Method for nanopore polypeptide signal homogenization and single-molecule protein sequencing

Abstract

Disclosed in the present invention is a method for nanopore polypeptide signal homogenization and single-molecule protein sequencing. In the method, an electrophoretic force (EPF) opposite to an electroosmotic flow (EOF) is used as a driving force to capture a polypeptide, and an electrical signal generated when a polypeptide to be tested passes through a pore channel of a nanopore is detected and analyzed via the nanopore to determine a sequence of the polypeptide to be tested. In the method of the present invention, by using the EPF to capture the polypeptide and overcoming the reverse EOF, an effect of stretching by two opposite forces is created, thereby successfully improving the nanopore electrical signal of the polypeptide, enhancing the discrimination of nanopore-based polypeptide analysis, and providing a new method for applications such as polypeptide discrimination, modification identification, and protein polypeptide spectrum analysis.

Description

A method for homogenizing nanoporous peptide signals and single-molecule protein sequencing

[0001] This application claims priority to Chinese patent application 2024117986473, filed on 2024 / 12 / 9. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical applications, specifically to a method for homogenizing nanoporous peptide signals and single-molecule protein sequencing. Background Technology

[0003] Post-translational modifications (PTMs) of proteins and peptides alter their biological functions and significantly increase the structural complexity and diversity of the human proteome. Efficient and accurate detection of PTMs is crucial for biomedical research, but remains a technological challenge. Post-translational modifications are numerous, with over 400 types even at relatively small volumes, such as phosphorylation, acetylation, and methylation. Novel PTMs with important biological functions are constantly being discovered, further increasing the complexity of protein and peptide structures. Mass spectrometry (MS) is the mainstream technique for proteomics and post-translational modification research, offering high-precision and high-throughput detection and analysis capabilities, and is a powerful tool for discovering novel post-translational modifications and potential modification sites. However, mass spectrometry also faces challenges in analyzing peptides with low abundance or low ionization rates, and the quantitative requirements for peptides in biomedical research further complicate mass spectrometry analysis. Therefore, technological innovation is an important research topic for mass spectrometry and other novel analytical techniques, aiming to meet the needs of proteomics research in the future.

[0004] Compared to indirect analysis and identification by hydrolyzing proteins into peptides, directly reading the amino acid sequence and structural information such as modifications of proteins represents the ideal state and ultimate goal of protein sequencing. This effectively improves the efficiency and spatiotemporal resolution of protein sequencing, identification, and structural analysis, holding significant importance and broad application prospects in fields such as life sciences, pharmaceutical science, and environmental and food analysis. However, protein sequencing technology has developed slowly and faces significant technical challenges, primarily due to the complex amino acid composition of proteins and the lack of effective amplification techniques.

[0005] Nanopore technology, a newly emerging single-molecule electroanalytical technique developed over the past two decades, exhibits high analytical sensitivity by analyzing only one molecule at a time. Currently, nanopore technology has successfully achieved nucleic acid sequencing, leading to the development of third-generation single-molecule DNA / RNA sequencers. Nanopore technology is also increasingly used for protein and polypeptide chain analysis. However, the more complex chemical compositions, varying electrical charges, and intricate higher-order structures of proteins and polypeptides present significant technical challenges for polypeptide analysis using nanopores, lagging behind its application in nucleic acid analysis. Uniformly capturing polypeptide chains with different electrical charges into nanopores, controlling their morphology and movement within the pores, and effectively reducing their permeation rate are current technical difficulties in nanopore polypeptide chain analysis and direct nanopore sequencing of proteins. For short peptides, the quality and distribution of the nanopore signal induced by the peptide are unpredictable and highly dependent on the peptide's sequence structure, thus limiting the development and application of nanopore peptide analysis technology. Specifically, the electrical signal induced by peptides typically exhibits a dispersed Gaussian distribution, sometimes with several peaks, thus weakening the ability of nanopore analysis to identify and differentiate peptides. To date, there remains a significant technical challenge in effectively and rationally designing and improving the electrical signals for nanopore peptide analysis. While deep learning-assisted data analysis has been widely applied to improve the distinction between peptides and post-translational modifications, accuracy still largely depends on the quality of the raw signals used for nanopore peptide detection and differentiation. Therefore, effective methods are needed to improve the signal uniformity and resolution of nanopore peptide analysis. For direct nanopore single-molecule protein sequencing analysis after protein unfolding, stretching the protein peptide chain, reducing the peptide chain permeation rate, and achieving unidirectional permeation are key technical challenges for single-molecule protein identification and sequence reading. Current analytical methods and reports still face substantial challenges in these areas, lacking effective solutions and breakthroughs. Therefore, direct protein analysis based on nanopores is still in its early stages of development and requires groundbreaking methodological innovation. Summary of the Invention

[0006] To address the above technical problems, this invention provides a method for homogenizing nanopore peptide signals and single-molecule protein sequencing.

[0007] Specifically, the present invention provides a method for homogenizing peptide signals and nanopore sequencing. The method uses an electrophoretic force EPF, which is opposite to the electroosmotic flow EOF, as the driving force to capture the peptide, and uses a nanopore to detect and analyze the electrical signal generated when the peptide passes through the pore of the nanopore to determine the sequence of the peptide.

[0008] In some implementations, the method uses an electrophoretic force EPF, which is opposite to the electroosmotic flow EOF, as the driving force to linearize the peptide, homogenize the signal, and then perform sequencing.

[0009] In some embodiments, the nanopore is a biological nanopore or a solid nanopore.

[0010] In some embodiments, the bio-nanopores include nanoporous proteins, and the solid nanopores include SiO2 nanopores and carbon nanotubes.

[0011] In some embodiments, the nanoporin is one or more of α-hemolysin mutant, aerolysin, Mycobacterium smegmatis porin A (MspA), fragility toxin C (FraC), and curly fimbriae assembly protein G (CsgG).

[0012] In some embodiments, the nanoporin is an α-hemolysin mutant.

[0013] In some embodiments, the α-hemolysin mutant has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and contains amino acid residues differing from the amino acid sequence shown in SEQ ID NO:1 at sites D13, E111, M113, N123, T125, or K147, wherein the positions of 13, 111, 113, 123, 125, and 147 refer to the amino acid residue numbers in SEQ ID NO:1.

[0014] In some embodiments, the amino acid residue differences at the D13 site include D13K; the amino acid residue differences at the E111 site include E111G, E111Y, and E111R; the amino acid residue differences at the M113 site include M113Y, M113W, M113F, M113R, M113D, and M113N; the amino acid residue differences at the N123 site include N123Y and N123W; the amino acid residue differences at the T125 site include T125Y and T125W; and the amino acid residue differences at the K147 site include K147G and K147E.

[0015] In some embodiments, the α-hemolysin mutant has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and includes one or more differences in amino acid residues at positions K8, D13, K21, D44, D45, E111, M113, N123, T125, D127, M131, T145, and K147 compared to the amino acid sequence shown in SEQ ID NO:1, wherein the positions of 8, 13, 21, 44, 45, 111, 113, 123, 125, 127, 131, 145, and 147 refer to the amino acid residue numbers in SEQ ID NO:1.

