JP2021050915A - CLEAVAGE METHOD, ANALYTICAL METHOD AND SPECIFIC CLEAVAGE REAGENT THAT SPECIFICALLY CLEAVE Cα-C BOND AND/OR SIDE CHAIN OF PEPTIDE BACKBONE - Google Patents

Abstract

To cleave the Cα-C bond and side chain of the peptide backbone to detect the product with high sensitivity.SOLUTION: The cleavage method is a cleavage method that specifically cleaves the Cα-C bond and/or side chain of the peptide main chain, and includes: preparing a sample containing a molecule having a peptide main chain in which multiple amino acids are peptide-bonded to each other; and performing laser irradiation of the sample in the presence of at least one of 3,5-dihydroxy-2-nitrobenzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid.SELECTED DRAWING: Figure 5

Description

The present invention relates to cleavage methods, analytical methods and specific cleavage reagents that specifically cleave the Cα-C bond and / or side chain of the peptide backbone.

After specifically cleaving a specific bond in a molecule such as a protein or peptide, the resulting molecule is analyzed. In such an analysis, the amino acid sequence can be easily determined by analyzing the data by utilizing the peculiarity of cleavage.

As an example of the above analysis, there is an analysis (MALDI-ISD analysis) that utilizes in-source decomposition during matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). The most widely used matrix in this MALDI-ISD analysis is 1,5-diaminonaphthalene (1,5-DAN). By specifically cleaving the N-Cα bond, 1,5-DAN mainly produces c-series ions and z-series ions as fragment ions (ISD fragment ions) generated by ISD. In addition to 1,5-DAN, 2,5-dihydroxybenzoic acid (2,5-DHB) and 5,1-aminonaphthol (5,1-ANL) are examples of matrices that specifically cleave N-Cα bonds. ) (Patent Document 1), 1,5-dihydroxynaphthalene (1,5-DHN), 5-aminosalicylic acid (5-ASA), 2-aminobenzoic acid (2-AA), and 2-aminobenzamide (2-aminobenzamide). AB) and the like have been reported (Non-Patent Documents 1, 2, 3, 4). However, in these matrices, it is difficult to distinguish between leucine and isoleucine, which are isomers with different side chain structures and the same mass, and c (n-1) obtained by lateral cleavage of proline (Pro) at the nth residue. ) There is a problem that series ions are not generated. In addition, the most widely used 1,5-DAN is known to have problems such as high toxicity, high volatility, easy oxidation, and unstable reagents.

On the other hand, a matrix that mainly produces a-series ions, x-series ions, and d-series ions can be used as ISD fragment ions by specifically cleaving the Cα-C bond. In this case, leucine and isoleucine can be distinguished by d-series ions, and fragment ions are also generated by cleavage across proline (Pro), so that amino acid sequence information can be obtained more reliably. As a matrix for ISD that specifically cleaves the Cα-C bond, 5-nitrosalicylic acid (5-NSA), 4-nitrosalicylic acid (4-NSA), 3-nitrosalicylic acid (3-NSA), 7,7 , 8,8-Tetracyanoquinodimethane (TCNQ), and 2,5-bis (2-hydroxyethoxy) -7,7,8,8-Tetracyanoquinodimethane (bisHE-TCNQ) have been reported. However, there is a problem that the sensitivity is generally lower than that of a matrix that specifically cleaves an N-Cα bond such as 1,5-DAN (Non-Patent Documents 1, 2, 3, 4).

In recent years, the inventors have made 3-hydroxy-4-nitrobenzoic acid (3H4NBA) and its isomers more sensitively as a-series ions and d-series ions as ISD fragment ions by Cα-C bond and side chain cleavage. (Patent Documents 2 and 3, Non-Patent Documents 5 and 6). It has been confirmed that these matrices generate a-series ions and d-series ions by Cα-C bond and side chain cleavage with sensitivity equal to or higher than 1,5-DAN. Until then, this 3H4NBA has been reported as a matrix that highly sensitively ionizes peptides modified with 2-nitrobenzenesulfenyl groups (Patent Documents 4 and Non-Patent Documents 7), and Cα in the peptide backbone of proteins and peptides. The property of specifically cleaving the -C bond and side chain was not known.

