WO2023030146A1 - Polypeptide composition analysis method based on copper ion modified mspa nanopores - Google Patents

Abstract

Provided is a polypeptide composition analysis method based on M2MspA-N91H nanopores, comprising the following steps: S1: uniformly mixing and reacting exopeptidase with a polypeptide to be tested, and obtaining a hydrolysate; S2: adding the hydrolysate into a nanopore system, where the nanopore system comprises: 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 a channel communicating the first medium and the second medium, and the hydrolysate is added into the first medium; S3: applying a driving force between the first medium and the second medium, free amino acids in the hydrolysate passing through the M2MspA-N91H nanopores and generating electrical signals; S4: performing data analysis on the electrical signals to obtain amino acid type information for the free amino acids.

Description

A method for analyzing peptide components based on copper ion-modified MspA nanopores

This application claims the priority of the Chinese invention patent application [CN2021110067648] filed on August 30, 2021, entitled "A method for detecting multiple amino acids based on nanopores and a method for analyzing polypeptide components". The method is incorporated in its entirety.

technical field

The invention belongs to the field of nanopore detection, and in particular relates to a method for analyzing polypeptide components based on copper ion-modified MspA nanopores.

Background technique

Nanopore sensing is a single-molecule sensing technology with a detection principle similar to that of a Kurt counter. The technique 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.

Due to the low signal-to-noise ratio and unpredictable conformational changes during translocation of amino acids through nanopores, it is still a challenge for nanopores to directly distinguish and detect amino acids. The existing technology cannot directly detect and distinguish the 20 kinds of amino acids, and often requires chemical modification of nanopores or derivatization of amino acids.

Contents of the invention

In view of this, the present invention provides a method for analyzing polypeptide components based on M2MspA-N91H nanopore, which is characterized in that it comprises the following steps:

S1 mixes the exopeptidase and the polypeptide to be detected evenly and reacts to obtain a hydrolyzate;

S2 adding the hydrolyzate into the nanopore system, the nanopore system comprising: M2MspA-N91H

nanopore, an insulating film, a first medium, and a second medium, wherein the M2MspA-N91H nanopore is embedded in the insulating film, and the M2MspA-N91H nanopore provides communication between the first medium and the second medium channel, the hydrolyzate is added to the first medium;

S3 applies a driving force between the first medium and the second medium, and free amino acids in the hydrolyzate pass through the M2MspA-N91H nanopore and generate an electrical signal;

S4 performs data analysis on the electrical signal to obtain amino acid type information of the free amino acid.

In one embodiment, the free amino acids include natural amino acids and unnatural amino acids.

In one embodiment, the natural amino acids include glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histamine One or more of acid, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline.

In one embodiment, the unnatural amino acid includes one or more of citrulline, L-norvaline, and amarine.

In one embodiment, the exopeptidase comprises aminopeptidase and/or carboxypeptidase.

In one embodiment, the M2MspA-N91H nanopore is bound to copper ions before the hydrolyzate is added to the nanopore system.

In one embodiment, the pH values of the first medium and the second medium comprise 6.5-7.5.

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

In one embodiment, the data analysis step described in S4 further includes:

i) Establish a standard signal database;

ii) Set signal filtering conditions;

iii) Excluding part of the electrical signal to obtain the retained electrical signal;

iv) judging the type of amino acid corresponding to the retained electrical signal according to the blocking rate of the standard signal in the standard signal database;

v) obtaining amino acid type information of the free amino acid.

In one embodiment, the signal filtering condition is to exclude electrical signals whose SD value is greater than the SD threshold, wherein the SD value of the electrical signal is calculated according to the following formula:

In the formula, x _i is the current value of each collection point of the signal, x is the average value of the signal current point, and n is the number of signal collection points.

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, so as to determine that the actual sequence of the polypeptide to be detected is different from that of the polypeptide to be detected. whether there is a difference in the expected sequence of ; or

S5 compares the amino acid type information of the free amino acid combined with the information of the exopeptidase with the polypeptide sequence information in the database to predict the actual sequence of the polypeptide to be detected.