[0016] In some embodiments, the amino acid residue differences at the K8 site include K8E and K8Q; the amino acid residue differences at the D13 site include D13K; the amino acid residue differences at the K21 site include K21Q; the amino acid residue differences at the D44 site include D44K; the amino acid residue differences at the D45 site include D45N; the amino acid residue differences at the E111 site include E111G, E111Y, and E111R; and the amino acid residue differences at the M113 site include M113Y and M113W. The amino acid residue differences at the N123 site include N123Y and N123W; the amino acid residue differences at the T125 site include T125Y and T125W; the amino acid residue differences at the D127 site include D127R; the amino acid residue differences at the M131 site include M131E; the amino acid residue differences at the T145 site include T145F; and the amino acid residue differences at the K147 site include K147G and K147E.

[0017] In some specific implementations, the amino acid residue differences are selected from the group consisting of:

[0018] (1)E111Y / R / G;

[0019] (2)M113Y / W / F / R / D / N; (3)K147E / G;

[0020] (4) D13K and D44K;

[0021] (5) E111Y and K147W;

[0022] (6) M113F and T145F;

[0023] (7) M131E and K147E;

[0024] (8) K8E, M131E and K147E;

[0025] (9) D13K, D44K and D45N;

[0026] (10)E111Q, M113F and K147N;

[0027] (11)E111Q, M113F and K147F;

[0028] (12) E111F, M113F and K147N;

[0029] (13)E111G, M113F and K147T;

[0030] (14)E111S, M113F and K147G;

[0031] (15)E111S, M113F and K147S;

[0032] (16)E111S, M113F and K147T;

[0033] (17) M113F, T145F and K147N;

[0034] (18)M113Y, K131E and K147E;

[0035] (19)M113W, K131E and K147E;

[0036] (20) K8E, M113Y, K131E and K147E;

[0037] (21) K8E, M113W, K131E and K147E;

[0038] (22) E111Q, M113F, T115F and K147N;

[0039] (23) E111Q, M113F, T145F and K147N;

[0040] (24) D44K, D45N, E111Q, M113F and K147N;

[0041] (25) D13K, D44K, D45N, D127R, E111Q, M113F and K147N

[0042] (26)N123Y / W;

[0043] (27)T125Y / W;

[0044] (28) K8Q and M113Y;

[0045] (29)E111R and K147G;

[0046] (30)E111Y and K147G;

[0047] (31)E111G and M113Y;

[0048] (32)M113Y and K147G;

[0049] (33)M113D and K147G;

[0050] (34) N123Y and K147G;

[0051] (35) N123W and K147G;

[0052] (36) K8Q, K21Q and M113Y;

[0053] (37)E111G, M113Y and K147G;

[0054] The " / " indicates that the site contains any of the listed amino acid mutations.

[0055] In some specific implementations, the α-hemolysin mutants are listed in Table 1.

[0056] In some implementations, the method adjusts the magnitudes of the EPF and EOF by adjusting the applied voltage.

[0057] In some implementations, the voltage is a negative voltage.

[0058] In some implementations, the negative voltage is -150mV to -20mV.

[0059] In some implementations, the negative voltage is -150mV to -80mV.

[0060] In some specific implementations, the negative voltage is -120mV to -80mV.

[0061] In some specific implementations, the negative voltage is -100mV.

[0062] In some implementations, the pH of the buffer solution is ≤7.5.

[0063] In some implementations, the pH of the buffer solution is >7.5.

[0064] In some implementations, the pH of the buffer solution is ≤3.8.

[0065] In some specific implementations, the pH of the buffer solution is 3.8.

[0066] In some implementations, the buffer solution of the method further includes salt ions.

[0067] In some specific implementations, the buffer solution is a citrate buffer or a formic acid buffer.

[0068] In some embodiments, the salt ion is selected from potassium ions, calcium ions, lithium ions, and sodium ions.

[0069] In some embodiments, the salt ions are derived from KCl, CaCl2, LiCl, and NaCl solutions.

[0070] In some specific implementations, the salt ions are derived from a KCl solution.

[0071] In some embodiments, the concentration of the salt ions is 50 mM to 4 M.

[0072] In some specific implementations, the concentration of the salt ions is 500 mM to 2 M.

[0073] In some specific implementations, the concentration of the salt ions is 1M or 2M.

[0074] In some embodiments, the buffer solution on both sides of the nanopore has a salt ion concentration difference.

[0075] In some embodiments, the salt ion concentration difference is 50 mM to 4 M.

[0076] In some embodiments, the salt ion concentration difference is 300 mM to 2 M.

[0077] In some specific implementations, the salt ion concentration difference is 500 mM-1 M.

[0078] In some embodiments, the polypeptide includes natural, unmodified polypeptides and post-translational modified polypeptides.

[0079] In some specific embodiments, the post-translational modification is selected from one or more of acetylation, phosphorylation, ubiquitination, methylation, glycosylation, lipidation, citrullination, and β-hydroxybutyrylation.

[0080] In some specific implementations, the polypeptide includes positively charged polypeptides, negatively charged polypeptides, and neutral polypeptides.

[0081] The present invention also provides an α-hemolysin mutant having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and having an amino acid sequence differing from the amino acid sequence shown in SEQ ID NO:1 in the amino acid sequence containing amino acid residues at positions D13, E111, M113, N123, T125, or K147, wherein the positions of 13, 111, 113, 123, 125, and 147 refer to the amino acid residue numbers in SEQ ID NO:1.

[0082] In some embodiments, the amino acid residue differences at the D13 site include D13K; the amino acid residue differences at the E111 site include E111G, E111Y, and E111R; the amino acid residue differences at the M113 site include M113Y, M113W, M113F, M113R, M113D, and M113N; the amino acid residue differences at the N123 site include N123Y and N123W; the amino acid residue differences at the T125 site include T125Y and T125W; and the amino acid residue differences at the K147 site include K147G and K147E.

[0083] In some embodiments, the α-hemolysin mutant has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and includes one or more differences in amino acid residues at positions K8, D13, K21, D44, D45, E111, M113, N123, T125, D127, M131, T145, and K147 compared to the amino acid sequence shown in SEQ ID NO:1, wherein the positions of 8, 13, 21, 44, 45, 111, 113, 123, 125, 127, 131, 145, and 147 refer to the amino acid residue numbers in SEQ ID NO:1.

[0084] In some embodiments, the amino acid residue differences at the M113 site include M113Y, M113W, M113F, M113R, M113D, and M113N.

[0085] In some embodiments, the amino acid residue differences at the K8 site include K8E and K8Q; the amino acid residue differences at the D13 site include D13K; the amino acid residue differences at the K21 site include K21Q; the amino acid residue differences at the D44 site include D44K; the amino acid residue differences at the D45 site include D45N; the amino acid residue differences at the E111 site include E111G, E111Y, and E111R; the amino acid residue differences at the N123 site include N123Y and N123W; the amino acid residue differences at the T125 site include T125Y and T125W; the amino acid residue differences at the D127 site include D127R; the amino acid residue differences at the M131 site include M131E; the amino acid residue differences at the T145 site include T145F; and the amino acid residue differences at the K147 site include K147G and K147E.