Japanese Unexamined Patent Publication No. 2013-130570 JP-A-2017-166999 Japanese Unexamined Patent Publication No. 2017-207312 Japanese Unexamined Patent Publication No. 2004-326391

Daiki Asakawa, Mitsuo Takayama "Ca -C Bond Cleavage of the Peptide Backbone in MALDI In-Source Decay Using Salicylic Acid Derivative Matrices" Journal of American Society for Mass Spectrometry (2011) Vol.22, pp1224-1233 Daiki Asakawa, Motoshi Sakakura, Mitsuo Takayama "Matrix Effect on In-Source Decay Products of Peptides in Matrix-Assisted Laser Desorption / ionization" Mass Spectrometry Vol.1 (2012). A0002. Daiki Asakawa "Principles of Hydrogen Radical Mediated Peptide / Protein Fragmentation during Matrix-Assisted Laser Desorption / Ionization Mass Spectrometry" Moss Spectrometry Reviews (2016) Vol.35, pp535-556 Daiki Asakawa "Developments in Matrix Molecules for MALDI-ISD Mass Spectrometry" Journal of Mass Spectrometry Society of Japan (2016), Vol. 64, No. 5, pp187-190 Yuko Fukuyama, Shunsuke Izumi, and Koichi Tanaka "3-Hydroxy-4-nitrobenzoic Acid as a MALDI Matrix for In-Source Decay" Analytical Chemistry (2016) Vol. 88, pp8058-8063 Yuko Fukuyama, Shunsuke Izumi, Koichi Tanaka "3-Hydroxy-2-Nitrobenzoic Acid as a MALDI Matrix for In-Source Decay and Evaluation of the Isomers" Journal of American Society for Mass Spectrometry (2018) Vol. 29, pp 2227-2236 Ei-ichi Matsuo, Chikako Toda, Makoto Watanabe, Noriyuki Ojima, Shunsuke Izumi, Koichi Tanaka, Susumu Tsunasawa, Osamu Nishimura "Selective detection of 2-nitrobenzene sulfenyl-labeled peptide by matrix-assisted laser desorption / ionization-time of flight mass spectrometry using a novel matrix "Proteomics (2006) Vol.6, pp2042-2049

There is a lack of a matrix that sensitively cleaves the Cα-C bonds and / or side chains of the peptide backbone.

本発明の第２の態様は、第１の態様の切断方法により前記分子を切断することと、前記照射により前記ペプチド主鎖のＣα−Ｃ結合および／または側鎖が切断されることに基づいて前記ペプチド主鎖のアミノ酸配列を決定することとを備える、分析方法に関する。
本発明の第３の態様は、ペプチド主鎖のＣ−Ｃα結合および／または側鎖を切断するための特異的切断試薬であって、下記構造式（１）で示される３，５−ジヒドロキシ−２−ニトロ安息香酸、および、下記構造式（２）で示される３，５−ジヒドロキシ−４−ニトロ安息香酸の少なくとも一つを含む、特異的切断試薬に関する。

The first aspect of the present invention is a method for specifically cleaving a Cα-C bond and / or a side chain of a peptide main chain, wherein a molecule having a peptide main chain in which a plurality of amino acids are peptide-bonded to each other is obtained. Prepare a sample containing the peptide, 3,5-dihydroxy-2-nitrobenzoic acid represented by the following structural formula (1), and 3,5-dihydroxy-4-nitrobenzoic acid represented by the following structural formula (2). The present invention relates to a cutting method comprising irradiating the sample with a laser in the presence of at least one acid.

A second aspect of the present invention is based on cleaving the molecule by the cleaving method of the first aspect and cleaving the Cα-C bond and / or side chain of the peptide backbone by the irradiation. The present invention relates to an analytical method comprising determining the amino acid sequence of the peptide backbone.
A third aspect of the present invention is a specific cleavage reagent for cleaving the C-Cα bond and / or side chain of the peptide main chain, which is represented by the following structural formula (1), 3,5-dihydroxy-. It relates to a specific cleavage reagent containing 2-nitrobenzoic acid and at least one of 3,5-dihydroxy-4-nitrobenzoic acid represented by the following structural formula (2).

