Molecule passage channel for amino acid sequence analysis, molecule passage membrane for amino acid sequence analysis, amino acid sequence analysis method, and amino acid sequence analysis device

Abstract

Provided are a molecule passage channel for amino acid sequence analysis, a molecule passage membrane for amino acid sequence analysis, an amino acid sequence analysis method, and an amino acid sequence analysis device that, compared to conventional methods, allow rapid and highly efficient analysis of amino acid sequences of peptide molecules from a small sample that is not highly refined. In the present invention, a molecule passage channel for amino acid sequence analysis, a molecule passage membrane for amino acid sequence analysis, an amino acid sequence analysis method, and an amino acid sequence analysis device are used for amino acid sequence analysis of peptide molecules. The molecule passage channel contains a protein having 80% or more homology with SecYEG protein.

Description

Molecular permeability channel for amino acid sequence analysis, molecular permeability membrane for amino acid sequence analysis, amino acid sequence analysis method, and amino acid sequence analysis device

[0001] The present invention relates to a molecule-permeable channel for amino acid sequence analysis, a molecule-permeable membrane for amino acid sequence analysis, an amino acid sequence analysis method, and an amino acid sequence analyzer used in a so-called sequencer for analyzing the amino acid sequences of peptide molecules such as proteins and oligopeptides. This application claims priority to Japanese Patent Application No. 2024-086612, filed May 28, 2024, the contents of which are incorporated herein by reference.

[0002] Amino acid sequence analysis, which obtains sequence information for peptide molecules such as proteins and oligopeptides, is essential for analyzing these biochemical substances. Identifying the amino acid sequences of peptides and proteins not only helps us understand their biological functions, but also leads to the development of new pharmaceuticals and their therapeutic applications. Short peptides, consisting of several dozen amino acids in length, are currently attracting considerable attention. For example, they are involved in signal transduction (e.g., insulin), apoptosis control, growth factors, immune responses, and neurotransmission (neuropeptide cell function control), and are expected to be used in applied research to develop antimicrobial peptides and DDS peptide drugs.

[0003] Currently, the conventional method for obtaining information on the amino acid sequence of peptide molecules has been widely used for a long time, combining Edman degradation, which identifies amino acids one by one from the N-terminus, with mass spectrometry, assuming that a certain amount of uniform sample can be obtained. However, these conventional methods are not only expensive to decode, but also require a long time to determine sequences that are only capable of determining lengths of a few dozen amino acids. Furthermore, it is difficult to determine the sequence of a mixture of peptides with heterogeneous sequences.

[0004] As a means of decoding nucleic acid sequences rather than peptide sequences, DNA sequencers using nanopore technology have been developed and are in use. Nanopore technology can directly read bases from the shielding current generated when DNA passes through a nano-sized pore to which a voltage is applied.

[0005] For example, Patent Document 1 discloses a nanopore structure having a thin film-like first metal member having a through-hole and a second metal member provided so as to narrow the diameter of the through-hole, the first metal member and the second metal member forming a nanopore having a pore diameter of 10 nm or less, and a base sequence analyzer using the same. This technology provides a nanopore structure formed of an inorganic material by combining multiple types of metal materials, and specifically, by providing a second metal member at the open end of the through-hole in the first metal member by an electroless plating reaction, it is possible to provide a nanopore structure having a nanopore having a pore diameter of 10 nm or less, and aims to provide a base sequence analyzer using the same.

[0006] Patent No. 7237388

[0007] Apart from the combined use of the Edman method and mass spectrometry, there is a strong demand for a method for more efficient and accurate sequence identification. However, peptide molecule analysis presents many challenges that are difficult to overcome. Peptide molecules are not only easily degraded by degradative enzymes, but also exist in small numbers. While nucleic acids can be amplified, for example, by PCR to increase the number of samples (analyte), no such amplification technology exists for peptide molecules. Therefore, detecting peptide molecules themselves and identifying their amino acid sequences is difficult.

[0008] The inventors of the present invention have focused on the possibility that if peptide molecules could be passed through a nanopore gradually, similar to the nanopore technology used in DNA sequencing, and information such as the charge of the sites at which they passed could be sequentially read, it would be possible to perform analysis with far greater efficiency than the conventional Edman method and mass spectrometry, and with small sample volumes. However, no technology had previously been developed that applies the nanopore technology used in DNA sequencing to the analysis of other molecules.

[0009] Many difficulties remain in applying nanopore technology, which is used in DNA sequencing, to peptide molecule sequencing. First, there is a major difference: unlike DNA, peptide molecules cannot be amplified. Furthermore, based on the nanopore technology used in DNA sequencing, it is thought that three core technologies will be required for this technology to be successful: rate-controlled channel proteins, parallelization, and AI waveform analysis, but none of these have been developed at all.

[0010] The inventors considered that the most important technology among these was the discovery of a rate-controlled channel protein that can gradually pass peptide molecules. When a rate-controlled channel protein is used to pass a molecule through a pore to which a voltage is applied, if the rate is not controlled, the molecule will pass through in an instant, making it impossible to obtain sufficient information. To solve this problem, DNA sequencers use molecular motors to slowly pass DNA through the pore. However, no rate-controlled channel proteins have yet been reported for peptide molecule sequence analysis.

[0011] Here, the present inventors focused on translocon-type nanopores. A translocon is a complex that transports peptides from intracellular substrates into the endoplasmic reticulum (ER) in vivo. It is known that in this translocon complex, a motor protein called SecA passes the peptide through the pore of the SecYEG complex in association with ATP hydrolysis. The present inventors focused on the possibility of applying this molecule to a molecular passage channel in a peptide sequencer and continued their intensive research.

[0012] The present invention has been made in consideration of the above-mentioned circumstances, and its object is to provide a molecule-permeable channel for amino acid sequence analysis, a molecule-permeable membrane for amino acid sequence analysis, an amino acid sequence analysis method, and an amino acid sequence analysis device that can analyze the amino acid sequences of peptide molecules with high efficiency, in a short time, with a small sample size, and with a low degree of purification, compared to conventional methods.