[0086] In some specific implementations, the amino acid residue differences are selected from the group consisting of:

[0087] (1)E111Y / R / G;

[0088] (2)M113Y / W / F / R / D / N; (3)K147E / G;

[0089] (4) D13K and D44K;

[0090] (5) E111Y and K147W;

[0091] (6) M113F and T145F;

[0092] (7) M131E and K147E;

[0093] (8) K8E, M131E and K147E;

[0094] (9) D13K, D44K and D45N;

[0095] (10)E111Q, M113F and K147N;

[0096] (11)E111Q, M113F and K147F;

[0097] (12) E111F, M113F and K147N;

[0098] (13)E111G, M113F and K147T;

[0099] (14)E111S, M113F and K147G;

[0100] (15)E111S, M113F and K147S;

[0101] (16)E111S, M113F and K147T;

[0102] (17) M113F, T145F and K147N;

[0103] (18)M113Y, K131E and K147E;

[0104] (19)M113W, K131E and K147E;

[0105] (20) K8E, M113Y, K131E and K147E;

[0106] (21) K8E, M113W, K131E and K147E;

[0107] (22) E111Q, M113F, T115F and K147N;

[0108] (23) E111Q, M113F, T145F and K147N;

[0109] (24) D44K, D45N, E111Q, M113F and K147N;

[0110] (25) D13K, D44K, D45N, D127R, E111Q, M113F and K147N

[0111] (26)N123Y / W;

[0112] (27)T125Y / W;

[0113] (28) K8Q and M113Y;

[0114] (29)E111R and K147G;

[0115] (30)E111Y and K147G;

[0116] (31)E111G and M113Y;

[0117] (32)M113Y and K147G;

[0118] (33)M113D and K147G;

[0119] (34) N123Y and K147G;

[0120] (35) N123W and K147G;

[0121] (36) K8Q, K21Q and M113Y;

[0122] (37)E111G, M113Y and K147G;

[0123] The " / " indicates that the site contains any of the listed amino acid mutations.

[0124] The present invention also provides an isolated nucleic acid encoding the α-hemolysin mutant described above.

[0125] The present invention also provides a recombinant expression vector comprising the aforementioned nucleic acid.

[0126] The present invention also provides a transformant comprising the nucleic acid or the recombinant expression vector described herein.

[0127] The present invention also provides a method for preparing an α-hemolysin mutant, comprising culturing the transformant to obtain a culture product containing the α-hemolysin mutant.

[0128] In some specific embodiments, the method further includes the step of purifying the culture product to obtain the α-hemolysin mutant.

[0129] This invention also provides the application of the α-hemolysin mutant in the characterization of protein post-translational modifications, detection of peptide biomarkers, protein polypeptide sequencing, and protein mutation analysis.

[0130] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0131] The reagents and raw materials used in this invention are all commercially available.

[0132] The positive and progressive effects of this invention are as follows:

[0133] The method of this invention utilizes electrophoretic force (EPF) to capture peptides and overcomes reverse electroosmotic flow (EOF), creating a stretching effect of two opposing forces. This successfully improves the nanopore electrical signal of peptides, enhancing the discrimination of peptides in nanopore analysis and providing a new method for peptide differentiation, modification identification, and protein-peptide profiling. Furthermore, this analytical method has been successfully applied to single-molecule per-pore sequencing analysis of proteins. After single-molecule proteins are unfolded by denaturing agents such as urea, they pass through the pore slowly, resulting in long-duration, characteristic single-molecule protein sequencing signals. The new method significantly reduces the protein per-pore rate compared to previously reported levels (2 to 3 orders of magnitude), overcoming the main technical bottleneck of nanopore single-molecule protein analysis. Simultaneously, it modifies the recognition site structure within the nanopore, characteristically amplifying the signals of positively charged amino acids and aromatic amino acids. Therefore, this invention provides a truly feasible direct protein sequencing analysis method. Attached Figure Description

[0134] Figures 1a-1d show the optimized αHL nanopore sensing system for peptide analysis.

[0135] Figures 2a-2e show how M113Y-aHL nanopores distinguish a series of P1 series peptides with different post-translational modifications.

[0136] Figures 3a-3e show the P2 series peptides distinguished by M113Y-aHL nanopores, which differentiate between citrullinated and β-hydroxybutyrylated peptides.

[0137] Figures 4a-4c show the signal homogenization of different charged peptides under pH 3.8 and 1M KCl conditions.

[0138] Figures 5a-5b show the Ires% distribution of peptides with different chemical and charge compositions detected by M113Y-aHL nanopores in 2M KCl buffer at pH 3.8, as well as the P1 and P3 series peptides distinguished by M113Y-aHL nanopores based on single aromatic amino acid substitutions.

[0139] Figures 6a and 6b show the voltage dependence and typical signal characteristics of positively charged peptides in 2M KCl buffer at pH 3.8.

[0140] Figure 7 is a schematic diagram showing how electrophoretic force (EPF) drives peptides to overcome reverse electroosmotic flow (EOF) through nanopores, homogenizing peptide electrical signals and highly sensitively distinguishing peptides with different PTMs.

[0141] Figures 8a-8c show trace plots and electrical signal diagrams of E111Y-aHL measurements of different long-chain substrates at -100 mV. (Figure 8a) Lower half: Characteristic signals of long-chain substrates passing through nanopores and voltage dependence of H2B residence time. The characteristic signals of long-chain substrates passing through nanopores are circled in red in Figures 8a-8c. Long-chain substrates were added to the cis side, and all electrophysiological measurements were performed in 2M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0142] Figures 9a-9c show traces and electrical signal diagrams of E111R-aHL measurements of different long-chain substrates at -80 mV. Long-chain substrates were added to the cis side, and all electrophysiological measurements were performed in 2 M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0143] Figures 10a-10c show traces and electrical signal diagrams of M113R-aHL measurements of different long-chain substrates at -80 mV. Long-chain substrates were added to the cis side, and all electrophysiological measurements were performed in 2 M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0144] Figures 11a-11c show traces and electrical signal diagrams of M113Y-aHL measurements of different long-chain substrates at -80 mV. The long-chain substrates were added to the cis side, and all electrophysiological measurements were performed in 2 M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0145] Figures 12a-12b show traces and electrical signal diagrams of M113W-aHL measurements of different long-chain substrates at -80 mV. Long-chain substrates were added to the cis side, and all electrophysiological measurements were performed in 2 M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz. Detailed Implementation

[0146] In this invention, we utilize αHL bionanopores with β-barrel-shaped transmembrane domains to detect a range of post-translational modifications (PTMs) in native peptides derived from RNA polymerases and histones. The PTMs tested in this invention include not only common types such as phosphorylation, acetylation, and methylation, but also some lesser-known or newly discovered modifications, such as citrullination and β-hydroxybutyrylation. Most importantly, we successfully improved the nanopore electrical signal of peptides by capturing them using electrophoretic force (EPF) and overcoming reverse electroosmotic flow (EOF), creating a stretching effect of two opposing forces. In this stretching scenario of two opposing forces, adjusting the applied voltage effectively modulates the signal blocking amplitude induced by the peptide (Ires% = Ib / Io * 100%), resulting in a more uniform signal distribution and thus better differentiation between different peptides and PTMs. Furthermore, by lowering the pH to 3.8 and increasing the ionic strength to 1 mCCl, a stronger reverse EOF was obtained. The trapping EPF electrophoretic force still effectively drove peptides with different original charge compositions (neutral, positive, and negative) into the nanopores for analysis. The stronger reverse EOF facilitated further stretching of the peptides, resulting in a more uniform peptide signal and better peptide differentiation. Our results demonstrate an effective method for improving the resolution of peptide analysis in nanopores.

