrafiltration; (II) Coupling of amino acids and load molecules: b1: concentrating the separated amino acid solution and removing the solvent; b2: reacting the amino acid to be tested, the bifunctional cross-linking reagent and the organic base 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 hours; the bifunctional cross-linking reagent is added in the form of a DMF solution, and the organic base is added in the form of a DMF solution; b3: reacting the amino acid-bifunctional cross-linking reagent complex with a load molecule to obtain a test amino acid molecule-load molecule composite probe; wherein the molar ratio of the amino acid-bifunctional cross-linking reagent complex to the load molecule is (1-100):1, and the reaction is carried out at room temperature for 1-8 hours; the load 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, 2,6-lutidine, imidazole and NMI; (III) Nanopore single channel recording: c1: Diphytylphosphatidylcholine was used to form a synthetic lipid bilayer with a diameter of 100-150 μm on a 25 μm thick polytetrafluoroethylene film; c2: embedding the nanopore into a phospholipid bilayer, the phospholipid bilayer divides the sample pool into two chambers, cis and trans, both chambers contain 1.0 mL of buffer solution; adding the amino acid molecule-load molecule composite probe to be tested into the cis chamber, and adding the main molecule into the cis chamber at the same time; The above experiments were performed in 0.8–4.0 M KCl, 10 mM citrate buffer at pH 5.0–8.0 and 25.0 ± 3.0 °C; The mass of the main molecule is more than 10 times the mass of the amino acid molecule-load molecule composite probe to be detected; c3: A fixed bias voltage in the range of +80–250 mV is applied to the nanopore single-channel test system and recorded. Driven by the host-guest interaction and electrophoretic force, the load molecules and the main molecules move toward the nanopore, 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 the Clampfit software to scan the recorded data and extract the block amplitude and block time length information of each characteristic current; d2: Calculate the current blockade ratio of the corresponding signal based on the baseline current; d3: Import the obtained data into Origin software, draw a signal block time length-current block ratio diagram, and perform frequency analysis on the signal current block ratio; d4: Determine the order in which the amino acids are hydrolyzed according to the frequency of the current blocking ratio from high to low, and determine the polypeptide sequence according to the type of protease used.

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