Beneficial effect

Nanopores of the prior art can only detect amino acids with specific functional groups, but experiments in the present invention show that M2MspA-N91H nanopores (porin A derived from Mycobacterium smegmatis) have versatility in detecting amino acids , which can robustly and sensitively detect and distinguish 20 common amino acids and unnatural amino acids at the single-molecule level. Furthermore, 20 common amino acids and unnatural amino acids can block M2MspA-N91H nanopores and cause corresponding blockage without additional introduction of enzymes into the M2MspA-N91H nanopore system and/or modification of amino acids (e.g., adding luminescent labels). current signal. The direct detection and differentiation of various amino acids can be realized by analyzing different current signals.

Based on this, combined with the hydrolysis properties of exopeptidases (such as carboxypeptidases), the present invention provides a method for analyzing polypeptide components based on M2MspA-N91H nanopores. According to the blocking rate of the standard signal in the established standard signal database, the amino acid type corresponding to the detected free amino acid signal can be judged, and then the component analysis of the polypeptide to be detected can be realized to a certain extent. The polypeptide component analysis method provided by the present invention is suitable for various application scenarios such as the expression of tumor neoantigens, the judgment of the synthesis quality of polypeptide drugs, and the prediction of unknown polypeptide sequences.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the description of the embodiments or the prior art. Apparently, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.

Figure 1 is a diagram of the structure and electrophysiological properties of M2MspA-N91H nanopores;

Fig. 2 is when adding different concentrations of copper ions, M2MspA-N91H nanopore single hole current signal diagram (1M KCl, pH 6);

Fig. 3 is the electric current signal figure that M2MspA-N91H nanopore carries out amino acid detection under the concentration of 3M KCl;

Fig. 4 is a scatter diagram of amino acid volume vs. current signal blocking rate (labeled letters are amino acid abbreviations);

Figure 5 is a diagram of blocking rate of amino acid current signal vs. blocking time (labeled letters are amino acid abbreviations);

Figure 6 is the experimental diagram of single amino acid signal extracted by Clampfit (A is the single-channel statistical result of Clampfit; B is the Amp S.D. In the second experiment, the blank group, the asp experimental group, and the asp+glu experimental group counted all the signals for 2 minutes); D is the source of the -5pA background signal; E is the source of the -12pA background signal);

Figure 7 is the current signal diagram measured after the carboxypeptidase hydrolyzate was added to the nanopore system (A is carboxypeptidase under the conditions of 0.5M KCl, 10mM MOPS, pH 7.5, 10U carboxypeptidase hydrolyzed 10mM EF polypeptide for 20 minutes and then added The current signal diagram detected by the nanopore system; B is a scatter diagram involving the blocking rate and passage time of all current signals obtained in the experiment; C is the nanopore detection of three polypeptides (P1, P2, P3) hydrolyzate);

Figure 8 is the experimental diagram of the M2MspA-N91H nanopore detection of unnatural amino acids (A is the statistical results of three unnatural amino acids; B is the current of the blank group (Blank) and the addition of amarin in the same experiment, within 1 minute blocking signal scatterplot);

Fig. 9 is the current trajectory diagram of the M2MspA-N91H nanopore after adding copper ions under three kinds of pH conditions;

Fig. 10 is a schematic diagram of the nanopore detection polypeptide hydrolyzate of the present invention;

FIG. 11 is a flowchart of establishing a standard signal database in an embodiment of the present invention;

Fig. 12 is a flow chart of data analysis in the embodiment of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be noted that, in this document, the term "comprising", "comprising" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements, It also includes other elements not expressly listed, or elements inherent in the process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising that element.

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

的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。 In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, range

The description should be read 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., and within this range Individual numbers such as 1, 2, 3, 4, 5 and 6. The above rules apply regardless of the breadth of the scope.

nanopore system

A "nanopore system" includes pores having nanoscale dimensions (referred to simply as "nanopores"), an insulating membrane, a first medium, and a second medium. In one embodiment of the present invention, the nanopores are M2MspA-N91H nanopores. The pores having nanoscale dimensions allow translocation of the analyte from one side of the insulating film to the other. In addition, those skilled in the art can also modify the above-mentioned M2MspA-N91H nanopore according to the actual situation (for example, any mutation, truncation, fusion, chemical modification, etc.) to obtain the corresponding variant, and the modification means are the present well known in the field.