[0147] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0148] Example 1: Overcoming reverse electroosmotic flow (EOF) by electrophoretic force (EPF) in peptide analysis effectively homogenizes the peptide analysis signal in nanopores.

[0149] We synthesized a polypeptide derived from RNA polymerase, named polypeptide 1 (P1). Post-translational modifications of P1 in polymerase are closely associated with protein dysfunction and many diseases, including cancer. Simultaneously, we synthesized a P1 with a tyrosine acetylated position 1 (P1-acY1) for preliminary optimization of analytical conditions. We selected α-hemolysin (αHL) nanopores for polypeptide analysis. Initially, we analyzed P1 and P1-acY1 polypeptides using wild-type α-hemolysin (WT-aHL) (Figure 1a) with a buffer condition of 500 mM KCl and pH 7.5, but no signal was observed regardless of whether a positive or negative voltage was applied to the trans side of the nanopore. We hypothesized that there might be insufficient interaction between the polypeptide and the nanopore, causing the peptide to pass through the pore too quickly to be sampled. Therefore, we mutated the αHL recognition site 113 to tyrosine, replacing the original methionine (M113Y), to create a narrower recognition site and potentially increase the interaction between the polypeptide and the nanopore (Figure 1a). As expected, we observed blockage induced by the P1 and P1-acY1 peptides through the M113Y-aHL nanopore. The P1 peptide sequence contains tyrosine residues, and the potential aromatic interaction with the tyrosine residue at position 113 in the nanopore lumen may also contribute to the peptide's longer residence time within the pore and the induction of signaling.

[0150] WT-αHL exhibits slight anion selectivity. Since no charged residues are substituted within the pores of the M113Y-αHL mutant, M113Y-αHL should also exhibit slight anion selectivity (Fig. 1b). We first applied a positive voltage to the working electrode (trans) and added the peptide to the cis (cis) side to create an electroosmotic flow (EOF) from cis to trans to capture the peptide (Fig. 1c, left), where the positively charged P1 peptide experiences an electrophoretic force (EPF) in the opposite direction. Under these conditions, we observed a heterogeneous peptide signal, which reflects the blocking Ires% (I B / I OA broad Gaussian distribution (*100%) was observed (Fig. 1d, top). Furthermore, adjusting the applied voltage failed to improve the signal, thus making it impossible to distinguish between P1 and P1-acY1 peptides (Fig. 1d, top). We then applied a negative voltage to the working electrode (trans), under which the driving force became EPF and opposite to the EOF direction (Fig. 1c, middle and right). We observed a more uniform peptide signal and a narrower Ires% distribution at higher voltages (Fig. 1d, middle top). At a voltage of -100 mV, complete differentiation of the two peptides was achieved simply by using Ires% (Fig. 1d, middle top). Therefore, this demonstrates that EPF trapping peptides in the opposite direction to the EOF provides an effective method for improving peptide signals and achieving peptide differentiation. We further optimized the pH and ionic strength of the buffer and detected the electrochemical signals of P1 and P1-acY1 in 1M and 2M KCl buffers at pH 3.8, respectively. We found that the P1 series peptides exhibited the most uniform signal in 2M KCl buffer at pH 3.8, and achieved high-sensitivity single-molecule resolution for both P1 and P1-acY1 (Figure 1c, bottom).

[0151] (Figure 1a) Left: Schematic diagram of αHL nanopores embedded in a lipid bilayer, with the M113 recognition site shown as purple spheres; Middle: Top view of the cavities of wild-type αHL and M113Y-αHL nanopores, with the residue at position 113 showing a spherical shape and side chain groups, namely methionine (yellow) and tyrosine (purple), respectively; Right: Peptide sequences and side chain structures of P1 and P1-acY1. (Figure 1b) IV curves of WT-αHL and M113Y-αHL in asymmetric salt concentration (trans 0.5M / cis 2M KCl) buffer, used to determine ion selectivity at different pH (7.5 / 3.8). (Figure 1c) Schematic diagram of the charge state of the cavity of M113Y-αHL nanopores, and schematic diagrams of electrophoretic force (EPF) and electroosmotic flow (EOF) under different voltage conditions. The dark blue markings in the right figure show the enhanced positive charge distribution within the nanopore cavity at pH 3.8. Positively charged peptides were added to the cis side, and a working voltage was applied to the trans side. (Fig. 1d) Top part: Histograms of Ires% distributions of P1 and P1-acY1 peptides measured under positive and negative voltages. Gaussian fitting was applied to each distribution to reflect the uniformity of the signal induced by the peptide. The peptide was added to the cis side. All electrophysiological measurements were performed in 500 mM KCl buffer at pH 7.5, and signals were collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz. Bottom part: Histograms of Ires% distributions of P1 and P1-acY1 peptides measured under negative voltage. Gaussian fitting was applied to each distribution to reflect the uniformity of the signal induced by the peptide. The peptide was added to the cis side. Electrophysiological measurements were performed in 1 M KCl and 2 M KCl buffers at pH 3.8, and signals were collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0152] Example 2 effectively modulates the signal of peptides by changing the applied voltage and achieves effective differentiation of complex peptide modifications.

[0153] Because we discovered that applying a reverse voltage modulates the signal of the P1 peptide, we explored whether this method could analyze more types of post-translational modifications. In addition to P1-acY1, we synthesized and detected P1 peptides with lysine acetylation at position 7 (P1-K7ac), double acetylation at positions 1 and 7 (P1-acY1-K7ac), phosphorylation of tyrosine at position 1 (P1-Y1p), and monomethylation of lysine at position 7 (P1-K7me1) (Figures 2a and 2b). P1-acY1 and P1-K7ac peptides belong to isomers with different acetylation positions. We first performed a comprehensive characterization of all peptides under a series of positive and negative voltages in a buffer condition of 500 mM KCl, pH 7.5. Similar to P1 and P1-acY1, the signals of all peptides were heterogeneous under positive voltage. Under negative voltage, peptides are captured into nanopores by overcoming reverse electroosmotic flow (EOF) driven by electrophoretic force (EPF), resulting in a signal with good uniformity (Figure 2c). Except for P1-K7me1, the best distinguishing effect was observed at -100 mV. The P1-K7me1 peptide showed similar Ires% to the P1 peptide at different voltages, therefore further optimization and methodological improvements are needed to distinguish this monomethylation modification. The other five peptides were well distinguished under these experimental conditions. Notably, the isomers P1-acY1 and P1-acK7, as well as the P1-acY1-acK7 peptide with two acetylated groups, were distinguished with an accuracy approaching 100%. This indicates that the M113Y-aHL nanopore system is well-suited for detecting complex post-translational modifications of peptides by utilizing novel electrophoretic force to capture and overcome reverse electroosmotic flow.

