A method for polypeptide component analysis based on copper ion modified MspA nanopore

Abstract

The application belongs to the field of nanopore detection, and particularly relates to a polypeptide component analysis method based on a copper ion modified MspA nanopore. The application provides a polypeptide component analysis method based on an M2MspA-N91H nanopore, which is characterized by comprising the following steps: S1, uniformly mixing an exopeptidase and a polypeptide to be detected and reacting to obtain a hydrolysis product; S2, adding the hydrolysis product into a nanopore system, the nanopore system comprising: an M2MspA-N91H nanopore, an insulating film, a first medium, and a second medium, wherein the M2MspA-N91H nanopore is embedded in the insulating film, the M2MspA-N91H nanopore provides a channel connecting the first medium and the second medium, and the hydrolysis product is added to the first medium; S3, applying a driving force between the first medium and the second medium, free amino acids in the hydrolysis product passing through the M2MspA-N91H nanopore and generating an electrical signal; and S4, performing data analysis on the electrical signal to obtain amino acid type information of the free amino acids.

Description

[0001] This application claims priority to Chinese invention patent application [CN2021110067648], filed on August 30, 2021, entitled "Method for Detecting Multiple Amino Acids Based on Nanopores and Method for Analyzing Peptide Composition", which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention belongs to the field of nanopore detection, specifically relating to a method for peptide component analysis based on copper ion-modified MspA nanopores. Background Technology

[0003] Nanopore sensing is a single-molecule sensing technology with a detection principle similar to that of a Kurt counter. This technology features real-time and direct monitoring at the single-molecule level and generally does not require labeling or modification of the analyte. These advantages make nanopores an emerging technology for biosensing and biodetection.

[0004] The low signal-to-noise ratio and unpredictable conformational changes during the translocation of amino acids through nanopores pose a challenge for the direct differentiation and detection of amino acids using nanopores. Current technologies cannot directly detect and effectively differentiate 20 amino acids, often requiring chemical modification of the nanopores or derivatization of the amino acids. Summary of the Invention

[0005] In view of this, the present invention provides a method for peptide component analysis based on M2MspA-N91H nanopores, characterized by comprising the following steps: S1 involves mixing the exopeptidase with the target polypeptide and reacting them to obtain the hydrolysis product. S2 adds the hydrolysis product to the nanoporous system, the nanoporous system comprising: M2MspA-N91H nanopores, an insulating film, a first medium, and a second medium, wherein the M2MspA-N91H nanopores are embedded in the insulating film, the M2MspA-N91H nanopores provide channels connecting the first medium and the second medium, and the hydrolysis product is added to the first medium; S3 applies a driving force between the first medium and the second medium, and the free amino acids in the hydrolysis product pass through the M2MspA-N91H nanopores and generate electrical signals. S4 performs data analysis on the electrical signal to obtain information on the amino acid types of the free amino acids.

[0006] In one embodiment, the free amino acids include natural amino acids and non-natural amino acids.

[0007] In one embodiment, the natural amino acid includes one or more of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline.

[0008] In one embodiment, the non-natural amino acid includes one or more of citrulline, L-valine, and amaurine.

[0009] In one embodiment, the exopeptidase includes aminopeptidase and / or carboxypeptidase.

[0010] In one embodiment, the M2MspA-N91H nanopores are bound to copper ions before the hydrolysis products are added to the nanopore system.

[0011] In one embodiment, the pH values ​​of the first medium and the second medium include 6.5-7.5.

[0012] In one embodiment, the final concentration of the polypeptide solution to be detected is greater than 10 μM.

[0013] In one implementation, the data analysis step in S4 further includes: i) Establish a standard signal database; ii) Set signal filtering conditions; iii) Eliminate part of the electrical signal to obtain the retained electrical signal; iv) Determine the type of amino acid corresponding to the retained electrical signal by using the blocking rate of the standard signal in the standard signal database; v) Obtain the amino acid type information of the free amino acid.

[0014] In one implementation, the signal filtering condition is to exclude electrical signals with an SD value greater than an SD threshold, wherein the SD value of the electrical signal is calculated according to the following formula: In the formula, x i Let x be the current value at each acquisition point of the signal, x be the average value of the signal current at each acquisition point, and n be the number of signal acquisition points.

[0015] In one embodiment, the method further includes S5 comparing the amino acid type information of the free amino acid with the expected sequence information of the polypeptide to be detected, to determine whether there is a difference between the actual sequence of the polypeptide to be detected and the expected sequence of the polypeptide to be detected; or S5 compares the amino acid type information of the free amino acid with the information of the exopeptidase and the polypeptide sequence information in the database to predict the actual sequence of the polypeptide to be detected.