[0013] In order to solve the above problems, the present invention has the following aspects. [1] A molecule passage channel for use in a method for analyzing the amino acid sequence of a peptide molecule, for passing the peptide molecule, comprising a protein having 80% or more homology to SecYEG protein. [2] A molecule passage channel for amino acid sequence analysis according to [1], further comprising SecA protein. [3] A molecule passage membrane for use in a method for analyzing the amino acid sequence of a peptide molecule, comprising the molecule passage channel for amino acid sequence analysis according to [1] or [2] and a lipid bilayer membrane. [4] A method for analyzing the amino acid sequence of a peptide molecule, comprising a step of passing the peptide molecule through a molecule passage channel for amino acid sequence analysis containing a protein. [5] The method for analyzing the amino acid sequence according to [4], wherein the protein in the molecule passage channel for amino acid sequence analysis containing a protein comprises a protein having 80% or more homology to SecYEG protein. [6] The amino acid sequence analysis method according to [4] or [5], wherein the protein in the amino acid sequence analysis molecule passage channel containing the protein further comprises SecA protein. [7] The amino acid sequence analysis method according to [4] to [6], wherein in the step of passing the peptide molecule through the amino acid sequence analysis molecule passage channel containing the protein, the passing speed of the peptide molecule is 1 millisecond to 10,000 milliseconds. [8] The amino acid sequence analysis method according to [4] to [7], wherein a voltage is applied to a site of the peptide molecule that has passed through the amino acid sequence analysis molecule passage channel and the ion current is measured to analyze the type of amino acid at the site. [9] An amino acid sequence analysis device comprising the amino acid sequence analysis molecule passage channel according to [1] or [2], configured to pass a peptide molecule through the amino acid sequence analysis molecule passage channel and analyze the type of amino acid at the site of the peptide molecule that has passed.

[10] The amino acid sequence analysis device according to [9], comprising the amino acid sequence analysis molecule passage membrane comprising the amino acid sequence analysis molecule passage channel and a lipid bilayer membrane.

[11] The amino acid sequence analysis device according to [9] or

[10] , comprising: a voltage application means for applying a voltage to a site of the peptide molecule that has passed through the amino acid sequence analysis molecule passage channel; and a current measurement means for measuring an ionic current at the site.

[0014] According to the present invention, it is possible to provide a molecule-permeable channel for amino acid sequence analysis, a molecule-permeable membrane for amino acid sequence analysis, an amino acid sequence analysis method, and an amino acid sequence analysis device that can analyze the amino acid sequences of peptide molecules with high efficiency, in a short time, and with a small sample size, with a low degree of purification, compared to conventional methods.

[0015] 1 is a schematic diagram showing a molecule-permeable membrane for amino acid sequence analysis including a molecule-permeable channel for amino acid sequence analysis according to the present embodiment.

[0034] FIG. 1 is a graph showing the results of measurements under the conditions of ATP(+), Mg(-), and prePhoA(-) in this example.

[0035] FIG. 2 is a graph showing measurements under the conditions of ATP(+), Mg(+), and prePhoA(-).

[0036] FIG. 3 is a graph showing measurements under the conditions of ATP(+), Mg(-), and prePhoA(+) in this example.

[0037] FIG. 4 is a further enlarged view of FIG. 5.

[0038] FIG. 5 is a graph showing the entire trace of FIG. 5 after passing through a 50 Hz low-pass filter.

[0039] FIG. 6 is a schematic diagram showing the relationship between the cutoff current and dwell time used in subsequent analysis of this example.

[0039] FIG. 7 is a graph showing verification of the ATP concentration dependency of waveform A in this example.

[0039] FIG. 8 is a graph showing waveform A in this example.

[0039] FIG. 9 is a graph showing waveform B in this example. 1 is a graph showing a comparison between waveform B and waveform C in this example. 2 is a schematic diagram summarizing the results of this example. 3 is a schematic diagram of the measurement of the nanopore signal of SecY(I403N)AEG. 4 is a graph showing the change in current when each component of SecY(I403N)AEG is added. 5 is a graph showing the proOmpA of the nanopore signal of SecY(I403N)AEG. １－３１ -(YGRG) １０ Schematic diagram of measurement using peptides. １－３１ -(YGRG) １０ 1 is a graph showing the change in current when each component including a peptide is added.１－３１ -(YGRG) １０ 1 is a graph showing the change in current when each component containing peptide but not containing urea was added. １－３１ -(YGRG) １０ 10 is another graph showing the change in current when each component containing peptide but not containing urea was added. １－３１ -(YGRG) １０ FIG. 10 is another graph showing the change in current when each component containing peptide but not containing urea is added.

[0016] The following describes embodiments of the molecule-permeable channel for amino acid sequence analysis, the molecule-permeable membrane for amino acid sequence analysis, the amino acid sequence analysis method, and the amino acid sequence analysis device according to the present invention, although the present invention is not limited to the following embodiments.

[0017] (Molecular passage channel for amino acid sequence analysis) The molecular passage channel for amino acid sequence analysis of this embodiment is used in a method for analyzing the amino acid sequence of a peptide molecule, and is a molecular passage channel for passing the peptide molecule, and includes a protein having 80% or more homology to the SecYEG protein.

[0018] FIG. 1 is a schematic diagram showing an amino acid sequence analysis molecule passage membrane including an amino acid sequence analysis molecule passage channel of this embodiment. As shown in the figure, an amino acid sequence analysis molecule passage membrane 100 includes an amino acid sequence analysis molecule passage channel 10 (molecule passage channel) and a lipid bilayer membrane 30. The amino acid sequence analysis molecule passage channel 10 is composed of a SecYEG protein 11 and a SecA protein 12. In this figure, a peptide molecule 20 is passing through the amino acid sequence analysis molecule passage channel 10. In the example shown in the figure, the peptide molecule 20 has a signal sequence 21 at its N-terminus. The N-terminal signal sequence 21 is incorporated into the amino acid sequence analysis molecule passage channel 10.

[0019] In this embodiment, peptide molecules, the subject of amino acid sequence analysis, broadly refer to molecules in which amino acids (amino acid residues) are bound together by peptide bonds, and include a wide range of polypeptides, from oligopeptides of several to several tens of residues to larger polypeptides. Polypeptides larger than oligopeptides include proteins. There is no particular limitation on the size of peptide molecules, and peptides with two or more amino acid residues and up to tens of thousands of residues can be used. The term "amino acid sequence analysis method" broadly refers to a method of analyzing a peptide sequence, which analyzes the amino acid sequence of a peptide molecule by identifying the amino acid residues that make up the peptide molecule. The term "amino acid sequence analysis method" also broadly refers to a method of measuring or estimating information such as the length (number of residues) of a peptide.