In one embodiment of the present invention, the nanoscale-sized pores are embedded in the insulating film, and the insulating film (also can be understood as a composite of the nanoscale-sized pores and the insulating film ) separates the first medium from the second medium, and the channels having nanometer-sized pores provide passages connecting the first medium and the second medium; After a driving force is applied between the second medium, the analyte located in the first medium interacts with the M2MspA-N91H nanopore to form a current (ie, an electrical signal). In the present invention, "first medium" refers to the medium in which the analyte is added to the nanopore system; "second medium" refers to the two parts of the medium separated by the insulating film. , the other side of the "first medium". In the present invention, the driving force refers to the force that drives the analyte to interact with the nanopore by means of potential, electroosmotic flow, concentration gradient and the like.

The first medium and the second medium may be the same or different, and the first medium and the second medium may comprise electrically conductive fluids. The conductive liquid is an aqueous alkali metal halide solution, specifically sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl), and sodium bromide (NaBr). In one embodiment of the present invention, the concentrations of the conductive liquid contained in the first medium and the second medium are different, in other words, the concentrations of the conductive liquid in the first medium and the second medium exist The difference, and then there is a difference in the osmotic pressure on both sides of the insulating film. The first medium and/or the second medium may further comprise a buffer, such as HEPES, 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).

An insulating membrane refers to a membrane that has the ability to host nanopores and block ionic currents passing through non-nanopores. The insulating film may include a phospholipid film and/or a polymer film. Exemplary phospholipid membranes include DPHPC, DOPC, E.coli lipid, and exemplary polymer membranes include triblock copolymer polymer membranes.

In a specific embodiment of the present invention, the nanopore system includes two electrolyte chambers, which are separated by an insulating membrane to form a trans (-trans) compartment and a cis (-cis) compartment, the The pores of the M2MspA-N91H channel are embedded in the insulating film, and only the M2MspA-N91H channel on the insulating film communicates with the above two electrolyte chambers. When a potential is applied to the above two electrolyte compartments, electrolyte ions in solution in the electrolyte compartments move by electrophoresis and pass through the M2MspA-N91H channel.

Analyte

The analyte is a charged substance. An analyte is charged if it has a net charge. The analyte can be negatively charged or positively charged. An analyte is negatively charged if it has a net negative charge. An analyte is positively charged if it has a net positive charge. Suitable analytes are preferably amino acids.

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-translationally modified variants, such as the 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.

In another embodiment of the present invention, the analyte may be an unnatural amino acid. "Unnatural amino acid" means an amino acid that is not a proteinogenic amino acid or a post-translationally modified variant thereof (i.e. an amino acid other than the 20 common amino acids or pyrrolysine or selenocysteine or a post-translationally modified variant thereof) . In a specific embodiment of the present invention, the unnatural amino acid is citrulline, L-norvaline, and amanitine.

The interaction between the nanopore and the analyte

The analyte may be in contact with either side of the nanopore on both sides of the insulating film. The analyte may be in contact with either side of the insulating film, so that the analyte passes through the channel of the nanopore to reach the other side of the insulating film. In this case, the analyte interacts with the nanopore as it passes through the insulating membrane via the passage of the pore. Alternatively, the analyte can be in contact with the side of the insulating film, and the side of the insulating film can make the analyte interact with the nanopore so that it is separated from the nanopore and stays in the nanopore. on the same side as the insulating film. The analyte can interact with the nanopore in any manner and at any location. The analyte can also hit the nanopore, interact with the nanopore, make it separate from the nanopore and stay on the same side of the insulating membrane.

During the interaction of the analyte with the nanopore, the analyte affects the current flowing through the nanopore in a manner specific to the analyte, that is, the current flowing through the nanopore The current is characteristic for a particular DUT. Control experiments can be performed to determine the effect of a particular analyte on the current flowing through the nanopore, and then to identify the particular analyte in the sample or to determine the presence of the particular analyte in the sample. More specifically, the presence or absence or concentration of the analyte can be identified based on the comparison of the current pattern obtained by detecting the analyte with a known current pattern obtained using a known analyte under the same conditions wait.

The nanopore system of the present invention may also include one or more measurement devices that measure the current flowing through the nanopore, such as patch clamp amplifiers or data acquisition devices.