[0016] Beneficial effects

[0017] Existing nanopore technologies often only detect amino acids with specific functional groups. However, experiments in this invention demonstrate that the M2MspA-N91H nanopore (derived from porin A of Mycobacterium smegmatis) is versatile in amino acid detection, robustly and sensitively detecting and distinguishing 20 common and non-natural amino acids at the single-molecule level. Furthermore, without the need for additional enzyme introduction or amino acid modification (e.g., adding luminescent labels) into the M2MspA-N91H nanopore system, all 20 common and non-natural amino acids can block the M2MspA-N91H nanopore and elicit corresponding blocking current signals. By analyzing these different current signals, direct detection and differentiation of multiple amino acids can be achieved.

[0018] Based on this, and considering the hydrolytic properties of exopeptidases (such as carboxypeptidases), this invention provides a peptide component analysis method based on M2MspA-N91H nanopores. By analyzing the blocking rate of standard signals in an established standard signal database, the type of amino acid corresponding to the detected free amino acid signal can be determined, thus achieving component analysis of the peptide to be detected to a certain extent. The peptide component analysis method provided by this invention is applicable to various scenarios such as the expression of tumor neoantigens, the assessment of the quality of synthesized peptide drugs, and the prediction of unknown peptide sequences. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0020] Figure 1 The structure and electrophysiological properties of the M2MspA-N91H nanopores are shown in the figure.

[0021] Figure 2 The single-pore current signal of M2MspA-N91H nanopores with different concentrations of copper ions added (1MKCl, pH6);

[0022] Figure 3 The current signal diagram for amino acid detection in M2MspA-N91H nanopores at a concentration of 3M KCl;

[0023] Figure 4 A scatter plot of amino acid volume versus current signal blocking rate (the letters are abbreviations for amino acids);

[0024] Figure 5 This is a graph showing the blocking rate vs. blocking time of amino acid current signals (the letters are abbreviations for amino acids).

[0025] Figure 6 Experimental graphs for extracting single amino acid signals using Clampfit (A: Clampfit single-channel statistical results; B: AmpSD value of the signal; C: Single-step signal in the scatter plot based on SD value (from top to bottom, all signals from the blank group, asp experimental group, and asp+glu experimental group for 2 minutes in the same experiment are counted); D: Source of -5pA background signal; E: Source of -12pA background signal).

[0026] Figure 7 The current signals measured after adding carboxypeptidase hydrolysates to the nanopore system are shown in the following graphs: (A is the current signal obtained after adding 10U of carboxypeptidase to the nanopore system after hydrolyzing 10mM EF peptide for 20 minutes under the conditions of 0.5MKCl, 10mM MOPS, and pH 7.5; B is a scatter plot of the blocking rate and pore passage time of all current signals obtained in the experiment involved in A; C is the detection of hydrolysates of three peptides (P1, P2, P3) in the nanopore system).

[0027] Figure 8 Experimental results for detecting non-natural amino acids using M2MspA-N91H nanopores (A shows the statistical results of three non-natural amino acids; B shows the scatter plot of current blocking signals within 1 minute in the blank group and after the addition of amaurine in the same experiment).

[0028] Figure 9 The current trajectory diagram of M2MspA-N91H nanopores after the addition of copper ions under three pH conditions;

[0029] Figure 10 This is a schematic diagram of the nanopore detection of polypeptide hydrolysis products according to the present invention;

[0030] Figure 11 This is a flowchart illustrating the process of establishing a standard signal database in an embodiment of the present invention;

[0031] Figure 12 This is a flowchart of data analysis in an embodiment of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0034] As used in this specification, the term "about" typically means + / - 5% of the value, more typically + / - 4%, more typically + / - 3%, more typically + / - 2%, even more typically + / - 1%, even more typically + / - 0.5% of the value.

[0035] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible subranges and independent numerical values ​​within those ranges. For example, range The description should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0036] Nanoporous systems

[0037] The "nanopore system" comprises pores with nanoscale dimensions (referred to as "nanopores"), an insulating film, a first dielectric, and a second dielectric. In one embodiment of the present invention, the nanoscale pores are M2MspA-N91H nanopores. These nanoscale pores allow the analyte to translocate from one side of the insulating film to the other. Furthermore, those skilled in the art can modify the aforementioned M2MspA-N91H nanopores (e.g., any mutation, truncation, fusion, chemical modification, etc.) to obtain corresponding variants, and the modification methods are well known in the art.