[0020] The peptide molecule may have a signal sequence recognized by the amino acid sequence analysis molecule passage channel. When the peptide molecule has a signal sequence, it may be contained at the end of the peptide molecule. Furthermore, the signal sequence is preferably located on the N-terminal side of the peptide molecule. Furthermore, it is preferable that about 5 to 40 sequences on the N-terminal side have homology to known signal sequences, and preferably about 20 sequences. A signal sequence of about 1 to 20 sequences in length, known as an oligopeptide, is sometimes called a signal peptide. When the amino acid sequence analysis molecule passage channel is a SecYEG protein, the signal peptide can be selected appropriately, but in this embodiment, the following sequence shown in SEQ ID NO: 1 is used. Signal sequence of this embodiment (SEQ ID NO: 1): MKQSTIALALLPLLFTPVTKA Furthermore, when analyzing a peptide molecule without a signal sequence, a signal sequence or a molecule having such a sequence may be added separately during amino acid sequence analysis.

[0021] The molecule passage channel for amino acid sequence analysis of this embodiment is a molecule passage channel for passing the peptide molecule, which is used in the method for analyzing the amino acid sequence of the peptide molecule. In summary, by passing the peptide molecule through the molecule passage channel for amino acid sequence analysis of this embodiment, information on the amino acid residues constituting the peptide molecule is obtained sequentially for each amino acid residue, and the amino acid sequence of the peptide molecule is analyzed.

[0022] The molecule passage channel for amino acid sequence analysis of this embodiment preferably contains a protein having 80% or more homology with the SecYEG protein.

[0023] SecYEG, also known as the Sec translocon, is a passive protein membrane channel conserved in many organisms. SecYEG is also known to function by binding to SecA. SecA is an ATPase that binds to SecYEG to drive membrane transport of peptide molecules. In vivo, the movement of SecA is thought to push peptide molecules through the membrane-embedded SecYEG, and this action is thought to cause membrane transport of peptide molecules.

[0024] SecYEG consists of a complex of three subunits: SecY, SecE, and SecG. In this specification, the term "SecYEG protein" refers to this complex of three subunits. In this embodiment, the SecYEG protein is primarily an assembly of three subunits, but it also broadly refers to conjugates such as fusion proteins of subunits. Each subunit of SecYEG is conserved in many biological species, and any subunit from any organism may be used as appropriate. In this embodiment, each subunit of SecYEG in E. coli is used. In E. coli, SecY is registered in GenBank: CAD6001560.1, SecE is registered in GenBank: CAD6022906.1, and SecG is registered in GenBank: CAD6002359.1.

[0025] The protein contained in the molecule passage channel for amino acid sequence analysis has 80% or more homology with the SecYEG protein. More specifically, it has 80% or more homology with any of the subunits of SecYEG in any of the above-mentioned biological species. Homology includes cases where any amino acid is substituted with an amino acid derivative or a substituted amino acid. The homology may be 90% or more, or may be 95% or more.

[0026] The proteins contained in the molecule passage channel for amino acid sequence analysis may further include a SecA protein. SecA is conserved in many biological species, and SecA from any organism may be used as appropriate. In this embodiment, SecA from E. coli is used. SecA from E. coli is registered in GenBank: CAD6005338.1.

[0027] The phrase "the protein contained in the molecule passage channel for amino acid sequence analysis includes SecA protein" may mean that SecA is contained in a state dissociated from SecYEG, or that SecA is contained in a state associated with SecYEG. In this specification, a state in which SecA is associated with or fused to SecYEG may be referred to as a SecA-SecYEG complex. In addition, in this specification, a fusion protein in which SecA is fused with any subunit of SecYEG may be particularly referred to as SecYAEG.

[0028] In the amino acid sequence analysis molecule passage channel of this embodiment, three SecYEG subunits are associated to form a substantially cylindrical shape. This cylindrical SecYEG is embedded in a manner that penetrates the lipid bilayer membrane of the amino acid sequence analysis molecule passage membrane described below. SecA is associated with SecYEG. When the peptide molecule has the signal sequence, the molecules of the amino acid sequence analysis molecule passage channel recognize the signal sequence as a result of the action of the amino acid sequence analysis molecule passage channel. SecA consumes ATP to push the peptide molecule into the hollow space within the cylindrical structure of SecYEG, causing it to penetrate the hollow space of the cylindrical structure of SecYEG. The rate at which SecA penetrates the cylindrical SecYEG structure by the peptide molecule, i.e., the rate at which the peptide molecule passes through the amino acid sequence analysis molecule passage channel, can be controlled by the amount of ATP, etc.

[0029] The channel for passing amino acid sequence analysis molecules may have another form that does not have SecA. Even if it does not have SecA, peptide molecules may pass through SecYEG.

[0030] (Molecular permeability membrane for amino acid sequence analysis) The molecular permeability membrane for amino acid sequence analysis of this embodiment is a molecular permeability membrane for use in a method for analyzing the amino acid sequence of a peptide molecule, and includes the molecular permeability channel for amino acid sequence analysis and a lipid bilayer membrane.

[0031] The lipid bilayer membrane may be any known one. In this embodiment, for example, a lipid bilayer membrane containing a lipid such as DOPC (dioleoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPG (dioleoylphosphatidylglycerol), or DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) can be used. In this embodiment, a lipid bilayer membrane containing DOPC, DOPE, and DOPG is used.

[0032] Furthermore, it is preferable that the lipid bilayer membrane contains a charged lipid. The lipid bilayer membrane is used in a state in which the protein molecules of the amino acid sequence analysis molecule passage channel penetrate the membrane, and if a charged lipid is mixed into the lipid bilayer membrane, the permeability of the molecules when they pass through the amino acid sequence analysis molecule passage channel is good. As for the charge of the lipid, it is preferable to use the above-mentioned lipids, taking into account, for example, the contribution of PE in the DOPE and the contribution of the negative charge of PG in the DOPG. In addition, anionic lipids, non-membrane lipids, etc. may be mixed.

[0033] When manufacturing the amino acid sequence analysis molecule permeable membrane of this embodiment, protein molecules of the amino acid sequence analysis molecule permeable channel are incorporated into the lipid bilayer membrane. The incorporation process preferably involves applying a voltage to incorporate the protein molecules of the amino acid sequence analysis molecule permeable channel into the lipid bilayer membrane. This incorporation process can be performed using the process described in Peng Jing et al., Mol. BioSyst., 2016, 12, 480-489, or elsewhere.

