JP7202554B2 - Cleavage methods, analytical methods and specific cleavage reagents specifically cleaving the Ca-C bonds and/or side chains of peptide backbones - Google Patents

Description

The present invention relates to cleaving methods, analytical methods and specific cleaving reagents that specifically cleave Cα-C bonds and/or side chains of peptide backbones.

After specifically cleaving a particular bond in a molecule such as a protein or peptide, the resulting molecule is analyzed. In such analysis, data analysis utilizing the specificity of cleavage facilitates determination of the amino acid sequence and the like.

An example of such an analysis is an analysis using in-source decomposition (MALDI-ISD analysis) during matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The most widely used matrix for this MALDI-ISD analysis is 1,5-diaminonaphthalene (1,5-DAN). 1,5-DAN specifically cleaves N—Cα bonds to generate mainly c-series ions and z-series ions as fragment ions generated by ISD (ISD fragment ions). In addition to 1,5-DAN, 2,5-dihydroxybenzoic acid (2,5-DHB), 5,1-aminonaphthol (5,1-ANL ) (Patent Document 1), 1,5-dihydroxynaphthalene (1,5-DHN), 5-aminosalicylic acid (5-ASA), 2-aminobenzoic acid (2-AA), and 2-aminobenzamide (2- AB), etc. have been reported (Non-Patent Documents 1, 2, 3, 4). However, with these matrices, it is difficult to distinguish between leucine and isoleucine, which are isomers with different side chain structures and the same mass. ) has the problem of not producing series ions. In addition, 1,5-DAN, which is the most widely used, is known to have problems such as high toxicity, high volatility, and instability as a reagent because it is easily oxidized.

On the other hand, by specifically cleaving the Cα-C bond, it is possible to use a matrix that mainly produces a-series ions, x-series ions, and further d-series ions as ISD fragment ions. In this case, it is possible to distinguish between leucine and isoleucine by d-series ions, and fragment ions are also produced by cleavage across proline (Pro), so that amino acid sequence information can be obtained more reliably. Matrices for ISD that specifically cleave Cα-C bonds include 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. (Non-Patent Documents 1, 2, 3, 4), but there is a general problem of lower sensitivity than matrices such as 1,5-DAN that specifically cleave N-Cα bonds.

Recently, the inventors have more sensitively identified 3-hydroxy-4-nitrobenzoic acid (3H4NBA) and its isomers as ISD fragment ions by Cα-C bond and side chain cleavage, a-series ions and d-series ions. (Patent Documents 2 and 3, Non-Patent Documents 5 and 6). These matrices have been shown to generate a-series and d-series ions from Cα-C bond and side chain cleavage with similar or greater sensitivity than 1,5-DAN. Until then, this 3H4NBA had been reported as a matrix for sensitive ionization of peptides modified with a 2-nitrobenzenesulfenyl group (Patent Document 4, Non-Patent Document 7), and Cα of the peptide backbone of proteins and peptides. The properties of specifically cleaving -C bonds and side chains were unknown.

JP 2013-130570 A JP 2017-166999 A JP 2017-207312 A JP-A-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-nitrobenzenesulfenyl-labeled peptide by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using a novel matrix" Proteomics (2006) Vol.6, pp2042-2049

A matrix that sensitively cleaves the Cα-C bonds and/or side chains of the peptide backbone is lacking.

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。

A first aspect of the present invention is a cleavage method for specifically cleaving Cα-C bonds and/or side chains of a peptide backbone, comprising: 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): irradiating said sample with a laser in the presence of at least one acid.

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

According to the present invention, Cα-C bonds and/or side chains of the peptide backbone can be cleaved and the products can be detected with high sensitivity.

FIG. 1 is a flow chart showing the flow of the analysis method of one embodiment. FIG. 2 is a conceptual diagram for explaining 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 kit for mass spectrometry. FIG. 5 shows the ISD spectra of N-acetyl-renin substrates using 35H2NBA (top), 35H4NBA (middle) and 3H4NBA (bottom) as ISD matrices. FIG. 6 is the ISD spectra of angiotensin I using 35H2NBA (top), 35H4NBA (middle) and 3H4NBA (bottom) as ISD matrices. FIG. 7 shows the ISD spectra of amyloid β(1-16) using 35H2NBA (top), 35H4NBA (middle) and 3H4NBA (bottom) as ISD matrices. FIG. 8 is the ISD spectrum of ACTH(18-39) using 35H2NBA as the ISD matrix. FIG. 9 is the ISD spectrum of amyloid β(1-40) using 35H2NBA as the ISD matrix.