[0038] In one embodiment of the present invention, the nanoscale pores are embedded in the insulating film, and the insulating film (which can also be understood as a composite of the nanoscale pores and the insulating film) separates the first medium from the second medium. The channels of the nanoscale pores provide a pathway connecting the first medium and the second medium. After a driving force is applied between the first medium and the second medium, the analyte located in the first medium interacts with the M2MspA-N91H nanopores to form a current (i.e., an electrical signal). In the present invention, the "first medium" refers to the medium in which the analyte is located when it is added to the nanopore system; the "second medium" refers to the other side of the "first medium" among the two media separated by the insulating film. In the present invention, the driving force refers to the force that drives the analyte to interact with the nanopores through potential, electroosmotic flow, concentration gradient, etc.

[0039] The first medium and the second medium may be the same or different, and both may include a conductive liquid. The conductive liquid is an aqueous solution of an alkali metal halide, specifically sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl), or sodium bromide (NaBr). In one embodiment of the invention, the concentrations of the conductive liquid in the first medium and the second medium are different; in other words, there is a difference in the concentration of the conductive liquid in the first medium and the second medium, resulting in a difference in osmotic pressure across the insulating membrane. The first medium and / or the second medium may also include a buffer solution, such as HEPES or MOPS. The concentration range of the first medium and / or the second medium may be 0.3M-3M. The pH range of the first medium and / or the second medium is 6-9 (preferably 6.5-7.5).

[0040] An insulating membrane is a membrane capable of carrying nanopores and blocking ionic currents passing through non-nanopores. The insulating membrane may include phospholipid membranes and / or polymer membranes. Exemplary phospholipid membranes include DPHPC, DOPC, and E. coli lipid, and exemplary polymer membranes include triblock copolymer polymer membranes.

[0041] In one specific embodiment of the present invention, the nanoporous system includes two electrolyte chambers separated by an insulating membrane to form a trans (-trans) chamber and a cis (-cis) chamber. The pores of the M2MspA-N91H channel are embedded in the insulating membrane, and the insulating membrane has only M2MspA-N91H channels to connect the two electrolyte chambers. When a potential is applied to the two electrolyte chambers, electrolyte ions in the solution in the electrolyte chambers move by electrophoresis and pass through the M2MspA-N91H channels.

[0042] Test object

[0043] The analyte is a charged substance. It is charged if it carries a net charge. The analyte can be negatively or positively charged. It is negatively charged if it carries a net negative charge, and positively charged if it carries a net positive charge. A suitable analyte is preferably an amino acid.

[0044] In one embodiment of the present invention, the amino acid may be a natural amino acid. Natural amino acids are naturally occurring amino acids and their post-translational modifications, such as 20 common amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine), etc.

[0045] In another embodiment of the present invention, the analyte may be a non-natural amino acid. "Non-natural amino acid" refers to an amino acid that is not a protein amino acid or a variant thereof with post-translational modifications (i.e., not one of the 20 common amino acids, or pyrrolidone, or selenocysteine, or a variant thereof with post-translational modifications). In a specific embodiment of the present invention, the non-natural amino acid is citrulline, L-valine, or amaurine.

[0046] The interaction between the nanopore and the analyte

[0047] The analyte can contact either side of the nanopore on the insulating membrane. Alternatively, the analyte can contact either side of the insulating membrane such that it passes through the channel of the nanopore to reach the other side of the insulating membrane. In this case, the analyte interacts with the nanopore as it passes through the channel of the pore into the insulating membrane. Or, the analyte can contact a side of the insulating membrane that allows it to interact with the nanopore, causing it to separate from the nanopore and remain on the same side of the insulating membrane. The analyte can interact with the nanopore in any manner and at any location. The analyte can also impact the nanopore, interacting with it and causing it to separate from the nanopore and remain on the same side of the insulating membrane.

[0048] During the interaction between the analyte and the nanopore, the analyte influences the current flowing through the nanopore in a analyte-specific manner; that is, the current flowing through the nanopore is characteristic of a particular analyte. Control experiments can be performed to determine the effect of a specific analyte on the current flowing through the nanopore, thereby identifying the specific analyte in the sample or determining its presence in the sample. More specifically, the presence or concentration of the analyte can be identified by comparing the current pattern obtained by detecting the analyte with a known current pattern obtained under the same conditions using a known analyte.

[0049] The nanopore system of the present invention may also include one or more measuring devices for measuring the current flowing through the nanopore, such as patch clamp amplifiers or data acquisition devices.