[0034] The amino acid sequence analysis molecule permeable membrane of this embodiment may have amino acid sequence analysis molecule permeable channels arranged in parallel and integrated. For example, in the amino acid sequence analysis molecule permeable membrane, the amino acid sequence analysis molecule permeable channels are arranged in parallel. By arranging measuring members in parallel, each of which has an element capable of measuring information (e.g., ion current) about the sample that has passed through the amino acid sequence analysis molecule permeable channel, depending on the arrangement position, information about a large number of samples can be obtained. This technique of parallelization and integration using a parallel arrangement and measuring member configuration has been conventionally used in nucleic acid sequence analysis, and similar techniques can be used.

[0035] (Amino Acid Sequence Analysis Method) The amino acid sequence analysis method of this embodiment is a method for analyzing the amino acid sequence of a peptide molecule, and includes the step of passing the peptide molecule through a channel for amino acid sequence analysis containing a protein.

[0036] The peptide molecules and the molecule passage channel for amino acid sequence analysis can be appropriately selected from those described above. The protein in the molecule passage channel for amino acid sequence analysis containing the protein preferably includes a protein having 80% or more homology with the SecYEG protein. The protein in the molecule passage channel for amino acid sequence analysis containing the protein preferably further includes the SecA protein.

[0037] In the step of passing the peptide molecules through the amino acid sequence analysis molecule passage channel, the speed at which the peptide molecules pass is preferably 1 millisecond to 10,000 milliseconds. Preferably, it is 2 milliseconds or more and less than 5,000 milliseconds. This speed is intended to allow a peptide of approximately 300 amino acids to pass through in a few seconds, and can be appropriately selected from a few milliseconds to a few seconds. The speed at which the peptide molecules pass can be adjusted by the molecules and the conditions in the step. For example, it can be adjusted by the structure of the amino acid sequence analysis molecule passage channel. For example, it can be adjusted by the ease with which the peptide molecules pass through the amino acid sequence analysis molecule passage channel, depending on the presence or absence of SecA protein and the structure or modification of SecYEG protein. Furthermore, the speed at which SecA protein passes the peptide molecules through the amino acid sequence analysis molecule passage channel can be adjusted by the conditions in which the amino acid sequence analysis molecule passage channel is present, such as the composition of the solution.

[0038] In the amino acid sequence analysis method, it is also preferable to analyze the type of amino acid at the site by applying a voltage to the vicinity of the amino acid sequence analysis molecule passage channel of the peptide molecule and measuring an ion current.

[0039] In the amino acid sequence analysis method, urea may be added when the peptide molecule passes through the amino acid sequence analysis molecule passage channel. Addition of urea can promote the unfolding of secondary or higher structures, such as a folded structure, of the peptide molecule. The amino acid sequence analysis molecule passage channel may also have the effect of promoting the unfolding of a folded structure when the peptide molecule passes through, so it is not necessarily necessary to add the promoting component from the outside. However, the promoting component, such as urea, may be added as appropriate to facilitate the passage of the peptide molecule depending on its structure or to adjust the passage rate. The concentration of urea may be selected as appropriate and may be, for example, 2 to 8 M.

[0040] The amino acid sequence analysis method will be described with reference to an example in Fig. 1. A molecule-passing channel for amino acid sequence analysis 10 having a SecYEG protein 11 site and a SecA protein 12 site is embedded in a lipid bilayer membrane 30 so that the cylindrical shape of the SecYEG protein 11 site penetrates the lipid bilayer membrane 30. Here, the site of the lipid bilayer membrane 30 where the SecA protein 12 is located is referred to as the outer membrane side 40, and the site sandwiching the membrane is referred to as the inner membrane side 50.

[0041] When a voltage is applied between the outer membrane side 40 and the inner membrane side 50, ions pass through the amino acid sequence analysis molecule passing channel 10 and move between the outer membrane side 40 and the inner membrane side 50. For example, a negative charge can be applied to the outer membrane side 40 and a positive charge can be applied to the inner membrane side 50. Here, the amount of ions flowing changes depending on the state of the amino acid sequence analysis molecule passing channel 10, for example, whether or not a larger molecule is passing through and the properties of that molecule, so that information on the state of the amino acid sequence analysis molecule passing channel 10, for example, information on the molecules in the amino acid sequence analysis molecule passing channel 10, can be obtained.

[0042] When ATP and Mg elements are present on the membrane outer side 40, the SecA protein 12 site recognizes a peptide molecule 20 having a signal sequence 21, consumes ATP (converts it to ADP), and allows the peptide molecule 20 to pass through the SecYEG protein 11 site. At this time, the information on the ions flowing between the membrane outer side 40 and the membrane inner side 50 changes depending on the information on the peptide molecule 20. Therefore, information on the peptide molecule 20 can be obtained by analyzing the information on the ions.

[0043] (Amino acid sequence analysis device) The amino acid sequence analysis device of this embodiment is an amino acid sequence analysis device equipped with the amino acid sequence analysis molecule passage channel, and is configured to pass a peptide molecule through the amino acid sequence analysis molecule passage channel and analyze the type of amino acid at the portion of the peptide molecule that has passed through the channel.

[0044] Specifically, the amino acid sequence analyzer of this embodiment may include the channel through which amino acid sequence analysis molecules pass, and may also include the membrane through which amino acid sequence analysis molecules pass.

[0045] The amino acid sequence analysis device of this embodiment may include a voltage application means for applying a voltage to a site of the peptide molecule that has passed through the amino acid sequence analysis molecule passage channel, and a current measurement means for measuring an ion current at the site. By applying a voltage to the vicinity of the amino acid sequence analysis molecule passage channel of the peptide molecule using the voltage application means and the current measurement means and measuring the ion current, the type of amino acid at the site can be analyzed, and the amino acid sequence analysis method can be appropriately performed.

[0046] The amino acid sequence analysis device of this embodiment may also include a sample addition means for adding peptide molecules to be analyzed as samples to the amino acid sequence analysis molecule permeable membrane. It may also include a molecule addition means for adding ATP or other molecules near the amino acid sequence analysis molecule permeable membrane. As described above, the addition of ATP or other molecules can control the conditions under which the peptide molecules pass through the amino acid sequence analysis molecule permeable channel, for example, the permeation speed. The amino acid sequence analysis device of this embodiment may also include other appropriate components of a molecular analysis device. For example, it may include a control means for controlling the voltage application means, current measurement means, and other operation or analysis means. It may also include an analysis means for analyzing information obtained by the current measurement means and other device conditions. It may also include a storage means for storing information obtained from the analysis means.