According to the present invention, the Cα-C bond and / or side chain of the peptide main chain can be cleaved to detect the product with high sensitivity.

FIG. 1 is a flowchart showing the flow of the analysis method of one embodiment. FIG. 2 is a conceptual diagram for explaining the cleavage of the peptide main chain. FIG. 3 is a conceptual diagram for explaining the generation of a-series ions, x-series ions, and d-series ions. FIG. 4 is a conceptual diagram showing a mass spectrometry kit. FIG. 5 is an ISD spectrum of an N-acetyl-renin substrate using 35H2NBA (upper), 35H4NBA (middle) and 3H4NBA (lower) as the ISD matrix. FIG. 6 is an ISD spectrum of angiotensin I using 35H2NBA (upper), 35H4NBA (middle) and 3H4NBA (lower) as the ISD matrix. FIG. 7 is an ISD spectrum of amyloid β (1-16) using 35H2NBA (upper), 35H4NBA (middle) and 3H4NBA (lower) as the ISD matrix. FIG. 8 is an ISD spectrum of ACTH (18-39) using 35H2NBA as the ISD matrix. FIG. 9 is an ISD spectrum of amyloid β (1-40) using 35H2NBA as the ISD matrix.

Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings.

FIG. 1 is a flowchart showing the flow of the analysis method of the present embodiment. In step S1001, a sample and a matrix reagent are prepared.

(About the sample)
The sample is not particularly limited as long as it contains or may contain a molecule having a peptide main chain containing a plurality of amino acids linked by peptide bonds (hereinafter, referred to as a molecule to be analyzed) such as a protein or a peptide. In the following, the molecule to be analyzed may be arbitrarily modified as long as it contains the Cα-C bond of the cleaving peptide backbone. The term peptide can be a molecule having a peptide backbone consisting of 2 or more and less than 50 amino acids, and the term protein can be a molecule having a peptide backbone consisting of 50 or more amino acids, with the exception of convention. Yes, the boundaries of the range of peptides and proteins are not limited to this.

(Matrix reagent)
The matrix reagents are of 3,5-dihydroxy-2-nitrobenzoic acid represented by the following structural formula (1) and 3,5-dihydroxy-4-nitrobenzoic acid represented by the following structural formula (2). Includes at least one. Hereinafter, 3,5-dihydroxy-2-nitrobenzoic acid is appropriately referred to as 35H2NBA, and 3,5-dihydroxy-4-nitrobenzoic acid is appropriately referred to as 35H4NBA.

As described below, 35H2NBA and 35H4NBA act as specific cleavage reagents that specifically cleave the peptide backbone and / or side chains. When step S1001 is completed, step S1003 is started.

In step S1003, a sample for mass spectrometry is prepared. In the following embodiments, an example of mass spectrometry (MALDI-MS) in which Matrix Assisted Laser Desorption / Ionization (MALDI) is performed will be described.

In the preparation of a sample for mass spectrometry, a droplet of a mixed solution containing a sample and a matrix reagent is formed on a sample plate for MALDI, and then the solvent is removed from the mixed solution to contain the molecule to be analyzed and the matrix. A mixed crystal is obtained as a sample for mass spectrometry.

The sample plate for MALDI is not particularly limited, and known ones and the like can be used. The sample plate for MALDI can be a conductive metal plate, for example, a stainless steel plate.

Solvents for preparing samples for mass analysis include water, acetonitrile (ACN), trifluoroacetic acid (TFA), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF) and dimethyl sulfoxide (DMSO) or these. It is selected from a solution obtained by mixing at least two of the above. More specifically, examples of the solvent include ACN aqueous solution, ACN-TFA aqueous solution, MeOH-TFA, MeOH aqueous solution, EtOH-TFA aqueous solution, EtOH aqueous solution, THF-TFA aqueous solution, THF aqueous solution, DMSO-TFA aqueous solution, or A DMSO aqueous solution or the like can be used, and more preferably, an ACN aqueous solution or an ACN-TFA aqueous solution can be used. The concentration of ACN in the ACN-TFA aqueous solution is, for example, 10 to 90% by volume, preferably 25 to 75% by volume, and the concentration of TFA is, for example, 0.05 to 1% by volume, preferably 0.05 to 0.1% by volume. Can be.