[0050] A method for peptide composition analysis based on M2MspA-N91H nanopores

[0051] This invention also provides a method for peptide component analysis based on M2MspA-N91H nanopores, characterized by comprising the following steps: S1 involves mixing the exopeptidase with the target polypeptide and reacting them to obtain the hydrolysis product. S2 adds the hydrolysis product to the nanoporous system, the nanoporous system comprising: M2MspA-N91H nanopores, an insulating film, a first medium, and a second medium, wherein the M2MspA-N91H nanopores are embedded in the insulating film, the M2MspA-N91H nanopores provide channels connecting the first medium and the second medium, and the hydrolysis product is added to the first medium; S3 applies a driving force between the first medium and the second medium, and the free amino acids in the hydrolysis product pass through the M2MspA-N91H nanopores and generate electrical signals. S4 performs data analysis on the electrical signal to obtain information on the amino acid types of the free amino acids.

[0052] A "polypeptide" is a peptide or protein containing two or more amino acids linked by peptide bonds. Polypeptides can contain natural, modified, or synthetic amino acids. Polypeptides can also be naturally modified (e.g., through post-translational processing) or chemically modified (e.g., amidation, acylation, cross-linking, etc.). In this invention, the sequence information of the polypeptide can be known or unknown.

[0053] "Exopeptidase" refers to a hydrolytic enzyme that removes the terminal amino acids of a polypeptide by cleaving peptide bonds. Terminal amino acids are approximately 10 amino acids located at the N-terminus or C-terminus of the polypeptide. Therefore, "exopeptidases" are divided into aminopeptidases that hydrolyze amino acids one by one from the N-terminus of the polypeptide and carboxypeptidases that hydrolyze amino acids one by one from the C-terminus of the polypeptide. Exemplary aminopeptidases include cystein-6-aminopeptidase, prolyl-6-aminopeptidase, and arginine-6-aminopeptidase. Exemplary carboxypeptidases include carboxypeptidase A, B, C, and Y. In this invention, the exopeptidase can be a carboxypeptidase and / or an aminopeptidase.

[0054] Under suitable conditions, the exopeptidase can completely or partially hydrolyze the polypeptide to be detected. For example, such as Figure 10 As shown, carboxypeptidase A1 can cleave C-terminal amino acids except for lysine (K), arginine (R), and proline (P) (i.e., the hydrolysis stops at R, K, and P). In other words, if the polypeptide to be tested does not contain any of these three amino acids, then the polypeptide will be completely hydrolyzed by carboxypeptidase A, and the amino acid composition of the free amino acids in the resulting hydrolysis products will be the amino acid composition of the polypeptide to be tested. If the polypeptide to be tested contains one or more of these three amino acids, then the polypeptide will be partially hydrolyzed by carboxypeptidase A (hydrolysis stops at the stop point), and the amino acid composition of the free amino acids in the resulting hydrolysis products will be the amino acid composition of the polypeptide to be tested before the stop point.

[0055] When the sequence of the polypeptide to be detected is known, the amino acid types of the free amino acids in the obtained hydrolysis product can be compared with the expected sequence information of the polypeptide to be detected to determine whether there is a difference between the actual sequence of the polypeptide to be detected and the expected sequence of the polypeptide to be detected (for example, some amino acids that should appear in the hydrolysis product do not appear, while some amino acids that should not appear in the hydrolysis product do appear). This is applicable to application scenarios such as the expression of tumor neoantigens and the judgment of the quality of polypeptide drug synthesis.

[0056] When the sequence of the polypeptide to be detected is unknown, the sequence of the polypeptide to be detected can be predicted or the possible range of the polypeptide to be detected can be narrowed to a certain extent by comparing the amino acid types of the free amino acids in the obtained hydrolysis products with the information of the exopeptidase used (e.g., carboxypeptidase or aminopeptidase, whether it has a "hydrolysis stop point") in the database through machine learning or other methods.

[0057] Example 1

[0058] Materials and Methods

[0059] Amino acid detection:

[0060] Single-channel experiments were performed using a classic vertical lipid bilayer apparatus (Warner Instruments), consisting of two chambers (cis-cis and trans-trans) connected by a 150 μm diameter pore. Two Ag-AgCl electrodes were used to apply the transmembrane voltage and measure the transmembrane ion current. The cis chamber was grounded. The buffer conditions for amino acid detection were as follows: 1) Buffer solution: 0.3M-3M KCl and NaCl salt solutions (preferably 1M KCl aqueous solution), 10 mg M OPS; 2) Copper ion concentration range: 1 nM to 200 μM; 3) pH range: 6-9 (preferably 6.5-7.5).