Embodiments for carrying out the present invention will be described below with reference to the drawings.

FIG. 1 is a flow chart showing the flow of the analysis method of this embodiment. At step S1001, a sample and a matrix reagent are prepared.

(Regarding the sample)
The sample is not particularly limited as long as it contains or may contain a molecule having a peptide backbone comprising multiple amino acids linked by peptide bonds, such as a protein or peptide (hereinafter referred to as a molecule to be analyzed). In the following, the molecule to be analyzed may have any modification including a cleavable peptide backbone Cα-C bond. The term peptide may refer to molecules with a peptide backbone of 2 or more but less than 50 amino acids and the term protein may refer to molecules with a peptide backbone of 50 or more amino acids, with the customary exceptions. Yes, and the boundary between peptides and proteins is not limited to this.

(matrix reagent)
The matrix reagents are 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). contains at least one In the following, 3,5-dihydroxy-2-nitrobenzoic acid is arbitrarily referred to as 35H2NBA and 3,5-dihydroxy-4-nitrobenzoic acid is arbitrarily referred to as 35H4NBA.

As described below, 35H2NBA and 35H4NBA act as specific cleaving reagents that specifically cleave peptide backbones and/or side chains. After step S1001 ends, 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) with Matrix Assisted Laser Desorption/Ionization (MALDI) will be described.

In preparing a sample for mass spectrometry, droplets of a mixture containing a sample and a matrix reagent are formed on a MALDI sample plate, and then the solvent is removed from the mixture to form a droplet containing the molecule to be analyzed and the matrix. Mixed crystals are obtained as samples for mass spectrometry.

The MALDI sample plate is not particularly limited, and known plates can be used. The MALDI sample plate can be a conductive metal plate, such as a stainless steel plate.

Water, acetonitrile (ACN), trifluoroacetic acid (TFA), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF) and dimethylsulfoxide (DMSO) or is selected from a solution or the like obtained by mixing at least two of 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 An aqueous DMSO solution or the like can be used, and more preferably an aqueous ACN solution or an aqueous ACN-TFA 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

A method for preparing a mixture containing a sample and a matrix reagent is not particularly limited. For example, a solvent is added to a sample to prepare a solution containing the sample (hereinafter referred to as sample solution). On the other hand, the solvent is added to the matrix reagent to prepare a matrix-containing solution (hereinafter referred to as matrix solution). The prepared sample solution and matrix solution can be dropped onto the MALDI sample plate in any order to form droplets of a mixture of the two. Such a method is called an on-target mix method because the sample and matrix are mixed on the MALDI sample plate. Alternatively, the matrix solution may be added to the sample, or the sample solution may be added to the matrix, and the resulting mixed solution of the sample and matrix may be dropped onto the MALDI sample plate to form droplets.
A sample for mass spectrometry may be prepared using a matrix additive. The type of such additive is not particularly limited as long as it has an arbitrary effect such as improvement of sensitivity. In preparing a sample for mass spectrometry containing an additive, a solvent can be added to the additive to prepare an additive solution, and this 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 droplets formed on the MALDI sample plate is not particularly limited. If wells are formed in the MALDI sample plate, droplets can be formed so as to fit in the wells. The droplet size can be, for example, between 0.1 μL and 2 μL, and can be, for example, about 0.5 μL.

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

The obtained mixed crystal has a substantially circular shape at the contact surface with the MALDI sample plate. That is, the outer edge of the mixed crystal is substantially circular. The average diameter of the mixed crystal may vary depending on the amount of the sample, the amount of matrix, the amount of the mixed liquid added dropwise, the composition of the solvent, etc., but it should be, for example, 0.1 to 3 mm, preferably 0.5 to 2 mm. can be done. The average diameter is the arithmetic mean of the length of the line segment cut off at the outer edge of the mixed crystal from the straight line passing through the center of gravity of the approximate circle in one mass spectrometry sample.

When an ordinary metal plate is used as the MALDI sample plate, the molecules to be analyzed are mainly present within the approximate circle. Therefore, the molecules to be analyzed can be easily ionized without specifying the irradiation position of the laser during ionization. After step S1003 ends, step S1005 is started.