A method for analyzing peptide components based on M2MspA-N91H nanopore

The present invention also provides a method for analyzing polypeptide components based on the M2MspA-N91H nanopore, which is characterized in that it comprises the following steps:

S1 mixes the exopeptidase and the polypeptide to be detected evenly and reacts to obtain a hydrolyzate;

S2 adding the hydrolyzate into the nanopore system, the nanopore system comprising: M2MspA-N91H

nanopore, an insulating film, a first medium, and a second medium, wherein the M2MspA-N91H nanopore is embedded in the insulating film, and the M2MspA-N91H nanopore provides communication between the first medium and the second medium channel, the hydrolyzate 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 hydrolyzate pass through the M2MspA-N91H nanopore and generate an electrical signal;

S4 performs data analysis on the electrical signal to obtain amino acid type information of the free amino acid.

"Polypeptide" refers to a peptide or protein comprising two or more amino acids linked by peptide bonds. Polypeptides may contain natural, modified or synthetic amino acids. Polypeptides can also be modified naturally (e.g., by post-translational processing) or chemically (e.g., amidation, acylation, cross-linking, etc.). In the present invention, the sequence information of the polypeptide may be known or unknown.

"Exopeptidase" refers to a hydrolase that removes terminal amino acids of polypeptides by cleaving peptide bonds. Terminal amino acids are amino acids within about 10 amino acids of the N- or C-terminus of a polypeptide. Accordingly, "exopeptidases" are classified into aminopeptidases that hydrolyze amino acids one by one from the N-terminus of polypeptides and carboxypeptidases that hydrolyze amino acids one by one from the C-terminus of polypeptides. Exemplary aminopeptidases include cysteinyl aminopeptidase, prolyl aminopeptidase, arginyl aminopeptidase. Exemplary carboxypeptidases include carboxypeptidases A, B, C, Y. In the present invention, the exopeptidase may be carboxypeptidase and/or aminopeptidase.

Under appropriate conditions, the exopeptidase can completely or partially hydrolyze the polypeptide to be detected. For example, as shown in Figure 10, carboxypeptidase A1 can cut C-terminal amino acids except lysine (K), arginine (R), proline (P) (that is, the stop point of hydrolysis is R, K, P). In other words, if the polypeptide to be detected does not contain the three amino acids R, K, and P, the polypeptide to be detected will be completely hydrolyzed by carboxypeptidase A, and the amino acid type information of the free amino acids in the obtained hydrolyzate is The amino acid composition of the polypeptide to be detected. If the polypeptide to be detected contains one or more of the three amino acids R, K, and P, the polypeptide to be detected will be partially hydrolyzed by carboxypeptidase A (stop hydrolysis at the stop point), and the obtained hydrolyzate The amino acid type information of the free amino acid is the amino acid composition of the polypeptide to be detected before the stop point.

When the sequence of the polypeptide to be detected is known, the amino acid type information of the free amino acids in the obtained hydrolyzate can be compared with the expected sequence information of the polypeptide to be detected to determine whether the actual sequence of the polypeptide to be detected is consistent with the expected sequence of the polypeptide to be detected. Describe whether there are differences in the expected sequence of the polypeptide to be detected (for example, some (some) amino acids that should appear in the hydrolyzate do not appear, and some (some) amino acids that should not appear in the hydrolyzate appear), which is suitable for tumor neogenesis Application scenarios such as the expression of antigens and the judgment of the synthetic quality of peptide drugs.

When the sequence of the polypeptide to be detected is unknown, it can be based on the amino acid type information of the free amino acids in the obtained hydrolyzate and in combination with the information of the exopeptidase used (for example, carboxypeptidase or aminopeptidase, whether there is a "hydrolysis stop point" ), compare in the database by means of machine learning, etc., and then predict the sequence of the polypeptide to be detected or narrow the possible range of the polypeptide to be detected to a certain extent (for example, starting at the N-terminus or C-terminus, amino acid Composition (including defined species of free amino acids and possible species corresponding to "hydrolysis stop points").

Embodiment one

Materials and Methods

Amino Acid Detection:

Single-channel experiments were performed using a classical vertical lipid bilayer setup (Warner Instruments), consisting of two chambers (cis in cis and trans in trans) connected by a 150 μm diameter aperture. Two Ag-AgCl electrodes are used to apply the transmembrane voltage and measure the transmembrane ionic current. The cis chamber is at voltage ground. The buffer conditions for amino acid detection are:

1) Buffer: 0.3M-3M salt solution such as KCl and NaCl (preferably 1M aqueous KCl solution), 10mM MOPS;

2) Copper ion concentration range: 1nM to 200μM;

3) pH range: 6-9 (preferably 6.5-7.5).