[0047] (Effects of this embodiment) According to this embodiment, it is possible to provide a molecule-permeable channel for amino acid sequence analysis, a molecule-permeable membrane for amino acid sequence analysis, an amino acid sequence analysis method, and an amino acid sequence analysis device that can analyze the amino acid sequences of peptide molecules with high efficiency, a small sample size, and low purity in a short time compared to conventional methods.

[0048] In this embodiment, the inventors focused on translocons, which have the function of actively transporting peptides. They reconstituted a translocon consisting of a SecA-fused SecYEG membrane protein, an ATP-hydrolyzing motor protein that pushes substrate peptides into the SecYEG pore from a specific direction in conjunction with the ATP hydrolysis process, into a lipid bilayer membrane, and were able to capture characteristic changes in current value that are thought to be due to the passage of peptide molecules. These current changes are thought to contain amino acid information, and it is thought that amino acid sequence information can be obtained using machine learning, etc. It is expected that sequencing will be developed that can directly determine the amino acid sequence of peptides that cannot be amplified by these translocon-based nanopore measurement methods at the single molecule level.

[0049] The development of a peptide sequencer using the translocon-type nanopore measurement method based on this embodiment is expected to have a tremendous impact. Because new sequence information can be obtained, comprehensive information on proteins as well as on trace peptides, including unknown short peptides, can be obtained in vivo. This is expected to be applied not only to the field of life science, but also to the fields of agriculture, nutrition, engineering, pharmacy, and medicine.

[0050] Although the embodiment of the present invention has been described above, the present invention is not limited to the above embodiment and various modifications can be made.

[0051] The effects of the present invention will be made clearer by the following examples and comparative examples. Note that the present invention is not limited to the following examples, and can be practiced by making appropriate changes within the scope of the present invention.

[0052] [Test Example 1] (Test Equipment) Tests were performed using an Orbit 16 TC (manufactured by Nanion Technologies) for lipid bilayer membrane formation and measurement. This equipment is capable of constructing a lipid bilayer membrane on a flat surface, inserting a channel protein into the membrane, and monitoring changes in ionic current values ​​in the presence of an applied voltage. The following reagents were prepared: 10 mM HEPES KOH pH 7.4 1 M KCl; mixed lipid (40% DOPC / 30% DOPE / 30% DOPG) 10 mg / mL; SecYAEG-liposome 8 μg / mL; ATP 100 mM; MgCl ２ 100 mM prePhoA 222.7 μg / mL (8 M Urea, 50 mM Tris-HCl pH 8, 10 mM 2-mercaptoethanol)

[0053] Here, SecYAEG is a fusion protein formed by genetically linking SecA and SecYEG. Because SecA easily dissociates from SecYEG, this study used SecYAEG, which is a fusion protein expressed in which the C-terminus of one of the SecYEG subunits, SecY, is linked to the N-terminus of SecA via a linker, and then associated with the other two subunits, SecE and SecG, which were separately expressed (PLoS One. 2017 Aug 18;12(8):e0183434.). SecYAEG-liposomes are liposomes into which SecYAEG has been introduced. SecYAEG and SecYAEG-liposomes were provided by the Tsukasaki Laboratory at the Nara Institute of Science and Technology.

[0054] The lipid mixture (40% DOPC / 30% DOPE / 30% DOPG) that forms the lipid bilayer membrane was used to improve the permeability of SecYEG by incorporating a charged lipid. PrePhoA was used as a test peptide molecule that passes through the SecYEG-containing amino acid sequence analysis molecule passage channel. PrePhoA is a 471-amino acid precursor peptide of alkaline phosphatase with a SecYEG passage signal sequence, known to be a peptide that permeates SecYEG. It is known to become a mature form by being cleaved at the 20 amino acids (signal sequence) on the N-terminal side.

[0055] The test procedure was as follows: First, the model cell was inserted into the Orbit 16 TC and calibrated. Then, the chip was inserted and 200 μL of buffer was added. The temperature controller was set to 37°C. Air trapped in the channels was removed, and a voltage of 10 mV was applied to confirm that all channels were overloaded.

[0056] A lipid bilayer was formed on the chip by adding 1 μL of the lipid mixture and rotating the stirrer. A strong voltage was applied with a Zap to break the membrane. The unbroken membrane was multilayered, so it was blown away by pipetting. This procedure was repeated until most of the channels were broken with a single Zap.

[0057] Then, 20 μL of SecYAEG-liposome was added. A voltage of 100 mV was applied and recording was started. 4 μL of ATP, MgCl ２ When a stepwise change in current was observed, 30 μL of prePhoA was added and measurements were performed. ２ Measurements were also carried out under conditions in which prePhoA was not added.

[0058] (Measurement Results) First, under the condition that ATP is not added, MgCl ２ Regardless of the presence or absence of prePhoA, the baseline remained at 0 and no current flowed even when voltage was applied (results not shown).

[0059] Figure 2 is a graph showing the measurement results under the conditions of ATP(+), Mg(-), and prePhoA(-). (a) shows the change in voltage every 0 to 15 seconds. (b) shows the change in current under the same conditions. (c) shows a partial enlargement of the time range indicated by c in (b) (5.00 to 5.10 seconds). Under the condition of only ATP, as shown in (b), it was confirmed that a corresponding current flows when the voltage is changed. In other words, the current value changes depending on the voltage, confirming that the passage pores of the molecule passage channel for amino acid sequence analysis are open and ions are passing through. However, even when a portion of the area where the voltage was increased was enlarged, no blocking current was observed, and no waveform indicating any kind of permeation was observed.

[0060] Figure 3 is a graph showing measurements under conditions of ATP(+), Mg(+), and prePhoA(-). (a) shows the change in voltage every 0 to 15 seconds. (b) shows the change in current under the same conditions. (c) shows a partial enlargement of (b) from the time range indicated by c (0.50 to 5.60 seconds). Under conditions of ATP and Mg, as shown in (b), it was confirmed that a corresponding current flows when the voltage is changed. In other words, the current value changes depending on the voltage, confirming that the passage pores of the molecule passage channel for amino acid sequence analysis are open and ions are passing through. However, even when a portion of the region where the voltage was increased was enlarged, no blocking current was observed, and no waveform indicating any kind of permeation was observed.