In step S1005, the sample for mass spectrometry prepared in step S1003 is irradiated with a laser to perform mass spectrometry. A sample for mass spectrometry including a sample and a matrix is irradiated with a laser to generate ions dissociated by In-Source Decay (ISD) (hereinafter referred to as fragment ions). The generated fragment ions are moved by the action of the electric field formed by the conductive MALDI sample plate 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 the mass spectrometer are detected by an ion detector. Data obtained by 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 ionization is performed by MALDI. Any one or more mass separation methods such as quadrupole, ion trap, and time-of-flight can be used in combination as appropriate. The mass spectrometer is not particularly limited as long as it is combined with an ion source for performing MALDI. The mass spectrometer is, for example, a MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectrometer, a MALDI-QIT (Matrix Assisted Laser Desorption Ionization-Quadrupole Ion Trap) mass spectrometer, a MALDI- QIT-TOF (matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight) mass spectrometer, MALDI-FTICR (matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance) mass spectrometer, etc. can be done.

The inventors found that the use of 35H2NBA or 35H4NBA described above as a matrix favorably produces a-series ions, x-series ions, and d-series ions in in-source decomposition.

FIG. 2 is a conceptual diagram for explaining cleavage of the peptide main chain. FIG. 2 shows structural formula F of a peptide formed by peptide bonding of four amino acids as a non-limiting example. 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 main chain, the ions corresponding to the N-terminal side of the molecule having the peptide main chain are a-series ions (a ₁ , a ₂ and a ₃ ), and the C-terminal side corresponds to ions are called x-series ions (x ₁ , x ₂ and x ₃ ). Similarly, when the C—N bond is broken, 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 called the 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 this embodiment. In the presence of 3,5-dihydroxy-2-nitrobenzoic acid (35H2NBA) as a matrix, a sample containing proteins, peptides, etc. is irradiated with a laser. Hydrogen radicals are abstracted by 3-hydroxy-4-nitrobenzoic acid from the molecule M to be analyzed by laser irradiation, ^and the molecules to be analyzed ([MH]) from which the hydrogen radicals are abstracted are generated (arrow A1). . The nitro group of 35H2NBA has a high ability to accept hydrogen radicals, and is likely to cause a hydrogen radical abstraction reaction from the molecule to be analyzed. When the hydrogen radical abstraction reaction from the amide site of the peptide backbone by the nitro group of 35H2NBA causes cleavage of the Cα-C bond of the peptide backbone, at least one of a-series and x-series ions is generated. (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 generated from the a-series ion species by side chain cleavage (arrow A3). 35H4NBA can generate a-series ions, x-series ions, and d-series ions by a similar mechanism. In the d-series ions generated in this way, different ions can be produced depending on whether the leucine side chain is cleaved or the isoleucine side chain is cleaved. For details, refer to Scheme 7 of Non-Patent Document 3 below. In a preferred embodiment, 35H2NBA or 35H4NBA cleaves the Cα-C bond of the peptide backbone or cleaves the Cα-C bond and side chains 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 desired a-series ions, x-series ions, or d-series ions are generated.

Returning to FIG. 1, after step S1005 ends, step S1007 is started. In step S1007, the data (measurement data) obtained by mass spectrometry in step S1005 are analyzed to determine the amino acid sequence of the molecule to be analyzed. The data analysis in step S1007 is preferably performed by a processor such as a CPU included in the above-described 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 produced by in-source decomposition.

These peaks are identified by utilizing that the peptide main chain and/or side chains are cleaved in the laser irradiation in step S1005. For example, in the example of FIG. 2, suppose a ₁ ion, a ₂ ion and a ₃ ion are detected. In this case, the mass difference between the a1 and _a2 ions corresponds to _two hydrogen atoms, two carbon atoms, one oxygen atom and the side chain _R2 . The mass difference between the a2 and _a3 ions corresponds to ₂ hydrogen atoms, ₂ carbon atoms, 1 oxygen atom and the side chain R3. The types of side chains are limited because they are based on about 20 representative amino acids. When at least one of the a-series ions and the x-series ions is detected, such regularity is used to assign the ion species of the optimum fragment ion to the peak pattern of the mass spectrum, The amino acid sequence of the protein or peptide from which the fragment ions are generated can be determined. From this point of view, and from the m/z of the assumed d-series ions, it is also possible to distinguish between leucine and isoleucine. For example, when a-series ions and d-series ions are detected, an amino acid sequence can be determined that can distinguish between leucine and isoleucine. Algorithms for determining these amino acid sequences are not particularly limited, and known software and the like can be used.