[0061] As the buffer salt concentration or pH further decreases, the detection efficiency and discrimination of amino acids may decline. All experimental temperatures can be set between 15℃ and 60℃ (preferably 22.0℃ ± 0.5℃). A planar lipid bilayer is formed by coating the well connecting the two chambers with a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (AvantiPolarLipids) film dissolved in decane (40 mg / ml). Protein solution is then added to the cis chamber; after the protein embeds into the phospholipid membrane to form a single channel, copper chloride is added to the trans chamber, and amino acids are added to the cis chamber. After gently mixing the solution, the current signal is recorded.

[0062] Data processing:

[0063] Single-channel current recordings were amplified using an Axopatch200B amplifier (MolecularDevices) and filtered at 2kHz using a built-in four-hole low-pass Bessel filter. The data was digitized by a Digidata1550B converter (MolecularDevices) at a sampling rate of 100kHz. The sampled current signal file was extracted to obtain amino acid signal data. Single amino acid signals can be identified and defined using data such as signal blocking rate, blocking time, and SD.

[0064] Background noise processing:

[0065] For the current value distribution of a single amino acid signal, its SD value is calculated to obtain the deviation of the current value of each sampling point of the signal from the overall average value, thereby distinguishing the single amino acid signal from the background signal and the superimposed signal of multiple amino acids.

[0066] Methods for analyzing peptide composition:

[0067] The buffer conditions for the experiment are as described above. The experimental temperature can be set to 15℃-60℃ (preferably 25℃). Outside the nanopore system, an exopeptidase (e.g., carboxypeptidase) and a polypeptide are mixed at a certain concentration ratio, such as 10U carboxypeptidase, 0.5M KCl, 10mM MPOPS, pH 7.5, and 10mM polypeptide (…). Figure 7 A) After reacting at 25℃ for 20 minutes, the sample was added to a nanoporous sample cell for detection. The peptide composition was determined based on the type and quantity of amino acid signals observed.

[0068] Example 2: Detection of 20 natural amino acids using M2MspA-N91H nanopores

[0069] Modifying the structure and properties of nanopores with copper ions:

[0070] like Figure 1 As shown, the M2MspA-N91H nanopores have an octamer structure, with the eight amino acid residues at the narrowest point of the pores oriented towards the central axis of the M2MspA-N91H nanopores. Cu 2+ Add it to the inverting chamber and observe the current trajectory, Cu 2+ It can significantly reduce current fluctuations. Figure 1 The decrease in current level is related to Cu. 2+ The concentration of the additive is related to ( Figure 2 The copper ion concentration can range from 1 nM to 200 μM.

[0071] Detection of 20 natural amino acids:

[0072] In this embodiment, 20 natural amino acids were detected using M2MspA-N91H nanopores. The amino acid sequence of the MspA monomer (MspA is assembled from eight identical monomers to form a channel) is: GLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEGTLELGYQIGFPWSLGVGINFSYTTPNILINHGNITAPPFGLNSVITPNLFPGVSISARLGNGPGIQEVATFSVRVSGAKGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDSVTTYGEPWNMN (SEQ ID NO:1)

[0073]

[0074] When the target analyte passes through Cu 2+ When M2MspA-N91H is chelated, a specific current signal is generated, such as... Figure 3 As shown in the figure. Current trajectory diagrams after adding copper ions under three different pH conditions are shown in the figure. Figure 9 As shown in the figure, the results indicate that copper ion binding is unstable at pH 7.9, while it is stable at pH 7.5 and 6.5. Given that a higher solution pH favors more amino acids carrying a negative charge, thus improving detection efficiency, this embodiment chose to add MOPS buffer salt to the solution and stabilize the solution pH to around 7.5 to obtain better detection results.

[0075] From the scatter plot of amino acid volume versus current signal blocking rate ( Figure 4 It can be seen that there is a positive correlation between the blocking rate caused by amino acids and its hydrophobic volume.