The method for preparing the mixed solution containing the sample and the matrix reagent is not particularly limited. For example, a solvent is added to the sample to prepare a solution containing the sample (hereinafter referred to as a sample solution). On the other hand, the above solvent is added to the matrix reagent to prepare a solution containing the matrix (hereinafter referred to as a matrix solution). The prepared sample solution and the matrix solution can be dropped onto the sample plate for MALDI in any order to form droplets of a mixed solution of both. Such a method is called an on-target mix method because the sample and the matrix are mixed on the sample plate for MALDI. Alternatively, the matrix solution may be added to the sample, or the sample solution may be added to the matrix, and the obtained mixed solution of the sample and the matrix may be dropped onto the sample plate for MALDI to form droplets.
A sample for mass spectrometry may be prepared using a matrix additive. The type of such an additive is not particularly limited as long as it has an arbitrary effect such as improvement of sensitivity. In the preparation of a sample for mass spectrometry containing an additive, a solvent is added to the additive to prepare an additive solution, and the additive solution can be mixed with a sample solution, a matrix solution, or a mixture thereof. Alternatively, a sample solution, a matrix solution, or a mixture thereof may be added to the additive.

The size of the droplets formed on the MALDI sample plate is not particularly limited. If a well is formed in the sample plate for MALDI, a droplet can be formed so as to fit in the well. The size of the droplet can be, for example, 0.1 μL to 2 μL, for example, about 0.5 μL.

The method for removing the solvent from the droplets formed on the sample plate for MALDI is not particularly limited, and for example, natural drying or drying under vacuum can be performed. The amount of the matrix contained in the mass spectrometric sample corresponding to one droplet can be, for example, 1 pmol to 1,000 nmol, and preferably 10 pmol to 100 nmol. The amount of molecules contained in the sample can be, for example, 1 amol to 100 pmol, 100 amol to 50 pmol, or the like with respect to 10 nmol of the matrix.

The obtained mixed crystal has a substantially circular shape on the contact surface with the sample plate for MALDI. That is, the outer edge of the mixed crystal has a substantially circular shape. The average diameter of the mixed crystals may vary depending on the amount of the sample, the amount of the matrix, the amount of the dropped mixed solution, the composition of the solvent, etc., but is, for example, 0.1 to 3 mm, preferably 0.5 to 2 mm. Can be done. The average diameter is an arithmetic mean of the lengths of line segments in which a straight line passing through the center of gravity of the substantially circle is cut off at the outer edge of the mixed crystal in one sample for mass spectrometry.

When a normal metal plate is used as the sample plate for MALDI, the molecule to be analyzed mainly exists in the above-mentioned approximately circle. Therefore, it is possible to easily ionize the molecule to be analyzed without specifying the laser irradiation position at the time of ionization. When step S1003 is completed, step S1005 is started.

In step S1005, the mass spectrometric sample prepared in step S1003 is irradiated with a laser to perform mass spectrometric analysis. A laser is irradiated to a sample for mass spectrometry including a sample and a matrix, and ions (hereinafter referred to as fragment ions) dissociated by in-source decomposition (ISD) are generated. The generated fragment ions move by the action from the electric field formed by the conductive sample plate for MALDI and the electrodes of the mass spectrometer (not shown), and are introduced into the mass spectrometer inside the mass spectrometer. Fragment ions mass-separated by a mass spectrometer are detected by an ion detector. The data obtained by the detection (hereinafter referred to as measurement data) is input to an information processing device such as a computer and stored in a storage medium such as a hard disk.