As the buffer salt concentration or pH is further reduced, amino acid detection efficiency and discrimination may decrease. All experimental temperatures can be set at 15°C-60°C (preferably 22.0°C±0.5°C). Formation of planar lipids by coating the well connecting the two chambers with a thin film of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti Polar Lipids) dissolved in decane (40 mg/ml) Double membrane. Then add the protein solution to the cis cavity, after the protein is embedded in the phospholipid membrane to form a single channel, add copper chloride to the trans cavity, and add amino acids to the cis cavity, mix the solution gently and record the current signal.

data processing:

Single-channel current recordings were amplified using an Axopatch 200B amplifier (Molecular Devices) and filtered at 2 kHz using a built-in four-hole low-pass Bessel filter. Data was digitized by a Digidata 1550B converter (Molecular Devices) at a sampling rate of 100kHz. Extract the current signal file obtained by sampling to obtain amino acid signal data. Single amino acid signals can be identified and defined by data such as signal blocking rate, blocking time, and SD.

Handling of background noise:

For the current value distribution of a single amino acid signal, the SD value is calculated to obtain the degree of deviation between the current value of each sampling point of the signal and the overall average value, so as to distinguish the single amino acid signal from the background signal and the superimposed signal of multiple amino acids.

Peptide composition analysis method:

The buffer conditions for the experiments were as described above. The experimental temperature can be set at 15°C-60°C (preferably 25°C). Outside the nanopore system, mix exopeptidase (such as carboxypeptidase) and polypeptide according to a certain concentration ratio, such as 10U carboxypeptidase, 0.5M KCl, 10mM MOPS, pH 7.5, 10mM polypeptide (Figure 7A), 25℃ After reacting for 20 minutes, add the nanopore sample tank for detection. Determine the peptide composition based on the type and quantity of amino acid signals that appear.

Example 2: Detection of 20 Natural Amino Acids Using M2MspA-N91H Nanopore

Use copper ions to modify the structure and properties of nanopores:

As shown in Figure 1, the M2MspA-N91H nanopore has an 8-mer structure, and the 8 amino acid residues at the narrowest part of the channel are oriented toward the central axis of the M2MspA-N91H nanopore. Adding Cu ²⁺ into the trans chamber and observing the current trajectory, Cu ²⁺ can significantly reduce the current fluctuation (Fig. 1). The reduction of the current level is related to the concentration of Cu ²⁺ added (Fig. 2), and the copper ion concentration can range from 1 nM to 200 μM.

Detection of 20 natural amino acids:

In this example, 20 natural amino acids were detected by using the M2MspA-N91H nanopore. Among them, the amino acid sequence of the MspA monomer (MspA is assembled by eight identical monomers to form a channel) is:

The amino acid sequence of M2MspA-N91H is:

When the target analyte passes through Cu ²⁺ chelated M2MspA-N91H, a specific current signal is generated, as shown in Figure 3. Under the conditions of three pH values, the current trajectory diagram after adding copper ions is shown in Fig. 9 . The results showed that copper ion binding was unstable at pH 7.9, while copper ion binding was stable at pH 7.5 and 6.5. In view of the fact that a higher solution pH value is conducive to more amino acids being negatively charged, thereby improving the detection efficiency. Therefore, in this embodiment, MOPS buffer salt is added to the solution and the pH value of the solution is stabilized to about 7.5, in order to obtain a better detection effect.

From the scatter plot of amino acid volume and current signal blocking rate (Fig. 4), it can be seen that there is a positive correlation between the blocking rate caused by amino acid and its hydrophobic volume.

From the amino acid current signal blocking rate and blocking time diagram (Figure 5), it can be obtained that the blocking rates caused by 20 natural amino acids are: 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 . Judging by the blocking rate of amino acids and their standard deviations, non-overlapping amino acids include G, S, A, T, N, R, V, Q, M, L, I, D, F, W, E, a total of 15 The signals of these amino acids do not overlap and can be distinguished from each other.