[0076] From the diagram of amino acid current signal blocking rate and blocking time ( Figure 5 The blocking rates caused by the 20 natural amino acids were as follows: glycine (G): 0.1207±0.0006; alanine (A): 0.1471±0.0025; valine (V): 0.1904±0.0012; leucine (L): 0.1995±0.0003; isoleucine (I): 0.2072±0.0003; phenylalanine (F): 0.2201±0.0007; tryptophan (W): 0.2274±0.0002; tyrosine (Y): 0.2127±0.0040; aspartic acid (D): 0.2140±0.0028; histidine (H): 0. 2465±0.0009; Asparagine (N): 0.1651±0.0002; Glutamic acid (E): 0.2452±0.0016; Lysine (K): 0.1687±0.0031; Glutamine (Q): 0.1882±0.0009; Methionine (M): 0.1977±0.0005; Arginine (R): 0.1714±0.0022; Serine (S): 0.1313±0.0002; Threonine (T): 0.1610±0.0004; Cysteine ​​(C): 0.1862±0.0023; Proline (P): 0.2183±0.0010. Based on the blocking rate and standard deviation of amino acids, the signals of 15 amino acids, namely G, S, A, T, N, R, V, Q, M, L, I, D, F, W, and E, are non-overlapping and can be distinguished from each other.

[0077] Background noise processing:

[0078] The aforementioned method for directly detecting 20 amino acids may introduce background noise that interferes with subsequent analysis. Therefore, the researchers of this invention calculated the SD value of the current value distribution of a single amino acid signal using the following formula, obtaining the deviation of the current value at each acquisition point from the overall average value. This allows for the differentiation between single amino acid signals and background signals or superimposed signals from multiple amino acids.

[0079]

[0080] In the formula, x i Let x be the current value at each acquisition point of the signal, x be the average value of the signal current at each acquisition point, and n be the number of signal acquisition points.

[0081] like Figure 6 As shown in Figure C, the top, middle, and bottom plots are scatter plots of the current blocking signals within 2 minutes after adding aspartic acid (Asp), and adding a mixture of aspartic acid and glutamate (Asp+Glu), respectively, in the same experiment. An SD threshold can be set to filter the signal; for example, the SD threshold can be set to 2. Signals with an SD value less than 2 (blue) are considered valid signals; signals with an SD value greater than 2 are considered background signals or superimposed signals generated by the simultaneous passage of multiple amino acids, and therefore can be filtered out. After filtering, looking only at the blue signals, the newly generated signal peaks after adding aspartic acid and glutamate can be clearly seen. Using this background noise processing method, amino acid signals can be identified and statistically analyzed more accurately.

[0082] To better illustrate the processing steps, the researchers used Clampfit software to extract single amino acid signals. First, by fitting the SD value, background and double-step signals were filtered out, retaining only the single amino acid signals. For example... Figure 6 As shown in Figure A, in the Clampfit single-channel statistical results (only level 0 and level 1 are set), the "AmpS.D." value represents the signal fitting effect. The specific SD values ​​corresponding to the signals are as follows: Figure 6 As shown in Figure B, among the six signals shown, it can be seen that the SD value of the double-step signal is generally greater than 4. A single-step signal refers to an electrical signal whose current density distribution curve exhibits a single-peak distribution, with no current distribution between 0 and 60% of the peak value. A double-step signal, on the other hand, refers to an electrical signal whose current density distribution curve exhibits a double-peak distribution.

[0083] Setting the SD threshold in the range of 0-2 can filter most multi-step signals. However, background signal interference still exists near -5pA and -12pA. Signal sources near -5pA include... Figure 6 As shown in D, because its corresponding level 0 deviates too much from the baseline, it can be filtered out later. Signals near -12pA ( Figure 6E) is a normal background signal with a short blocking time. The background signal only interferes with the signals of nearby gly, ser, and ala amino acids, and the signals of these three amino acids are distinguishable.

[0084] Based on this, a standard signal database can be established using known detection data. The specific process is as follows: Figure 11 As shown: First, free amino acids of the standard are detected using M2MspA-N91H nanopores to obtain electrical signal data. Second, filtering conditions are set (e.g., setting the SD threshold of the electrical signal to 2). Third, according to the filtering conditions, some electrical signals are excluded to obtain the retained electrical signals. Fourth, it is determined whether the retained electrical signals are single-step signals: if the retained electrical signals are single-step signals, they are included in the standard signal database and regarded as the standard signal for that amino acid; if the retained electrical signals are not single-step signals (e.g., double-step signals), the filtering conditions are reset (e.g., increasing the SD threshold), and the retained electrical signals are obtained again, and then it is determined again whether the newly obtained retained electrical signals are single-step signals. Finally, a standard signal database is established using the single-step electrical signals.

[0085] Example 3: Detection of three non-natural amino acids using M2MspA-N91H nanopores

[0086] Based on the above method, this embodiment further detected three representative non-natural amino acids: citrulline, L-valine, and amaurine. Figure 8 As shown in Figure A, the blocking rates caused by these three non-natural amino acids are: citrulline: 0.1912±0.0011; L-valine: 0.1806±0.0017; and amaminine: 0.2333±0.0029. Based on the amino acid blocking rates and their standard deviations, the signals of the three non-natural amino acids do not overlap and can be distinguished from each other.