[0061] Next, measurements were performed on an example in which the peptide molecule prePhoA was added. Figure 4 is a graph showing measurements under the conditions of ATP(+), Mg(-), and prePhoA(+). (a) shows the change in voltage every 0 to 26 seconds. (b) shows the change in current under the same conditions. (c) shows a partially enlarged view of the time range indicated by c in (b) (13.02 to 13.12 seconds). Under the conditions of ATP and prePhoA, as shown in (b), it was confirmed that the pore channel of the amino acid sequence analysis molecule passage channel was open and ions were passing through. When the obtained waveform was enlarged as shown in (c), a spike-like waveform with a short dwell time was observed. Half the baseline was set as a threshold, and events exceeding this threshold (indicated by the arrow in the figure) were extracted and analyzed. The waveform obtained under these conditions is hereafter referred to as "waveform A."

[0062] Figure 5 is a graph showing measurements under conditions of ATP(+), Mg(+), and prePhoA(+). (a) shows the change in voltage every 0 to 25 seconds. (b) shows the change in current under the same conditions. (c) shows an enlarged view of a portion of (b) from the time range indicated by c (0.4 to 1.4 seconds). Under conditions of ATP, Mg, and prePhoA, as in (b), it was confirmed that the pore channel of the amino acid sequence analysis molecule passage channel was open and ions were passing through. When the obtained waveform was enlarged as in (c), two different types of waveforms (on the left and right of the dashed line in (c)) were confirmed, even though the voltage was the same.

[0063] Figure 6 is a further enlarged view of Figure 5. (a) is the same graph as (c) in Figure 5, showing further enlarged regions (b, c). (b) shows a partial enlargement of the time range indicated by b in (a) (0.42 seconds to 0.52 seconds), and (c) shows a partial enlargement of the time range indicated by c in (a) (0.84 seconds to 0.94 seconds). In Figure 6(b), which corresponds to the area to the left of the dashed line in Figure 5(c), a short spike waveform like waveform A was observed. This will be referred to as "waveform B" hereafter. In Figure 6(c), which corresponds to the area to the right of the dashed line in Figure 5(c), a previously unseen, oscillating waveform was observed. This will be referred to as "waveform C" . Events exceeding the threshold (respectively indicated by arrows) were similarly extracted for waveforms B and C.

[0064] Figure 7 is a graph of the entire trace of Figure 5 after passing through a 50 Hz low-pass filter. (a) shows 0 to 62 seconds, and (b) shows 62 to 125 seconds. The area where the baseline is high is the area where waveform B can be seen, and the area where the baseline is low is the area where waveform C can be seen. The boundary between waveform B and waveform C is indicated by a dashed line in the figure. It can be seen that the area where waveform B can be seen lasts for several seconds, and the area where waveform C can be seen lasts for several tens of seconds.

[0065] 8 is a schematic diagram showing the relationship between the interruption current and dwell time used in the subsequent analysis. The dwell time, which is the duration of one event, and the maximum current value ΔI that falls below the baseline I0 are used as feature quantities of the interruption current.

[0066] Figure 9 is a graph showing the ATP concentration dependence of waveform A. It is believed that as the ATP concentration (vertical axis, density in the figure) decreases, SecA activity decreases and the dwell time (horizontal axis) becomes longer. If this waveform indicates a permeation reaction driven by SecA, it is believed that lowering the ATP concentration would decrease SecA activity and lengthen the permeation time. However, since there was no significant change in the dwell time distribution even when the ATP concentration was changed, it is believed that waveform A represents a phenomenon that is independent of ATP concentration.

[0067] FIG. 10A is a graph showing waveform A. In the graph, dots represent waveform A (Mg(-)). FIG. 10B is a graph showing waveform B. In the graph, dots represent waveform B (Mg(+)). In the graph, a Mann-Whitney U test was performed on dwell time, with a p-value of 0.3866. The horizontal axis represents the logarithm of dwell time. The vertical axis represents delta I divided by baseline I0 and converted to a percentage, representing the rate of decline relative to the baseline. The plots of waveforms A and B overlap, and a Mann-Whitney U test was performed on dwell time, leading to the conclusion that these two waveforms do not represent different phenomena. Therefore, these waveforms may represent the same phenomenon.

[0068] FIG. 11 is a graph comparing waveform B and waveform C. In the figure, gray dots represent waveform B, and black dots represent waveform C. A Wilcoxon signed rank test was performed on the dwell time, with a p-value of << 0.01. A similar plot was created as above, and it was found that waveform C had more events with longer dwell times than waveform B, with less deep dwelling. A Wilcoxon signed rank test was performed on the dwell times of these two waveforms, and a significant difference was observed. Therefore, it is believed that these two waveforms represent different phenomena.

[0069] 12 is a schematic diagram summarizing the results of the example. As shown in (i) of the figure, under the condition where ATP was not added, no current flowed, i.e., no ions passed through, which is thought to be why no ion-passing pores were formed in the channel for passing molecules for amino acid sequence analysis.

[0070] As shown in Figure (ii), in the absence of magnesium ions, waveform A has an extremely short dwell time compared to previous studies, and is therefore not considered to be a waveform that indicates a peptide permeation reaction. Therefore, without magnesium ions, ATP is not hydrolyzed, SecA does not function, and peptide permeation does not occur. It can be inferred that waveform A probably represents a short-term reaction in which the peptide blocks and releases the SecYAEG hole. Furthermore, since it was confirmed that the channel is open under conditions with ATP present but no peptide, it is thought that SecYAEG undergoes a conformational change and adopts an open conformation upon ATP binding.

[0071] As shown in Figure (iii), when ATP, Mg, and peptide are all added, waveform B, like waveform A, is not considered to be a waveform that indicates peptide permeation. Waveform C persists for several tens of seconds, which is the same scale as the peptide permeation time in previous studies. Waveform B, like waveform A, is presumably the entry stage of permeation, where the peptide blocks or dissociates from the hole, while waveform C indicates peptide permeation. Therefore, it is thought that only when both ATP and magnesium ions are present can ATP be hydrolyzed, causing SecA to move and drive peptide permeation. In summary, this suggests that ATP is required for SecYAEG to adopt an open conformation, and that Mg ions are also required for SecA to function as a molecular motor and drive peptide permeation.