The type of mass spectrometry is not particularly limited as long as it is ionized by MALDI. Any one or more mass separation methods such as a quadrupole type, an ion trap type, and a time-of-flight type can be appropriately combined and used. The mass spectrometer is not particularly limited as long as it is a combination of ion sources for performing MALDI. Mass analyzers include, for example, MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass analyzer, MALDI-QIT (Matrix Assisted Laser Desorption Ionization-Quadrupole Ion Trap) mass analyzer, MALDI- Use a QIT-TOF (Matrix Assisted Laser Desorption Ionization-Quadrupole Ion Trap-Time of Flight) mass analyzer, MALDI-FTICR (Matrix Assisted Laser Desorption Ionization-Fourier Transformed Ion Cyclotron Resonance) mass analyzer, etc. Can be done.

The inventors have found that by using the above-mentioned 35H2NBA or 35H4NBA as a matrix, a-series ions, x-series ions, and d-series ions are preferably generated in insource decomposition.

FIG. 2 is a conceptual diagram for explaining the cleavage of the peptide main chain. FIG. 2 shows, as a non-limiting example, the structural formula F of a peptide formed by peptide bonds of four amino acids. In the figure, R ₁ , R ₂ , R ₃ and R ₄ indicate side chains of amino acids. When the Cα-C bond is cleaved in the peptide backbone, the ions corresponding to the N-terminal side of the molecule having the peptide backbone correspond to the a-series ions (a ₁ , a ₂ and a ₃ ) and the C-terminal side. Ions are called x-series ions (x ₁ , x ₂ and x ₃ ). Similarly, when the C-N bond is cleaved, the ions corresponding to the N-terminal side of the molecule having the peptide main chain are b-series ions (b ₁ , b ₂ and b ₃ ), and the ions corresponding to the C-terminal side. Are called y-series ions (y ₁ , y ₂ and y ₃ ). Similarly, when the N-Cα bond is cleaved, the ions corresponding to the N-terminal side of the molecule having the peptide main chain are c-series ions (c ₁ , c ₂ and c ₃ ), and the ions corresponding to the C-terminal side. Are referred to as z-series ions (z ₁ , z ₂ and z ₃ ).

FIG. 3 is a conceptual diagram showing an example of the generation process of a-series ions, x-series ions, and d-series ions assumed in the present embodiment. In the presence of 3,5-dihydroxy-2-nitrobenzoic acid (35H2NBA) as a matrix, a sample containing a protein, peptide or the like is irradiated with a laser. Hydrogen radicals are extracted from the molecule M to be analyzed by laser irradiation to 3-hydroxy-4-nitrobenzoic acid, and the molecule to be analyzed ([MH] ^· ) from which the hydrogen radicals have been extracted is generated (arrow A1). .. 35H2NBA has a high ability to accept hydrogen radicals of nitro groups, and easily causes a hydrogen radical abstraction reaction from the molecule to be analyzed. When the Cα-C bond cleavage of the peptide backbone is triggered by the extraction reaction of hydrogen radicals from the amide site of the peptide backbone by the nitro group of 35H2NBA, at least one of a-series ions and x-series ions is produced. (Arrow A2). In the example of FIG. 3, since the Cα-C bond is cleaved so that the a-series ions have radicals, d-series ions can be further generated from the a-series ion species by cleaving the side chain (arrow A3). For 35H4NBA, a-series ions, x-series ions, and d-series ions can be generated by the same mechanism. In the d-series ions generated in this way, different ions may be generated depending on whether the side chain of leucine is cleaved or the side chain of isoleucine is cleaved. For details, refer to Scheme 7 and the like in Non-Patent Document 3 below. In a preferred embodiment, 35H2NBA or 35H4NBA cleaves the Cα-C bond of the peptide backbone or the Cα-C bond and side chain of the peptide backbone.
Note that FIG. 3 is an example of the reaction, and the mechanism is not particularly limited as long as the target a-series ion, x-series ion or d-series ion is generated.

Returning to FIG. 1, when step S1005 is completed, step S1007 is started. In step S1007, the data (measurement data) obtained in the mass spectrometry of step S1005 is analyzed, and the amino acid sequence of the molecule to be analyzed is determined. The data analysis in step S1007 is preferably performed by a processor such as a CPU included in the above-mentioned information processing device, but is not particularly limited, and can be performed by any processing device or manual calculation.