[0087] like Figure 8 As shown in Figure B, the top and bottom graphs are scatter plots of the current blocking signal within 1 minute in the same experiment, for the blank group and after the addition of anamenine, respectively. Signals with an SD value less than 2 are considered valid signals. After filtering, a new signal peak can be clearly seen after the addition of anamenine.

[0088] Peptides can serve as substrates in enzymatic reactions. The above-mentioned method for detecting amino acids can be used to continuously monitor enzymatic reactions: by detecting the types of free amino acids in the reaction system, it is possible to determine whether an enzymatic reaction has occurred and the progress of the enzymatic cascade reaction.

[0089] Example 4: A method for peptide composition analysis based on M2MspA-N91H nanopores:

[0090] Immediately after the M2MspA-N91H nanoporous protein was inserted, the voltage was adjusted to +50 mV and recorded for 10 minutes. Next, 1 μL of 20 mM copper chloride solution was added to the inverted side of the sample well, and the recording was repeated for 10 minutes. Simultaneously, 8 μL of a 2 mM peptide solution was placed in a PCR tube and incubated at 37°C for 5 minutes. Then, 2 μL of carboxypeptidase A1 solution was added, mixed thoroughly, and reacted at 37°C for 5 minutes. After the reaction was complete, all the reaction product was added to the cis-side of the sample well, mixed thoroughly, and recorded at +50 mV for 2 hours. The detection principle is as follows. Figure 10 As shown, carboxypeptidase A1 (a type of exopeptidase) can hydrolyze amino acids one by one from the C-terminus of a polypeptide. The free amino acid products obtained from hydrolysis are captured by nanopores and thus detected. The polypeptide composition can be determined based on the type and quantity of amino acid signals that appear.

[0091] Since 1 U of exopeptidase can cleave the terminal amino acid of 1 μmol of a polypeptide within 1 min, the final concentration of the polypeptide solution can be calculated using the following formula (assuming the polypeptide can be completely hydrolyzed):

[0092]

[0093] In the formula, a is the length of the polypeptide, b is the amount of substance (μmol), d is the exopeptidase activity (U), and c is the reaction time (min).

[0094] In nanoporous systems, the detection effect is better when the concentration of free amino acids is 1 μM. If the final concentration of free amino acids is lower than 1 μM, the signal of free amino acids may be reduced, thus affecting the detection effect. Considering that the hydrolysis products (reaction products) may be diluted by at least 10 times after being added to the M2MspA-N91H nanoporous system, based on the above formula, the final concentration of the peptide solution in the reaction system should be greater than 10 times 1 μM (i.e., 10 μM).

[0095] The terminal amino acids of a peptide are hydrolyzed using an exopeptidase to obtain hydrolysis products (reaction products). These reaction products are then added to an M2MspA-N91H nanopore system under a driving force. The free amino acids in the reaction products pass through the M2MspA-N91H nanopores, generating electrical signals, and the corresponding electrical signal data are obtained. The specific data analysis process is as follows: Figure 12As shown: First, the reaction products were detected using M2MspA-N91H nanopores to obtain electrical signal data. Second, a standard signal database was established (as described above). Third, filtering conditions (SD threshold of the electrical signals) were set. Fourth, based on the filtering conditions, some electrical signals were excluded to obtain the retained electrical signals. Fifth, the amino acid types corresponding to the retained electrical signals were determined by the blocking rate of the standard signals in the standard signal database. Sixth, the amino acid type information of the free amino acids was obtained.

[0096] Figure 7 The analysis shows the composition of four different peptides based on the peptide composition analysis methods and data analysis methods described above. Figure 7 A is the current signal obtained by adding the reaction solution to the nanopore system after hydrolyzing 10 mMEF peptide with 10 U carboxypeptidase for 20 minutes under the conditions of 0.5 MKCl, 10 mM MOPS, and pH 7.5. Figure 7 B is Figure 7 A is a scatter plot of the blocking rate and via time of all current signals obtained in the experiment. It can be observed that the signal (red) with SD value between 1 and 2 has 3 distribution peaks. The signal with a blocking rate of around 0.4 is the signal of EF peptide. The two peaks on the left are closest to the blocking rates of glutamic acid (E) and phenylalanine (F), so they are identified as the signals of amino acids E and F.