[0154] All peptides except P1-acY1-acK7 show Ires% (I) at higher negative voltages. B / I O The *100% value also increased accordingly, while P1-acY1-acK7 showed a relatively constant Ires% (Fig. 2d), which may reflect that most peptides were stretched more and became more linear at higher voltages. However, the stretching effect observed on P1-acY1-acK7 was less pronounced, as its N-terminal amino and lysine primary amine groups were acetylated and contained no positively charged groups, making it almost neutral. Therefore, the electrophoretic force EPF it experienced was greatly reduced, resulting in a stable Ires% at different voltages. We also calculated the voltage dependence of peptide capture frequency. With increasing negative voltage, the capture frequency of all peptides decreased, which may be due to the enhanced opposite EOF at higher voltages, reducing peptide capture into the pores.

[0155] The Ires% distribution of these P1 peptides also reveals the importance of charge interactions in influencing the shape and behavior of peptides within nanopores (Fig. 2c). P1, P1-acY1, and P1-K7me1 peptides with positively charged lysine residues all elicited less homogeneous signaling. However, lysine-aminoacetylated P1-K7ac and P1-acY1-K7ac exhibited more homogeneous blockage. This suggests that interactions between lysine residues and the nanopore wall may contribute to increasing peptide residence time within the pore, but may also alter shape, thereby reducing peptide linearization. These interactions can be electrostatic or cation-π interactions. Our results indicate that, under stretching methods and scenarios such as electrophoretic forces and reverse electroosmosis, adjusting the applied voltage to modulate the size of EPF and EOF helps stretch and linearize peptides and overcome interference from unfavorable peptide-nanopore interactions.

[0156] By optimizing the analytical conditions, we found that the P1 series peptides exhibited the most uniform signal in a 2M KCl buffer at pH 3.8, and fine-tuning the applied negative voltage altered the Ires% value. Except for P1-K7me1, the best distinguishing effect was observed at -30 mV, and these peptides were efficiently distinguished in mixed systems (Figure 2e).

[0157] (Figure 2a) Sequences, modified residues, and chemical structures of different post-translational modifications of P1 series peptides, including acetylation, phosphorylation, and monomethylation. (Figure 2b) List of peptide sequences and molecular weights. (Figure 2c) Histograms of Ires% and scatter plots of residence time versus Ires% for different peptides measured at different negative voltages. (Figure 2d) Voltage dependence of peptide Ires%, capture frequency, and residence time. Error is the standard deviation of values ​​from at least three independent replicates. Peptides were added to the cis side, and all electrophysiological measurements were performed in 500 mM KCl buffer at pH 7.5, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz. (Figure 2e) Histograms of Ires% distribution and scatter plots of residence time versus Ires% for P1 series peptides measured at different negative voltages; mixed differentiation of P1 series peptides in a single recording at -30 mV. From left to right: scatter plot of residence time (ms) versus Ires% for peptides P1-Y1p, P1, P1-acY1, P1-K7ac, and P1-acY1-K7. Peptides were added to the cis side. All electrophysiological measurements were performed in buffer (100 mM citrate, 125 mM Tris, and 2 M KCl) at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0158] Example 3: Acidic pH and high salt concentration helped to further distinguish P2 peptides from citrullinated and β-hydroxybutyrylated modifications.

[0159] Previously, peptide sequences had a significant, unpredictable, and difficult-to-optimize impact on nanopore analysis results. Therefore, we further explored the use of the aforementioned analytical methods to detect peptides with different sequences, verifying the possibility of obtaining universally applicable results. Histones have important biological functions and are susceptible to various post-translational modifications. Citrulline substitution for arginine is known to lead to autoimmune and inflammatory diseases, such as rheumatoid arthritis. Citrulline and arginine differ in molecular weight by only 1 Dalton (Figure 3a), making accurate detection by mass spectrometry challenging. Furthermore, β-hydroxybutyrylation (Kbhb) of histone lysine is a newly discovered epigenetic modification closely related to ketone body metabolism (Figure 3a). Therefore, we synthesized a second peptide sequence derived from H4 histone, named P2, and P2-R5(cit) and P2-K2bhb modified with citrulline and β-hydroxybutyrylation (Figure 3b).

[0160] We tested peptide P2 under the original experimental conditions (500 mM KCl, pH 7.5). Similar to peptide P1, the signal of peptide P2 was not uniform when a positive voltage (trans) was applied. While the uniformity of the peptide signal was better when a negative voltage was applied, peptides P2-R5(cit) and P2-K2bhb could not be distinguished even under different negative voltages. This indicates that the original experimental conditions, including EPF and reverse EOF forces, could not effectively stretch the peptide sequence, affecting peptide differentiation. Therefore, we anticipate that lowering the solution pH and increasing the ionic strength of the solution could enhance reverse electroosmotic flow (EOF), potentially leading to further stretching of the peptide.

[0161] We tested these three peptides under new pH 3.8, 1M KCl solution conditions. The homogeneity of the peptide signal was further improved (Figure 3c), and changing the applied negative voltage, similar to the previous analysis of peptide P1, effectively regulated the homogeneity and Ires% value of peptide P2, achieving efficient differentiation of the three peptides at -50 mV. Notably, under the new experimental conditions, the Ires% value of peptide P2 was significantly higher than under the old solution conditions, demonstrating that peptide P2 was stretched and linearized more effectively. This also proves that a stronger reverse EOF does indeed help stretch the peptide. Analysis of peptides P2 and P1 using M113Y-αHL nanopores yielded similar biophysical properties (Figure 3d), further validating that the electrophoretic force trapping and overcoming reverse electroosmotic flow analysis mode is an efficient and feasible method for achieving signal homogenization for different peptide sequences, effectively stretching and linearizing peptide structures, and obtaining high-resolution peptide differentiation capabilities. Similarly, optimal differentiation was observed at -70 mV in 2M KCl buffer at pH 3.8, and it was able to efficiently distinguish P2 series peptides in mixed systems (Figure 3e). Including citrullinated modifications with a molecular weight difference of only 1 Da, these results demonstrate the high sensitivity of nanoporous single-molecule peptide analysis under this novel analytical modality.