From the measurement data, data corresponding to the mass spectrum in which the m / z (corresponding to the mass-to-charge ratio) of the detected molecule and the amplitude (intensity) of the signal when detected are associated (hereinafter referred to as mass spectrum data). Is created. The mass spectrum obtained in this embodiment may contain peaks corresponding to various fragment ions generated by insource decomposition.

In the laser irradiation in step S1005, the peptide main chain and / or side chain are cleaved to identify these peaks. For example, in the example of FIG. 2, and _{a 1} ions, it is _{a 2} ions and _{a 3} ions are detected. In this case, the mass difference between a ₁ ion and a ₂ ions, two hydrogen atoms, two carbon atoms, corresponding to one oxygen atoms and side chain R _2. mass difference between a ₂ ion and a ₃ ions, two hydrogen atoms, two carbon atoms, corresponding to an oxygen atom one and side chain R _3. The types of side chains are finite because they are based on about 20 typical amino acids. When at least one of a-series ions and x-series ions is detected, such regularity is used to assign the optimum fragment ion ion species to the peak pattern of the mass spectrum. The amino acid sequence of the protein or peptide from which the fragment ion is produced can be determined. From this point of view, it is also possible to distinguish between leucine and isoleucine from the assumed d-series ion m / z. For example, when a-series ions and d-series ions are detected, it is possible to determine the amino acid sequence capable of distinguishing between leucine and isoleucine. The algorithm for determining these amino acid sequences is not particularly limited, and known software or the like can be used.

(Mass spectrometry kit)
A mass spectrometric kit that is suitably used for the analysis method according to the present embodiment is provided. By providing the mass spectrometry kit, it is possible to more easily procure articles such as matrix reagents necessary for the above-mentioned analysis.

FIG. 4 is a conceptual diagram showing a mass spectrometry kit according to the present embodiment. The mass spectrometry kit 1 includes a matrix reagent 10 as a specific cleavage reagent for cleaving the C-Cα bond and / or side chain of the peptide main chain, and a container containing the matrix reagent 10 (hereinafter, reagent container 11). (Called). The matrix reagent 10 contains at least one of 3,5-dihydroxy-2-nitrobenzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid. Matrix reagent 10 is used as an ISD matrix that causes insource degradation.
The mass spectrometric kit 1 may include other articles used in mass spectrometric analysis, such as the above-mentioned solvent added to the matrix additive or the matrix reagent 10. The shape of the reagent container 11 is not particularly limited as long as the matrix reagent 10 can be stored.

(Aspect)
It will be understood by those skilled in the art that the plurality of exemplary embodiments or variations thereof described above are specific examples of the following embodiments.

(Item 1) The cleavage method according to one embodiment is a cleavage method for specifically cleaving a Cα-C bond and / or a side chain of a peptide main chain, in which a plurality of amino acids are peptide-bonded to each other. The sample is irradiated with a laser in the presence of at least one of 3,5-dihydroxy-2-nitrobenzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid. Be prepared to do. This allows the Cα-C bond and / or side chain of the peptide backbone to be cleaved and the product to be detected with high sensitivity.

(Item 2) In the analysis method according to one aspect, the molecule is cleaved by the cleaving method according to the first aspect, and the Cα-C bond and / or side chain of the peptide main chain is cleaved by the irradiation. It includes determining the amino acid sequence of the peptide backbone based on the above. This makes it possible to determine the amino acid sequence more accurately.

(Section 3) In the analysis method according to the other aspect, the analysis method according to the second aspect may include performing matrix-assisted laser desorption / ionization mass spectrometry, and the matrix-assisted laser desorption / ionization. In mass spectrometry, the mass spectrometric sample containing the sample and a matrix containing at least one of 3,5-dihydroxy-2-nitrobenzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid is irradiated. You may. As a result, the measurement data can be easily analyzed by MALDI, which easily generates monovalent ions, so that the amino acid sequence can be determined more accurately.