[0097] Figure 7 C shows the detection of three peptides (P1:APRLR) in the M2MspA-N91H nanopore. FYSL ;P2:DRVYIHP FHL ;P3:RPVKVYPNGAEDESAEAFP LEF The underlined parts indicate the experimental results of hydrolysis products (where the amino acids at the C-terminus of the polypeptide can be hydrolyzed into free amino acids by carboxypeptidase A1). The above analytical results demonstrate that the polypeptide composition analysis method provided by this invention can analyze the amino acid composition at the terminal end of polypeptides.

[0098] The polypeptide component analysis method provided by this invention can be applied to various scenarios, such as the identification of known polypeptides and the prediction of unknown polypeptides.

[0099] Tumor neoantigens, which are antigens not expressed in normal tissues but only in tumor tissues, are ideal targets for immunotherapy. Each patient has a unique neoantigen profile, and the aforementioned peptide composition analysis method can be used to screen for tumor neoantigen peptides: First, using a highly specific extraction method, known tumor cell surface antigen peptides are extracted and purified. The purified mixture theoretically contains only a small amount of known antigen peptides. Second, the purified mixture is analyzed using the aforementioned peptide composition analysis method. Finally, by comparing the types of amino acids detected in the test results with those of the known antigen peptides, it can be determined whether certain amino acids are produced or lacking, thereby determining whether tumor neoantigens may exist on the surface of tumor cells.

[0100] Currently, some peptide drugs have been developed and applied clinically. However, due to limitations in the synthesis process, the product quality and purity of these peptide drugs often fail to meet requirements. The aforementioned peptide composition analysis method can be used to detect the purity of peptide drugs: by comparing the amino acid composition results of the peptide drug under test with the expected types of amino acids synthesized, the synthesis quality of the peptide drug can be assessed to a certain extent.

[0101] Furthermore, the polypeptide composition analysis method provided by this invention can also be combined with machine learning to predict unknown polypeptides: the unknown polypeptide to be detected is hydrolyzed with an exonuclease and the free amino acids are detected; by using machine learning methods, combining the type of exonuclease, hydrolysis characteristics and amino acid composition detection results, the sequence of the unknown polypeptide can be predicted to a certain extent, and at the same time, the range of unknown polypeptides can be narrowed down.

[0102] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for peptide component analysis based on M2MspA-N91H nanopores, characterized in that, Includes the following steps: S1. The exopeptidase and the polypeptide to be detected are mixed evenly and reacted to obtain the hydrolysis product; the exopeptidase includes aminopeptidase and / or carboxypeptidase; the final concentration of the polypeptide to be detected is greater than 10 μM. S2. The hydrolysis product is added to a nanoporous system comprising: M2MspA-N91H nanopores, an insulating film, a first medium, and a second medium, wherein the M2MspA-N91H nanopores are embedded in the insulating film and provide channels connecting the first medium and the second medium; the hydrolysis product is added to the first medium; before the hydrolysis product is added to the nanoporous system, the M2MspA-N91H nanopores are bound to copper ions; the pH values ​​of the first medium and the second medium are between 6.5 and 7.

5. S3 applies a driving force between the first medium and the second medium, allowing free amino acids in the hydrolysis product to pass through the M2MspA-N91H nanopores and generate electrical signals; the free amino acids include natural amino acids and / or non-natural amino acids; the natural amino acids include one or more of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline; the non-natural amino acids are one or more of citrulline, L-valine, and amaurine. S4 performs data analysis on the electrical signal to obtain information on the amino acid types of the free amino acids; the data analysis step in S4 further includes: i) Establish a standard signal database; ii) Set signal filtering conditions; iii) Eliminate part of the electrical signal to obtain the retained electrical signal; iv) Determine the type of amino acid corresponding to the retained electrical signal by using the blocking rate of the standard signal in the standard signal database; v) Obtain the amino acid type information of the free amino acid; The signal filtering condition is to exclude electrical signals with an SD value greater than an SD threshold, wherein the SD value of the electrical signal is calculated according to the following formula: SD = In the formula, The current value at each acquisition point of the signal. is the average value of the signal current points, and n is the number of signal acquisition points; S5. The amino acid type information of the free amino acids is compared with the expected sequence information of the polypeptide to be detected to determine whether there is a difference between the actual sequence of the polypeptide to be detected and the expected sequence of the polypeptide to be detected; or S5 compares the amino acid type information of the free amino acid with the information of the exopeptidase and the polypeptide sequence information in the database to predict the actual sequence of the polypeptide to be detected.

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