[0162] (Figure 3a) Sequences of P2 series peptides, and chemical structures of modified residues and different post-translational modifications. (Figure 3b) List of peptide sequences and molecular weights. (Figure 3c) Histograms of Ires% for different peptides measured at different negative voltages and scatter plots of residence time versus Ires%. (Figure 3d) Voltage dependence of peptide Ires%, capture frequency, and residence time. Error represents the standard deviation of at least three independent replicates. Peptides were added to the cis side, and all electrophysiological measurements were performed in 1M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz. (Figure 3e) Histograms of Ires% distribution for P2 series peptides measured at different negative voltages and scatter plots of residence time versus Ires%, and mixed differentiation of P2 series peptides in a single recording at -70 mV. From left to right: scatter plots of residence time (ms) versus Ires% for P2-K2bhb, P2-R5(cit), and P2 peptides. The peptide was added to the cis side. All electrophysiological measurements were performed in a buffer solution at pH 3.8 (100 mM citric acid, 125 mM Tris, and 2 M KCl), and signals were collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0163] Example 4: Methods for peptide signal homogenization and effective stretching and linearization of peptides can be applied to the analysis of peptides with different charges.

[0164] We used voltage adjustment to alter the EPF and EOF forces to achieve peptide stretching and linearization. To further validate the universality of this method, we selected peptides with different charges for testing. It has been demonstrated that the EOF passing through the nanopore is affected by the buffer pH and ionic strength; the buffer pH modulates the charge state within the nanopore cavity. M113Y-αHL exhibits slight anion selectivity, which at low pH helps reduce the negative charge within the pore, resulting in stronger anion selectivity. A stronger reverse EOF was obtained when a negative charge was applied (Figure 4a). Simultaneously, increasing the ionic strength further increased the electroosmotic flow. This explains why we found better peptide linearization and a more uniform nanopore signal at 1M KCl and pH 3.8. Therefore, we used these analytical conditions to further analyze peptides with different charges.

[0165] For positively charged peptides, we selected P1 and P1-K7me1. Monomethylation resulted in a molecular weight difference of only 14 Daltons, and both peptides were positively charged. We compared the analytical results at 1M KCl, pH 3.8, and 500mM KCl, pH 7.5, showing that at 1M KCl, pH 3.8, with stronger reverse EOF, the homogeneity of the signals for both P1 and P1-K7me1 peptides was improved (Figure 4b). We also tested neutral peptides (P1-K7ac) and negatively charged peptides (P4), and similarly observed that the peptide signals exhibited high homogeneity at 1M KCl, pH 3.8 (Figure 4c), possibly the most homogeneous signals obtained for peptide analysis in nanopores. This indicates that stronger reverse EOF at acidic pH and high salt conditions helps stretch and linearize the peptides. To further verify the universality of the analytical method, we selected a series of peptides with different chemical and charge compositions for testing in 2M KCl buffer at pH 3.8. The results show that our method can obtain highly uniform nanopore signals when detecting additional positively charged peptide P3 and neutral peptide P7 (Fig. 5a). Peptides with single aromatic amino acid substitutions (P1-Y, P1-F, and P1-W, as well as P3-Y, P3-F, and P3-W) can also be effectively distinguished (Fig. 5b), further demonstrating the high sensitivity of the analytical method. However, for peptides that are still negatively charged at pH 3.8 (such as P5 and P6), the signal uniformity is not as good as that of positively charged peptides (Fig. 5a). This may be because the internal negatively charged residues affect the permeation process and morphology of the peptide within the pore, resulting in a broader signal distribution. These results demonstrate that under acidic and high-salt conditions, using electrophoretic force (EPF) to capture peptides and overcome reverse electroosmotic flow (EOF) is a universal and efficient method for homogenizing peptide signals and stretching and linearizing peptides with different chemical compositions and charges.

[0166] Based on the above experimental results, we conducted an in-depth analysis of the factors affecting the uniformity of peptide signals in nanopores. First, we calculated the voltage dependence of Ires%, residence time, and event frequency for P1 and P2 series peptides in different buffers to evaluate the potential mechanisms. We found that using electrophoretic forces to counteract electroosmotic trapping helps prolong the residence time of peptides in nanopores. With increasing applied potential, the residence time of some peptides (P1, P1-acY1, and P1-Y1p) increases, which may prevent them from translocating through the nanopores, but this phenomenon may be beneficial for peptide differentiation (Figure 6a). Simultaneously, we investigated the blocking signals of positively charged peptides under positive and negative potentials, finding that compared to positive voltage, the blocking induced under negative voltage (EPF counteracting EOF) was not only more uniform but also smoother and less noisy (Figure 6b). This study demonstrates that utilizing electrophoretic forces to counteract electroosmotic flow trapping of peptides can even stabilize or stretch the peptide chain, thereby improving signal uniformity. Secondly, residues with different charges within the peptide chain can lead to inconsistent peptide chain motion, causing velocity differences and structural distortion, thus reducing signal uniformity (Figure 5a). Furthermore, residues with different charges within the peptide chain and the interactions between the peptide and the nanopore (such as electrostatic, aromatic, and hydrophobic interactions) also significantly impact signal uniformity. Nanopore peptide signaling is a complex biophysical process involving the trajectory and morphology of the peptide chain within the confined space of the nanopore. Further experimental research, combined with molecular simulations, is needed to deeply investigate the interaction mechanisms between peptides and nanopores, the pore morphology and dynamics of the nanopores, thereby guiding and optimizing nanopore peptide analysis techniques.

[0167] (Figure 4a) Schematic diagram of the charge, electrophoretic force, and electroosmotic flow state within the αHL nanopore cavity under different solution conditions. (Figure 4b) Ires% distribution of positively charged P1 and P1-k7me1 peptide signals under different conditions. (Figure 4c) Ires% distribution of neutral and negatively charged peptide signals under different conditions. Peptides were added to the cis side, and signals were collected using a sampling rate of 10 kHz and a Bessel filter of 2 kHz in all electrophysiological measurements.

[0168] (Figure 5a) Ires% distribution of P1-P7 peptides at -50 mV. (Figure 5b) Top part: Comparison of Ires% distribution of P1-Y, P1-F, and P1-W peptides with single aromatic amino acid substitutions at different negative voltages; Bottom part: Comparison of Ires% distribution of P3-Y, P3-F, and P3-W peptides with single aromatic amino acid substitutions at different negative voltages. Peptides were added to the cis side, and all electrophysiological measurements were performed in 2M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz.

[0169] (Figure 6a) Top section: Voltage dependence of Ires%, capture frequency, and residence time for P1 series peptides. Error is the standard deviation of values ​​from at least three independent replicates. Peptides were added to the CIS side, and all electrophysiological measurements were performed in 2M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz. Bottom section: Voltage dependence of Ires%, capture frequency, and residence time for P2 series peptides. Error is the standard deviation of values ​​from at least three independent replicates. Peptides were added to the CIS side, and all electrophysiological measurements were performed in 2M KCl buffer at pH 3.8, with signals collected at a sampling rate of 10 kHz and a Bessel filter of 2 kHz. (Figure 6b) Typical current trajectories of the specified peptides at +50 mV and -50 mV, respectively.