(Item 4) The analysis method according to the other aspect is the analysis method according to the third aspect, and in the matrix-assisted laser desorption / ionization mass spectrometry, the a-series ions and x-series ions obtained by the irradiation. At least one of may be detected. Thereby, the amino acid sequence can be accurately determined based on the regularity of these ions.

(Clause 5) The analysis method according to the other aspect is the analysis method according to the third aspect, and in the matrix-assisted laser desorption / ionization mass spectrometry, the a-series ions and d-series ions obtained by the irradiation. May be detected. This makes it possible to accurately determine the amino acid sequence while distinguishing between these leucines and isoleucines.

(Section 6) The specific cleavage reagent according to one embodiment is a specific cleavage reagent for cleaving the Cα-C bond and / or side chain of the peptide main chain, and is 3,5-dihydroxy-2-nitro. It contains at least one of benzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid. This allows the Cα-C bond and / or side chain of the peptide backbone to be cleaved and the product to be detected with high sensitivity.

The present invention is not limited to the contents of the above embodiments. Other aspects conceivable within the scope of the technical idea of the present invention are also included within the scope of the present invention.

Examples are shown below, but the present invention is not limited to the analysis conditions and the like in the following examples.

<Method>
1. As a sample solution, N-acetyl-renin substrate, angiotensin I, amyloid β (1-16), adrenocorticotropic hormone (ACTH) (18- A 20 pmol / μL 50% ACN 0.1% TFA aqueous solution containing 39) and amyloid β (1-40) was prepared. The numbers in parentheses indicate the sequence numbers of the amino acids from the N-terminal on the amino acid sequence.
2. As a matrix solution, a 10 mg / mL 50% ACN 0.1% TFA aqueous solution of 35H2NBA (Hiroshima University synthesis), 35H4NBA (Hiroshima University synthesis), and 3H4NBA (Hiroshima University synthesis, see Patent Document 2) was prepared. At this time, a saturated solution was used for 3H4NBA.
3. 0.5 μL of the sample solution was added dropwise onto the MALDI plate, and then 0.5 μL of the matrix solution was immediately added dropwise (on-target mix method). Then, it was air-dried to prepare a sample / matrix mixed crystal.
4. The MALDI plate was inserted into the laser ionized time-of-flight mass analyzer AXIMA Performance (manufactured by Shimadzu / Kratos), evacuated, and then MALDI-ISD analysis was performed on each crystal on the MALDI plate. This ISD analysis was performed in linear mode and positive ion mode.

<Result>
FIG. 5 shows the ISD matrix (35H2NBA (upper) and 35H4NBA (middle)) that specifically cleaves the Cα-C bond and side chain in the above-described embodiment, and the conventional Cα-C bond and side chain specifically. It is the ISD spectrum of the N-acetyl-renin substrate (MW 1801.1) using the ISD matrix 3H4NBA (bottom) to be cleaved. On the right side of FIG. 5, an enlarged view of the peak of a10-ion among a-series ions, its resolution and S / N ratio (signal / noise ratio) are shown. The resolution was obtained by dividing m / z corresponding to the peak intensity by the half width of the peak. The signal-to-noise ratio was obtained by dividing the peak intensity by the baseline noise. The ISD spectrum is a mass spectrum obtained by detecting fragment ions generated by insource decomposition. The horizontal axis of the ISD spectrum is the m / z of the detected ions, and the vertical axis shows the relative intensity (% Int.) Of the detected ions, which is the same for each of the following ISD spectra.

FIG. 6 shows the ISD matrix (35H2NBA (upper) and 35H4NBA (middle)) that specifically cleaves the Cα-C bond and side chain in the above-described embodiment, and the conventional Cα-C bond and side chain specifically. It is an ISD spectrum of angiotensin I (MW 1296.5) using the ISD matrix 3H4NBA (bottom) to be cleaved. On the right side of FIG. 6, an enlarged view of the peak of a6-ion among a-series ions, its resolution and S / N ratio are shown.