(mass spectrometry kit)
A mass spectrometry kit suitable for use in the analysis method according to the present embodiment is provided. By providing a mass spectrometry kit, it is possible to more easily procure articles such as matrix reagents necessary for the above-described analysis.

FIG. 4 is a conceptual diagram showing the kit for mass spectrometry according to this embodiment. A mass spectrometry kit 1 comprises a matrix reagent 10 as a specific cleaving reagent for cleaving a C-Cα bond and/or a side chain of a peptide main chain, and a container storing the matrix reagent 10 (hereinafter referred to as a reagent container 11 ). Matrix reagent 10 includes 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 to cause in-source degradation.
The mass spectrometry kit 1 may also contain other items used in mass spectrometry, such as the matrix additive or the above-described solvent added to the matrix reagent 10 . Moreover, the shape of the reagent container 11 is not particularly limited as long as the matrix reagent 10 can be stored.

(Mode)
It will be appreciated by those skilled in the art that the multiple exemplary embodiments described above, or variations thereof, are specific examples of the following aspects.

(Section 1) A cleavage method according to one aspect is a cleavage method for specifically cleaving a Cα-C bond and/or a side chain of a peptide main chain, wherein a plurality of amino acids are peptide-bonded to each other. and irradiating the sample with a laser in the presence of at least one of 3,5-dihydroxy-2-nitrobenzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid Prepare to do. This cleaves the Cα-C bond and/or side chains of the peptide backbone and allows sensitive detection of the product.

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

(Section 3) In an analysis method according to another aspect, in the analysis method according to the aspect of Clause 2, it may comprise performing matrix-assisted laser desorption/ionization mass spectrometry, wherein the matrix-assisted laser desorption/ionization In mass spectrometry, a sample for mass spectrometry comprising 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. may This makes it easier to analyze the measurement data by MALDI, which tends to generate monovalent ions, so that the amino acid sequence can be determined more accurately.

(Section 4) An analysis method according to another aspect is the analysis method according to the aspect of Clause 3, wherein in the matrix-assisted laser desorption/ionization mass spectrometry, a-series ions and x-series ions obtained by the irradiation At least one of may be detected. Thus, the amino acid sequence can be accurately determined based on the regularity of these ions.

(Section 5) An analysis method according to another aspect is the analysis method according to the aspect of Clause 3, wherein in the matrix-assisted laser desorption ionization mass spectrometry, a-series ions and d-series ions obtained by the irradiation may be detected. As a result, the amino acid sequence can be accurately determined while distinguishing between leucine and isoleucine.

(Section 6) A specific cleaving reagent according to one aspect is a specific cleaving reagent for cleaving a Cα-C bond and/or side chain of a peptide backbone comprising 3,5-dihydroxy-2-nitro At least one of benzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid. This cleaves the Cα-C bond and/or side chains of the peptide backbone and allows sensitive detection of the product.

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 in 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 examples below.

<Method>
1. Sample solutions include N-acetyl-renin substrate, angiotensin I, amyloid β (1-16), adrenocorticotropic hormone (ACTH) (18- 39), and 20 pmol/μL 50% ACN 0.1% TFA aqueous solution containing amyloid β (1-40) was prepared. Numbers in parentheses above indicate sequence numbers of amino acids from the N-terminus on the amino acid sequence.
2. 10 mg/mL 50% ACN 0.1% TFA aqueous solutions of 35H2NBA (synthesized at Hiroshima University), 35H4NBA (synthesized at Hiroshima University), and 3H4NBA (synthesized at Hiroshima University, see Patent Document 2) were prepared as matrix solutions. At this time, a saturated solution of 3H4NBA was used.
3. Immediately after dropping 0.5 μL of the sample solution onto the MALDI plate, 0.5 μL of the matrix solution was dropped (on-target mix method). After that, it was air-dried to prepare a sample/matrix mixed crystal.
4. The MALDI plate was inserted into a laser ionization time-of-flight mass spectrometer AXIMA Performance (manufactured by Shimadzu/Kratos), and after evacuation, each crystal on the MALDI plate was subjected to MALDI-ISD analysis. This ISD analysis was performed in linear mode and positive ion mode.