Handling of background noise:

The above-mentioned method of directly detecting 20 kinds of amino acids may generate background noise and interfere with subsequent analysis. Based on this, the experimenters of the present invention calculated the SD value of the current value distribution of a single amino acid signal using the following formula, and obtained the degree of deviation between the current value of each collection point of the signal and the overall average value. In this way, the single amino acid signal can be distinguished from the background signal and the superimposed signal of multiple amino acids.

In the formula, x _i is the current value of each collection point of the signal, x is the average value of the signal current point, and n is the number of signal collection points.

As shown in Figure 6C, the upper, middle and lower figures are the blank group (Blank), the addition of aspartic acid (Asp) and the addition of a mixture of aspartic acid and glutamic acid (Asp+Glu) in the same experiment, respectively. Scatterplot of current blocking signal over 2 minutes. The SD threshold can be set to filter the signal, for example, the SD threshold can be set to 2, where the signal (blue) with an SD value less than 2 is considered to be a valid signal; the signal with an SD value greater than 2 is considered to be a background signal Simultaneous passage of one or more amino acids produces superimposed signals, which can therefore be filtered. After filtering, only look at the blue signal, and you can clearly see the newly generated signal peak after adding aspartic acid and glutamic acid. Using the background noise processing method, amino acid signals can be identified and counted more accurately.

In order to better demonstrate the processing process, the experimenters of the present invention used Clampfit software to extract single amino acid signals. First, using the fitted SD value, the background signal and double-step signal were filtered out, and the single amino acid signal was retained. As shown in Figure 6A, in the Clampfit single-channel statistical results (only level 0 and level 1 are set), the "Amp S.D." value indicates the signal fitting effect. The SD value corresponding to the specific signal is shown in Figure 6B; it can be seen from the six signals shown that the SD value of the double-step signal is generally greater than 4. A single-step signal means that the current density distribution curve of the electrical signal presents a single-peak distribution, and the current has no distribution between 0 and 60% of the peak value. The double-step signal means that the current density distribution curve of the electrical signal presents a bimodal distribution.

The SD threshold is set in the range of 0-2, which can filter most multi-step signals. However, background signal interference still exists around -5pA and -12pA. The signal source near -5pA is shown in Figure 6D, because its corresponding level 0 deviates too much from the baseline and can be filtered out in the later stage. The signal around -12pA (Fig. 6E) is a normal background signal with a short blocking time. The background signal only interferes with the nearby signals of gly, ser, and ala, and the signals of these three amino acids can be distinguished.

Based on this, known detection data can be used to establish a standard signal database, and the specific process is shown in Figure 11: First, standard free amino acids are detected through M2MspA-N91H nanopores to obtain electrical signal data. Second, filter conditions are set (for example, the SD threshold of the electrical signal is set to 2). Thirdly, according to the filter condition, some electrical signals are excluded to obtain retained electrical signals. Fourth, judge whether the reserved electrical signal is a single-step electrical signal: if the reserved electrical signal is a single-step electrical signal, then include the single-step electrical signal into the standard signal database, and regard the single-step electrical signal as the amino acid Standard signal; if the retained electrical signal is not a single-step electrical signal (such as a double-step electrical signal), reset the filter condition (for example, increase the SD threshold), and regain the retained electrical signal, and then judge again the retained Whether the electrical signal is a single-step electrical signal. Finally, a standard signal database is established using single-step electrical signals.

Example 3: Using M2MspA-N91H nanopore to detect three kinds of unnatural amino acids

Based on the above method, three representative unnatural amino acids were further detected in this example, namely citrulline, L-norvaline and amarine. As shown in Figure 8A, the blocking rates caused by these three unnatural amino acids were: citrulline: 0.1912±0.0011; L-norvaline: 0.1806±0.0017; amarine: 0.2333±0.0029. According to the amino acid blocking rate and its standard deviation, the signals of the three unnatural amino acids do not overlap and can be distinguished from each other.

As shown in FIG. 8B , the upper and lower graphs are the scatter diagrams of the current blocking signal within 1 minute of the blank group (Blank) and the addition of amarin in the same experiment, respectively. Signals with SD values less than 2 were considered valid signals, and after filtering, it was clearly seen that a new signal peak was generated after the addition of amarine.