[0072] [Test Example 2] Previously, electrical measurements of SecYEG reconstituted in a membrane have been reported. For example, it has been reported that simply reconstituting wild-type SecYEG in a membrane does not open the channel (J. Bio. Chem, 289, 24611-24616). Under low ionic strength, the channel opens in the presence of secA, a signal peptide with a signal sequence, a translocation protein, and ribosomes, exhibiting a gating signal. However, under high ionic strength, in the presence of a signal peptide, a step signal indicating a gradual transition to an open state is exhibited (Cell, Vol. 69, 677-664). Mutations that repeatedly open and close SecYEG have also been identified (Molecular Cell, 26, 501-509). However, no clear peptide translocation signal has yet been reported. Therefore, we created a mutant (I403N) in which Ile403, the site predicted to be involved in SecY channel gating, was replaced with Asn, and the translocation activity was examined. I403N was prepared by the method described in Molecular Cell 26, 501-509, etc., and was confirmed by Western blotting to have the same molecular weight and activity toward ATP as the wild type (WT) (not shown).

[0073] Figure 13 is a schematic diagram of the measurement of the nanopore signal of SecY(I403N)AEG. The first experimental condition shown in the figure was a SecYEG (Sec complex) containing a short RH peptide consisting of a repeating sequence of arginine and histidine, and the I403N mutant SecY in the presence of a signal peptide. After the Sec complex was inserted into the membrane, the RH peptide and signal peptide were added, and the signal observed after the addition of ATP was examined. As shown in the time series, the addition order was roughly SecYAEG, RH, signal peptide, and ATP.

[0074] The components are summarized below: Buffer: 10 mM Hepes pH 7.4, 1 M KCl, 5 mM MgSO ４(Buffer A) Planar Membrane Lipids: DPhPC Peptide (RH): RHRHRHRRHHRRHHRRRHHHRRRHHHH (R: arginine, H: histidine) Peptide (signal peptide): MKQSTIALALLPLLFTPVTKA

[0075] 14 is a graph showing the change in current when each component of SecY(I403N)AEG was added. The current was measured in the same manner as in Test Example 1. (a) is a measurement diagram with a scale of 0.2 nA and 100 sec, and (b) is a partially enlarged view of the graph in (a) after the addition of ATP, with a scale of 0.1 nA and 5 sec.

[0076] As shown in the figure, a clear and strong signal reaction occurs immediately after the addition of ATP. In particular, in the shaded area in (a), signals fluctuating up and down were observed about four times. These are thought to reflect peptide translocation, and when enlarged in (b), multiple such fluctuating signals were observed within the signal. These fluctuations are thought to be due to the repeated sequence. As shown by the downward-sloping arrow in (b), the signal as a whole tended to gradually decrease.

[0077] Test Example 3 To further investigate the action of SecY(I403N)AEG, a peptide molecule containing many repeating sequences was synthesized as the peptide molecule to be detected, and this was used for verification. １－３１ -(YGRG) １０ The amino acid sequence of this gene is shown in SEQ ID NO: 2. The first 31 amino acids are the proOmpA sequence including the signal peptide sequence, followed by 10 repeats of Tyr-Gly-Arg-Gly (YGRG), and finally the His-Myc Tag sequence. 1-31 -(YGRG) 10(SEQ ID NO: 2): MKQSTIALALLPLLFTPVTKARTPEMPVLENYGRGYGRGYGRGYGRGYGRGYGRGYGRGYGRGYGRGYGRGHHHHHHHIDEEQKLISEEDLLRKR This sequence was synthesized using Escherichia coli, purified, and the molecular weight was confirmed by SDS-PAGE (not shown). Note that peptides with repeats of YGLG, YGTG, or YGSG could not be purified efficiently, so a peptide with a repeat of YGRG was used.

[0078] Figure 15 shows the proOmpA nanopore signal of SecY(I403N)AEG. １－３１ -(YGRG) １０ This is a schematic diagram of measurements using peptides. Since translocation was confirmed in the reconstituted liposome experimental system, we investigated what kind of signal would be obtained using a nanopore measurement system. The method was the same as in Test Example 2: after introducing SecYAEG, peptide and ATP were added separately, and the change in signal was measured.

[0079] FIG. 16 shows the proOmpA of SecY(I403N)AEG. １－３１ -(YGRG) １０ 1 is a graph showing changes in current upon addition of each component, including a peptide. Current measurements were performed in the same manner as in Test Example 1. (a) is a measurement diagram with a scale of 1 nA and 4 min in the diagram, (b) is a partially enlarged view of the diagram in (a) after addition of ATP with a scale of 400 pA and 3 sec in the diagram, and (c) is a partially enlarged view of a portion of the signal in (b) with a scale of 400 pA and 0.3 sec in the diagram.

[0080] As shown in the figure, after the addition of ATP, an increase in signal was observed followed by a gradual decrease. As shown in the shaded area in (a), after the addition of ATP, the signal did not return to the baseline immediately after the addition of peptide alone, and unknown noise continued after the shaded area. Furthermore, when the decreasing signal was enlarged, periodic fluctuations were observed, suggesting that the repeat sequence portion of the peptide was reflected.

[0081] [Test Example 4] ProOmpA, a peptide molecule having repeats used in Test Example 3 １－３１-(YGRG) １０ The influence of urea was investigated using the above method. Under many measurement conditions, such as those in the above test examples, a phenomenon was observed in which the signal traces shown up to that point did not return to the baseline even after the signal increased after the addition of ATP. In these tests, there was a possibility that the urea added for conditions such as unfolding the secondary structure of the peptide molecule had an effect. To investigate the cause of this, the experiment was attempted again by minimizing the concentration of urea taken up when the peptide was added. The proOmpA of the nanopore signal of SecY(I403N)AEG in Test Example 3 １－３１ -(YGRG) １０ Measurements were performed using peptides, but without adding urea to the measurement buffer.

[0082] For chips, 10 mM HEPES-KOH (pH 7.4), 1 M KCl buffer, 5 mM MgSO ４ A buffer solution containing 8 M urea, 50 mM Tris-Cl (pH 8), 0.3 M NaCl, and 10 mM 2-mercaptoethanol (7.5 μl) was added to prepare a flat membrane, and SecYAEG was added to observe current fluctuations at 20 mV. If fluctuations were observed, 2.5 μl of the peptide was added and measurements were taken, followed by the addition of ATP.