[0170] Example 5 demonstrates how electrophoretic force (EPF) drives the unwinding of proteins into single polypeptide chains, overcoming reverse electroosmotic flow (EOF) through the pores to achieve long-duration single-molecule protein pores and single-molecule signal maps for protein identification.

[0171] As shown in Figures 8a to 12b, under the test conditions of pH 3.8 and 2M KCl, human histone H2B and model proteins Poly KS-2 and Poly SKW-1 were added to the cis terminus of mutant aHL nanopores such as M113Y. By applying different negative voltages to the trans terminus, electrophoretic forces drove the proteins to overcome reverse electroosmotic flow and enter the pores slowly (up to tens of seconds). This yielded very uniform single-molecule protein signals, which can serve as long-term characteristic signals for proteins.

[0172] Figures 8a-8c show trace plots and electrical signal diagrams of E111Y-aHL measurements of different long-chain substrates at -100mV. (Figure 8a) Bottom left: Characteristic signal of long-chain substrates passing through nanopores at -180mV; Bottom right: Voltage dependence of residence time of long-chain substrate H2B. This demonstrates that substrate H2B can pass through the nanopores at high voltages and achieve slow passage at -100mV, facilitating sequencing analysis. The red boxes in Figures 8a-8c highlight the characteristic signals of long-chain substrates passing through nanopores.

[0173] Example 6: Nanopore single-molecule protein sequencing analysis, which reads out specific amino acids, can be used for the rapid identification of unknown proteins.

[0174] As shown in Figures 8a to 12b, under the test conditions of pH 3.8 and 2M KCl, human histone H2B and model proteins Poly KS-2 and Poly SKW-1 were added to the cis terminus of the aHL nanopore. By applying different negative voltages to the trans terminus, electrophoretic forces drove the proteins to overcome reverse electroosmotic flow and enter the pore. While achieving a long permeation time, specific recognition sites within the aHL nanopore (such as positions 113, 111, and 147) were optimized to introduce aromatic amino acids and charged amino acids. These interact strongly with specific amino acids, such as charged and aromatic amino acids, on the polypeptide chains of test proteins like H2B, thereby enabling the reading of specific amino acids, including charged and aromatic amino acids. By sequentially reading the three amino acids from a single protein chain and comparing them with a protein database, proteins can be identified with high accuracy, thus achieving rapid identification of proteins through single-molecule nanopore sequencing.

[0175] For the three substrates we tested, all had isoelectric points greater than 10 and were positively charged in a 2M KCl buffer at pH 3.8. Human histone H2B contains multiple aromatic amino acids and charged amino acids, the model protein Poly-KS contains the positively charged amino acid lysine, and the model protein Poly-SKW-1 contains the aromatic amino acid tryptophan and the positively charged amino acid lysine. By optimizing specific recognition sites (e.g., 113, 111) within the aHL nanopore, aromatic and charged amino acids were introduced. These amino acids interacted strongly with specific amino acids, such as charged and aromatic amino acids, on the polypeptide chains of test proteins like H2B, allowing for the readout of specific amino acids. Our tests in a 2M KCl, pH 3.8 buffer solution showed that R mutations (positively charged mutations) at positions 111 and 113 within the aHL nanopore significantly enhanced the interaction with test proteins such as substrate H2B, thus generating characteristic signals (Figures 9a-9c, 10a-10c). Meanwhile, we also observed that the W and Y mutations (aromatic amino acid mutations) at position 113 produce stable signals when interacting with the substrate, facilitating the differentiation and identification of different substrate proteins based on Ires% (Fig. 11a-11c, Fig. 12a-12b). Interestingly, the Y mutation at position 111 produces a characteristic signal when the substrate passes through the pore (Fig. 8a-8c), which may be related to the position of 111 within the nanopore, making E111Y-aHL more sensitive.

[0176] Long-chain substrate sequence:

[0177] H2B (molecular weight 14729.93 Da, pI: 10.21, 132 amino acids)

[0178] Poly KS-2 (molecular weight 12492.82 Da, pI: 12.85, 113 amino acids)

[0179] Poly SKW-1 (molecular weight 13102.66 Da, p1: 11.65, 113 amino acids)

[0180] in conclusion

[0181] In this invention, we demonstrate an effective method for improving the accuracy and distinguishing sensitivity of nanopore peptide analysis by using M113Y-αHL nanopores to capture peptides via electrophoretic force (EPF) and overcome reverse electroosmotic flow (EOF) to homogenize the peptide analysis signal, stretch and linearize the peptides passing through the nanopores. This method also allows for adjustment of the peptide nanopore signal by simply adjusting the applied voltage. Furthermore, under acidic and high-salt conditions (e.g., pH 3.8 and 1M KCl), stronger reverse EOF can help further stretch the peptide to obtain a very uniform peptide signal. Using this novel method, we achieved efficient single-molecule distinction of different peptide sequences and a series of complex post-translational modifications. By testing peptides with different charges, we also demonstrated that this analytical method is applicable to the high-precision analysis of peptides with different charges, making it a universal new method for nanopore peptide analysis. This method is applicable to other different nanopore analysis systems, especially other α-hemolysin mutants, as listed in Table 1 below. Nanoporous peptide analysis technology, with its high precision and sensitivity, will greatly promote biomedical applications, such as the characterization of protein post-translational modifications and the highly sensitive detection and diagnosis of peptide biomarkers.

[0182] Table 1. α-hemolysin (αHL) mutants of this application.

[0183] Wild-type α-hemolysin (αHL) sequence

[0184] Table 2. Peptide sequence list

[0185] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for homogenizing polypeptide signals and sequencing nanopores, characterized in that, The method captures the polypeptide by using an electrophoretic force EPF, which is opposite to the electroosmotic flow (EOF), as the driving force, and determines the sequence of the polypeptide by detecting and analyzing the electrical signal generated when the polypeptide passes through the pores of the nanopore through a nanopore.

2. The method as described in claim 1, characterized in that, The nanopores are biological nanopores or solid nanopores; Preferably, the bio-nanopores include nanoporous proteins, and the solid nanopores include SiO2 nanopores and carbon nanotubes; More preferably, the nanoporous protein is selected from one or more of α-hemolysin mutant, Aerolysin, MspA, FraC and CsgG, and is even more preferably α-hemolysin mutant.