Polypeptides can be used as substrates to participate in enzymatic reactions, and the above methods 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 the enzymatic reaction occurs and the progress of the enzymatic cascade reaction .

Example 4: Method for analyzing polypeptide components based on M2MspA-N91H nanopore:

Immediately after M2MspA-N91H nanopore intercalation, the voltage was adjusted to +50 mV and recorded for 10 min. Next, add 1 μL of 20 mM copper chloride solution to the trans side of the sample tank, and record for 10 minutes. At the same time, 8 μL of the peptide solution with a concentration of 2 mM was placed in a PCR tube, and incubated at 37° C. for 5 minutes. Add 2 μL carboxypeptidase A1 solution, mix thoroughly, and react at 37°C for 5 minutes. After the reaction is completed, add all the reaction products to the cis side of the sample tank, mix thoroughly, and record at +50mV for 2 hours. The detection principle is shown in Figure 10. Carboxypeptidase A1 (a type of exopeptidase) can hydrolyze amino acids one by one from the C-terminus of the polypeptide, and the free amino acid products obtained by hydrolysis are captured by nanopores to be detected. According to the appearance of amino acid signals The type and quantity can determine the peptide composition.

Since 1 U of exopeptidase can cut 1 amino acid at the end of 1 μmol of polypeptide within 1 min, the final concentration of the polypeptide solution can be calculated according to the following formula (assuming that the polypeptide can be completely hydrolyzed):

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

In the nanopore system, when the concentration of free amino acids is 1 μM, the detection effect is better; if the final concentration of free amino acids is lower than 1 μM, the signal of free amino acids may be reduced, thereby affecting the detection effect. Considering that the hydrolyzate (reaction product) may be diluted at least 10 times after being added to the M2MspA-N91H nanopore system, therefore, based on the above formula, the final concentration of the polypeptide solution in the reaction system should be greater than 10 times of 1 μM (ie 10 μM) .

The terminal amino acid of the polypeptide is hydrolyzed by exopeptidase to obtain a hydrolyzate (reaction product). After the above reaction product is added to the M2MspA-N91H nanopore system and a driving force is applied, free amino acids in the reaction product pass through the M2MspA-N91H nanopore to generate an electrical signal, and obtain corresponding electrical signal data. The specific data analysis process is shown in Figure 12: First, the above reaction products are detected through the M2MspA-N91H nanopore to obtain electrical signal data. Second, build a standard signal database (as described above). Third, filter conditions (SD threshold of electrical signals) are set. Fourth, according to the filter condition, some electrical signals are excluded to obtain retained electrical signals. Fifth, determine the type of amino acid corresponding to the retained electrical signal based on the blocking rate of the standard signal in the standard signal database. Sixth, the amino acid type information of the free amino acid is obtained.

Fig. 7 shows the analysis of the composition of four different polypeptides based on the above polypeptide composition analysis method and data analysis method. Figure 7A is the current signal diagram obtained by adding the reaction solution to the nanopore system after hydrolyzing 10mM EF polypeptide with 10U carboxypeptidase under the conditions of 0.5M KCl, 10mM MOPS, and pH 7.5 for 20 minutes. FIG. 7B is a scatter diagram of FIG. 7A related to the blocking rate and hole-through time of all current signals obtained in the experiment. It can be observed that the signal (red) with SD value between 1-2 has 3 distribution peaks, and the signal of EF polypeptide near the blocking rate of 0.4; the left two peaks are related to glutamic acid (E) and phenylalanine ( The blocking rate of F) is the closest, so it is judged as the signal of amino acids E and F.

Fig. 7 C shows that M2MspA-N91H nanopore detects respectively three kinds of polypeptides (P1: APRLR FYSL ; P2: DRVYIHP FHL ; P3: RPVKVYPNGAEDESAEAFP LEF ; wherein underline represents that these amino acids of polypeptide C terminal can be hydrolyzed as free amino acids by carboxypeptidase A1) Experimental results of hydrolyzate. The above analysis results show that the polypeptide component analysis method provided by the present invention can analyze the terminal amino acid composition of the polypeptide.