FIG. 7 shows the ISD matrix (35H2NBA (upper) and 35H4NBA (middle)) that specifically cleaves the Cα-C bond and side chain in the above-described embodiment, and the conventional Cα-C bond and side chain specifically. It is an ISD spectrum of amyloid β (1-16) (MW 1955.1) using the ISD matrix 3H4NBA (lower) to be cleaved. On the right side of FIG. 7, an enlarged view of the peak of a10-ion, its resolution and S / N ratio are shown.

FIG. 8 is an ISD spectrum of ACTH 18-39 (MW 2465.7) using an ISD matrix (35H2NBA) that specifically cleaves Cα-C bonds and side chains in the above embodiments. The right side shows an enlarged view of the peak of a11-ion among the a-series ions, and its resolution and S / N ratio.

FIG. 9 is an ISD spectrum of amyloid β 1-40 (MW 4329.8) using the ISD matrix (35H2NBA) that specifically cleaves the Cα-C bond and side chains in the embodiment described above.

When 35H2NBA and 35H4NBA were used as the matrix, ISD analysis of proteins or peptides detected below m / z 2000, both a-series ions and a-series ions resulting from specific cleavage of Cα-C bonds and side chains. D-series ions were generated (Figs. 5-7). At this time, the resolution and S / N of the ISD fragments by 35H2NBA and 35H4NBA were similar to or higher than those of the conventional matrix 3H4NBA (Figs. 5 to 7). In particular, the resolution of ISD fragments by 35H2NBA and 35H4NBA was higher than that of the conventional matrix 3H4NBA (Figs. 5 to 7).

In the ISD of ACTH 18-39 detected at m / z 2400 and above, ISD fragments were obtained with relatively high resolution and S / N when 35H2NBA was used as a matrix (Fig. 8). When 35H2NBA was used as a matrix, ISD fragments were obtained with relatively high sensitivity even for amyloid β 1-40 detected at m / z 4330 (Fig. 9).

From the above results, it was confirmed that 35H2NBA and 35H4NBA are effective as a novel ISD matrix that specifically cleaves the Cα-C bond and side chain. In particular, for proteins or peptides detected at m / z 2000 or less, all compounds showed high resolution and S / N, and it was confirmed that they are effective as a highly sensitive and highly accurate ISD matrix. .. In particular, it was confirmed that 35H2NBA can obtain highly sensitive and highly accurate ISD fragments even for proteins or peptides detected at m / z 2400 or higher.

1 ... Mass spectrometry kit, 10 ... Matrix reagent, 11 ... Reagent container.

Claims (6)

A cleavage method that specifically cleaves the Cα-C bond and / or side chain of the peptide backbone.
To prepare a sample containing a molecule having a peptide main chain in which multiple amino acids are peptide-bonded to each other.
In the presence of at least one of 3,5-dihydroxy-2-nitrobenzoic acid represented by the following structural formula (1) and 3,5-dihydroxy-4-nitrobenzoic acid represented by the following structural formula (2). , A cutting method comprising irradiating the sample with a laser.

To cleave the molecule by the cleaving method according to claim 1,
An analytical method comprising determining the amino acid sequence of the peptide backbone based on the cleavage of the Cα-C bond and / or side chain of the peptide backbone by the irradiation. In the analysis method according to claim 2,
With matrix-assisted laser desorption / ionization mass spectrometry
In the matrix-assisted laser desorption / ionization mass spectrometry, for mass spectrometry including a matrix containing at least one of 3,5-dihydroxy-2-nitrobenzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid and the sample. An analytical method in which the sample is irradiated. In the analysis method according to claim 3,
In the matrix-assisted laser desorption / ionization mass spectrometry, an analysis method for detecting at least one of a-series ions and x-series ions obtained by the irradiation. In the analysis method according to claim 3,
In the matrix-assisted laser desorption / ionization mass spectrometry, an analysis method for detecting a-series ions and d-series ions obtained by the irradiation. A specific cleavage reagent for cleaving the C-Cα bond and / or side chain of the peptide backbone.
It contains at least one of 3,5-dihydroxy-2-nitrobenzoic acid represented by the following structural formula (1) and 3,5-dihydroxy-4-nitrobenzoic acid represented by the following structural formula (2). Specific cleavage reagent.

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