[0083] FIG. 17 shows the proOmpA of SecY(I403N)AEG. １－３１ -(YGRG) １０ Graphs showing changes in current upon addition of each component containing peptide but not urea. (a) is a graph after adding 30 pmol of SecYAEG (I403N), (b) is a graph after further adding 30 pmol of YGRG, (c) is an enlarged view of a portion of the peak in (b), (d) is a graph after further adding ATP, and (d) is an enlarged view of the peak in the shaded portion in (c).

[0084] FIG. 18 shows the proOmpA of SecY(I403N)AEG. １－３１ -(YGRG) １０1A and 1B are graphs showing changes in current upon addition of each component containing peptide but not containing urea, where (a) is a graph subsequent to the graph in FIG. 1, and (b), (c), and (d) are enlarged views of the peaks in the shaded areas in (a).

[0085] From these figures, the signal returns to baseline, and the effect of urea on membrane lipids and the Sec translocon disappears. All of the signals in the figures are considered to be peptide transit signals. In other words, in the case of the peptide in this test example, the peptide transit signal can be detected without adding urea, and since there is no effect of urea on the signal, it can be detected more clearly. From these results, it is considered effective to appropriately select the presence or concentration of urea in the detector depending on the structure of the peptide molecule.

[0086] [Test Example 5] Based on the verification of Test Example 4, it was verified whether analysis using wild-type (non-mutated) SecYAEG was possible in a test system without adding urea. The chip was prepared using a 10 mM HEPES-KOH (pH 7.4), 1 M KCl buffer, and 5 mM MgSO ４ A buffer solution containing 190 μl of 8 M urea, 50 mM Tris-Cl (pH 8), 0.3 M NaCl, and 10 mM 2-mercaptoethanol (7.5 μl) was added to prepare a flat membrane, and SecYAEG (non-mutated) was added to observe current fluctuations at 20 mV. If fluctuations were observed, 2.5 μl of the peptide was first added and measurements were taken, followed by the addition of ATP. The rest of the procedure was the same as in Test Example 4.

[0087] FIG. 19 shows the proOmpA of SecYAEG (WT). １－３１ -(YGRG) １０ This is another graph showing the change in current when each component containing peptide but not urea was added. (a) is a graph showing the entire measurement with YGRG and ATP added, while (b), (c), (d), and (e) are enlarged views of some peaks in (a). The scales in (b) to (d) are the same as those in (e) (vertical axis: 200 pA, horizontal axis: 30 s).

[0088] As shown in the figure, even with wild-type (WT) SecYAEG, signals returned to baseline without the addition of urea. Several repeating changes were observed in these signals. These condition studies are likely to provide useful insights for future development of detection methods capable of detecting differences in the amino acid sequences of peptides.

[0089] Test Example 6: We verified whether the peptide molecule under analysis needed to be bound to a signal sequence. ProOmpA (a wild-type peptide with a signal sequence) and proOmp(Δss) (a peptide in which the signal sequence was removed from proOmpA) were subjected to a membrane permeation test with SecYAEG in the same manner as in Test Example 1, and detected by Western blotting using an anti-proOmpA antibody (results not shown).

[0090] In Western blotting using anti-proOmpA antibodies, for samples to which ATP had been added, both proOmpA and proOmp(Δss) showed bands of proOmp that had passed through the membrane. However, the band of proOmp(Δss) was slightly fainter after 30 minutes than that of proOmpA, which has a signal sequence. These results suggest that for the molecule passage channel for amino acid sequence analysis of this embodiment, it may not be essential that the peptide molecule to be analyzed has a signal sequence. On the other hand, having a signal sequence may improve the speed, efficiency, etc. of passage through the channel.

[0091] Although the embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims.

[0092] According to the present invention, it is possible to provide a molecule-permeable channel for amino acid sequence analysis, a molecule-permeable membrane for amino acid sequence analysis, an amino acid sequence analysis method, and an amino acid sequence analysis device that can analyze the amino acid sequences of peptide molecules with high efficiency, in a short time, and with a small sample size, with a low degree of purification, compared to conventional methods.

Claims

1. A molecule passage channel for amino acid sequence analysis, used in a method for analyzing the amino acid sequence of a peptide molecule, for passing said peptide molecule, comprising a protein having 80% or more homology with the SecYEG protein.

2. The molecule-passing channel for amino acid sequence analysis according to claim 1, further comprising SecA protein.

3. A molecule-permeable membrane for use in a method for analyzing the amino acid sequence of a peptide molecule, comprising the molecule-permeable channel for amino acid sequence analysis according to claim 1 or 2 and a lipid bilayer membrane.

4. A method for analyzing the amino acid sequence of a peptide molecule, comprising the step of passing the peptide molecule through a channel for passing molecules for amino acid sequence analysis that contains a protein.

5. The amino acid sequence analysis method according to claim 4, wherein the protein of the molecule-transport channel for amino acid sequence analysis containing the protein comprises a protein having 80% or more homology with the SecYEG protein.

6. The method for analyzing an amino acid sequence according to claim 4 or 5, wherein the protein of the molecule-passing channel for amino acid sequence analysis containing the protein further comprises SecA protein.

7. The amino acid sequence analysis method according to claim 4 or 5, wherein in the step of passing the peptide molecules through a molecule passage channel for amino acid sequence analysis containing the protein, the speed at which the peptide molecules pass is 1 millisecond to 10,000 milliseconds.

8. The amino acid sequence analysis method according to claim 4 or 5, wherein a voltage is applied to a portion of the peptide molecule that has passed through the amino acid sequence analysis molecule passage channel, and the type of amino acid at that portion is analyzed by measuring the ionic current.

9. An amino acid sequence analyzer equipped with a molecule passage channel for amino acid sequence analysis according to claim 1 or 2, configured to pass a peptide molecule through said molecule passage channel for amino acid sequence analysis and analyze the type of amino acid at the site of said peptide molecule that has passed through said channel.

10. The amino acid sequence analyzer according to claim 9, comprising a membrane for passing amino acid sequence molecules, the membrane comprising the channel for passing amino acid sequence molecules and a lipid bilayer membrane.

11. The amino acid sequence analysis device according to claim 9, comprising: a voltage application means for applying a voltage to the site of the peptide molecule that has passed through the amino acid sequence analysis molecule passage channel; and a current measurement means for measuring the ionic current at the site.

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