3. The method as described in claim 2, characterized in that, The α-hemolysin mutant has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and includes one or more differences in amino acid residues at positions K8, D13, K21, D44, D45, E111, M113, N123, T125, D127, M131, T145, and K147 compared to the amino acid sequence shown in SEQ ID NO:1, wherein the positions of 8, 13, 21, 44, 45, 111, 113, 123, 125, 127, 131, 145, and 147 refer to the amino acid residue numbers in SEQ ID NO:1; Preferably, the amino acid residue differences at the K8 site include K8E and K8Q; the amino acid residue differences at the D13 site include D13K; the amino acid residue differences at the K21 site include K21Q; the amino acid residue differences at the D44 site include D44K; the amino acid residue differences at the D45 site include D45N; the amino acid residue differences at the E111 site include E111G, E111Y, and E111R; and the amino acid residue differences at the M113 site include M113Y, M113W, and M1 13F, M113R, M113D, and M113N; the amino acid residue differences at the N123 site include N123Y and N123W; the amino acid residue differences at the T125 site include T125Y and T125W; the amino acid residue differences at the D127 site include D127R; the amino acid residue differences at the M131 site include M131E; the amino acid residue differences at the T145 site include T145F; and the amino acid residue differences at the K147 site include K147G and K147E. More preferably, the amino acid residue difference is selected from the group consisting of: (1)E111Y / R / G; (2)M113Y / W / F / R / D / N; (3)K147E / G; (4) D13K and D44K; (5) E111Y and K147W; (6) M113F and T145F; (7) M131E and K147E; (8) K8E, M131E and K147E; (9) D13K, D44K and D45N; (10)E111Q, M113F and K147N; (11)E111Q, M113F and K147F; (12) E111F, M113F and K147N; (13)E111G, M113F and K147T; (14)E111S, M113F and K147G; (15)E111S, M113F and K147S; (16)E111S, M113F and K147T; (17) M113F, T145F and K147N; (18)M113Y, K131E and K147E; (19)M113W, K131E and K147E; (20) K8E, M113Y, K131E and K147E; (21) K8E, M113W, K131E and K147E; (22) E111Q, M113F, T115F and K147N; (23) E111Q, M113F, T145F and K147N; (24) D44K, D45N, E111Q, M113F and K147N; (25) D13K, D44K, D45N, D127R, E111Q, M113F and K147N (26)N123Y / W; (27)T125Y / W; (28) K8Q and M113Y; (29)E111R and K147G; (30)E111Y and K147G; (31)E111G and M113Y; (32)M113Y and K147G; (33)M113D and K147G; (34) N123Y and K147G; (35) N123W and K147G; (36) K8Q, K21Q and M113Y; (37)E111G, M113Y and K147G.

4. The method according to any one of claims 1-3, characterized in that, The method adjusts the magnitudes of EPF and EOF by adjusting the applied voltage; Preferably, the voltage is a negative voltage, which is -150mV to -20mV, more preferably -150mV to -80mV, and even more preferably -120mV to -80mV, for example -100mV.

5. The method according to any one of claims 1-4, characterized in that, The buffer solution of the method has a pH of ≤7.5, preferably ≤3.8, for example 3.8; the buffer solution of the method also includes salt ions; Preferably, the buffer solution is a citrate buffer or a formic acid buffer; and / or, the salt ions are selected from potassium ions, calcium ions, lithium ions and sodium ions, preferably derived from KCl, CaCl2, LiCl and NaCl solutions; and / or, the concentration of the salt ions is 50 mM to 4 M, preferably 500 mM to 2 M, for example 1 M or 2 M.

6. The method as described in claim 5, characterized in that, The buffer solution on both sides of the nanopore has a salt ion concentration difference; Preferably, the salt ion concentration difference is 50mM to 4M, more preferably 300mM to 2M, and even more preferably 500mM to 1M.

7. The method according to any one of claims 1-6, characterized in that, The polypeptide includes natural, unmodified polypeptides and post-translational modified polypeptides; preferably, the post-translational modification is selected from one or more of acetylation, phosphorylation, ubiquitination, methylation, glycosylation, lipidation, citrullination, and β-hydroxybutyrylation; and / or, the polypeptide includes positively charged polypeptides, negatively charged polypeptides, and neutral polypeptides.

8. An α-hemolysin mutant, characterized in that, The α-hemolysin mutant has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:1, and includes one or more differences in amino acid residues at positions K8, D13, K21, D44, D45, E111, M113, N123, T125, D127, M131, T145, and K147 compared to the amino acid sequence shown in SEQ ID NO:1, wherein the positions of 8, 13, 21, 44, 45, 111, 113, 123, 125, 127, 131, 145, and 147 refer to the amino acid residue numbers in SEQ ID NO:1; The amino acid residue differences at the M113 site include M113Y, M113W, M113F, M113R, M113D, or M113N; Preferably, the amino acid residue differences at the K8 site include K8E and K8Q; the amino acid residue differences at the D13 site include D13K; the amino acid residue differences at the K21 site include K21Q; the amino acid residue differences at the D44 site include D44K; the amino acid residue differences at the D45 site include D45N; the amino acid residue differences at the E111 site include E111G, E111Y, and E111R; the amino acid residue differences at the N123 site include N123Y and N123W; the amino acid residue differences at the T125 site include T125Y and T125W; the amino acid residue differences at the D127 site include D127R; the amino acid residue differences at the M131 site include M131E; the amino acid residue differences at the T145 site include T145F; and the amino acid residue differences at the K147 site include K147G and K147E. More preferably, the amino acid residue differences are selected from the group consisting of: (1)E111Y / R / G; (2)M113W / F / D / N; (3)K147E / G; (4) D13K and D44K; (5) E111Y and K147W; (6) M113F and T145F; (7) M131E and K147E; (8) K8E, M131E and K147E; (9) D13K, D44K and D45N; (10)E111Q, M113F and K147N; (11)E111Q, M113F and K147F; (12) E111F, M113F and K147N; (13)E111G, M113F and K147T; (14)E111S, M113F and K147G; (15)E111S, M113F and K147S; (16)E111S, M113F and K147T; (17) M113F, T145F and K147N; (18)M113Y, K131E and K147E; (19)M113W, K131E and K147E; (20) K8E, M113Y, K131E and K147E; (21) K8E, M113W, K131E and K147E; (22) E111Q, M113F, T115F and K147N; (23) E111Q, M113F, T145F and K147N; (24) D44K, D45N, E111Q, M113F and K147N; (25)D13K, D44K, D45N, D127R, E111Q, M113F and K147N (26)N123Y / W; (27)T125Y / W; (28) K8Q and M113Y; (29)E111R and K147G; (30)E111Y and K147G; (31)E111G and M113Y; (32)M113Y and K147G; (33)M113D and K147G; (34) N123Y and K147G; (35) N123W and K147G; (36) K8Q, K21Q and M113Y; (37)E111G, M113Y and K147G.

9. An isolated nucleic acid encoding the α-hemolysin mutant as described in claim 8.

10. A recombinant expression vector comprising the nucleic acid as described in claim 9.

11. A transformant comprising the nucleic acid as described in claim 9, or the recombinant expression vector as described in claim 10.

12. A method for preparing an α-hemolysin mutant, comprising culturing the transformant as described in claim 11 to obtain a culture product containing the α-hemolysin mutant; Preferably, the method further includes the step of purifying the culture product to obtain the α-hemolysin mutant.

13. The application of the α-hemolysin mutant as described in claim 8 in the characterization of protein post-translational modifications, detection of polypeptide biomarkers, protein polypeptide profiling and protein mutation analysis.

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