The above-mentioned polypeptide composition analysis method provided by the present invention can be applied to many different scenarios, such as the identification of known polypeptides and the prediction of unknown polypeptides:

Tumor neoantigens, which are not expressed in normal tissues but only expressed in tumor tissues, are ideal targets for immunotherapy. Each patient has its unique neoantigen spectrum, and the above-mentioned peptide component analysis method can be used to screen tumor neoantigen peptides: first, use a highly specific extraction method to extract and purify known tumor cell surface antigen peptides, and the purified The mixture theoretically contains only a small amount of antigenic peptides with known components; secondly, use the above-mentioned peptide composition analysis method to detect the purified mixture; finally, compare the amino acid species detected in the test results with the amino acid species of known antigenic peptides In contrast, it can be judged whether certain amino acids are produced or lacked, and then judged whether neoplastic antigens may exist on the surface of tumor cells.

At present, some peptide drugs have been developed and applied clinically, but due to the limitation of the synthesis process, the product quality and purity of peptide drugs cannot meet the requirements. Through the above-mentioned polypeptide composition analysis method, the detection of the purity of the polypeptide drug can be realized: comparing the detection results of the amino acid composition of the polypeptide drug to be detected with the expected synthetic amino acid type can judge the synthetic quality of the polypeptide drug to a certain extent .

In addition, the polypeptide composition analysis method provided by the present invention can also be combined with machine learning to predict unknown polypeptides: hydrolyze the unknown polypeptide to be detected with exonuclease and detect free amino acids; use machine learning methods to combine the type of exonuclease , hydrolysis characteristics, and amino acid composition detection results, compared with known peptides in the database, can predict the sequence of the unknown peptide to a certain extent, and at the same time play a role in narrowing the range of unknown peptides.

Embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementations, and the above-mentioned specific implementations are only illustrative, rather than restrictive, and those of ordinary skill in the art will Under the enlightenment of the present invention, many forms can also be made without departing from the gist of the present invention and the protection scope of the claims, and these all belong to the protection of the present invention.

Claims (10)

A method for analyzing polypeptide components based on M2MspA-N91H nanopore, characterized in that it comprises the following steps: S1 mixes the exopeptidase and the polypeptide to be detected evenly and reacts to obtain a hydrolyzate; S2 adds the hydrolyzate to the nanopore system, the nanopore system includes: M2MspA-N91H nanopore, insulating film, first medium, second medium, wherein the M2MspA-N91H nanopore is embedded in the insulating film Among them, the M2MspA-N91H nanopore provides a channel connecting the first medium and the second medium, and the hydrolyzate 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 hydrolyzate pass through the M2MspA-N91H nanopore and generate an electrical signal; S4 performs data analysis on the electrical signal to obtain amino acid type information of the free amino acid. The method according to claim 1, wherein the free amino acids include natural amino acids and unnatural amino acids. The method according to claim 2, wherein said natural amino acid comprises glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, Aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline One or more; the unnatural amino acid may optionally include one or more of citrulline, L-norvaline, and amarine. The method of claim 1, wherein the exopeptidase comprises aminopeptidase and/or carboxypeptidase. The method of claim 1, wherein said M2MspA-N91H nanopore is bound to copper ions before said hydrolyzate is added to said nanopore system. The method according to claim 1, wherein the pH values of the first medium and the second medium include 6.5-7.5. The method according to claim 1, characterized in that the final concentration of the polypeptide solution to be detected is greater than 10 μM. The method according to claim 1, wherein the data analysis step in S4 further comprises: i) Establish a standard signal database; ii) Set signal filtering conditions; iii) Excluding part of the electrical signal to obtain the retained electrical signal; iv) judging the amino acid type corresponding to the retained electrical signal through the blocking rate of the standard signal in the standard signal database; v) obtaining amino acid type information of the free amino acid. The method according to claim 8, wherein the signal filtering condition is to exclude electrical signals whose SD value is greater than the SD threshold, wherein the SD value of the electrical signal is calculated according to the following formula:

In the formula, x _i is the current value of each collection point of the signal, x is the average value of the signal current point, and n is the number of signal collection points. The method according to claim 1, characterized in that, the method further comprises 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 the actual sequence information of the polypeptide to be detected. Whether the sequence differs from the expected sequence of the polypeptide to be detected; or S5 compares the amino acid type information of the free amino acid combined with the information of the exopeptidase with the polypeptide sequence information in the database to predict the actual sequence of the polypeptide to be detected.

Images