<Results>
FIG. 5 shows ISD matrices (35H2NBA (top) and 35H4NBA (middle)) that specifically cleave Cα-C bonds and side chains in the above embodiment and conventional Cα-C bonds and side chains that are specifically cleaved. ISD spectrum of N-acetyl-rennin substrate (MW 1801.1) with cleaving ISD matrix 3H4NBA (bottom). The right side of FIG. 5 shows an enlarged view of the peak of a10- ion among the a-series ions, and its resolution and S/N ratio (signal/noise ratio). Resolution was obtained by dividing the m/z corresponding to the peak intensity by the half-width of the peak. S/N ratio was obtained by dividing peak intensity by baseline noise. An ISD spectrum is a mass spectrum obtained by detecting fragment ions produced by in-source decomposition. The horizontal axis of the ISD spectrum is the m/z of the detected ions, and the vertical axis is the relative intensity (% Int.) of the detected ions.

FIG. 6 shows ISD matrices (35H2NBA (top) and 35H4NBA (middle)) that specifically cleave Cα-C bonds and side chains in the above embodiment and conventional Cα-C bonds and side chains that are specifically cleaved. ISD spectrum of angiotensin I (MW 1296.5) using the cutting ISD matrix 3H4NBA (bottom). The right side of FIG. 6 shows an enlarged view of the peak of the a6- ion among the a-series ions, and its resolution and S/N ratio.

FIG. 7 shows ISD matrices (35H2NBA (top) and 35H4NBA (middle)) that specifically cleave Cα-C bonds and side chains in the above embodiment and conventional Cα-C bonds and side chains that are specifically cleaved. ISD spectrum of amyloid β(1-16) (MW 1955.1) using the cleaving ISD matrix 3H4NBA (bottom). The right side of FIG. 7 shows an enlarged view of the a10- ion peak, its resolution and S/N ratio.

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 embodiment described above. 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 an ISD matrix (35H2NBA) that specifically cleaves Cα-C bonds and side chains in the embodiment described above.

When 35H2NBA and 35H4NBA were used as matrices, ISD analysis of proteins or peptides detected at m/z 2000 or less showed both a-series ions and d-series ions were produced (Figs. 5-7). At this time, the resolution and S/N of ISD fragments by 35H2NBA and 35H4NBA were comparable to or higher than those of 3H4NBA, the conventional matrix (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. 5-7).

For ISD of ACTH 18-39 detected at m/z 2400 or higher, 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, an ISD fragment was obtained with relatively high sensitivity even for amyloid β 1-40 detected at m/z 4330 (Fig. 9).

These results confirmed that 35H2NBA and 35H4NBA are effective as novel ISD matrices that specifically cleave Cα-C bonds and side chains. In particular, all compounds exhibited high resolution and S/N for proteins or peptides detected at m/z 2000 or less, confirming their effectiveness as highly sensitive and highly accurate ISD matrices. . In particular, 35H2NBA was confirmed to yield highly sensitive and highly accurate ISD fragments even for proteins or peptides detected at m/z 2400 or higher.

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

Claims (6)

A cleavage method for specifically cleaving Cα-C bonds and/or side chains of a peptide backbone, comprising:
providing a sample containing a molecule having a peptide backbone in which a plurality of 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) and irradiating the sample with a laser.

cleaving the molecule by the cleaving method of claim 1;
determining the amino acid sequence of the peptide backbone based on the cleavage of Cα-C bonds and/or side chains of the peptide backbone by the irradiation. In the analysis method according to claim 2,
performing matrix-assisted laser desorption ionization mass spectrometry;
In said matrix-assisted laser desorption/ionization mass spectrometry, a matrix for mass spectrometry comprising said sample and a matrix comprising at least one of 3,5-dihydroxy-2-nitrobenzoic acid and 3,5-dihydroxy-4-nitrobenzoic acid A method of analysis, wherein the sample is subjected to said irradiation. In the analysis method according to claim 3,
The analysis method, wherein at least one of a-series ions and x-series ions obtained by the irradiation is detected in the matrix-assisted laser desorption/ionization mass spectrometry. In the analysis method according to claim 3,
The method of analysis, wherein the matrix-assisted laser desorption/ionization mass spectrometry detects a-series ions and d-series ions obtained by the irradiation. A specific cleaving reagent for cleaving C-Cα bonds and/or side chains of a peptide backbone, comprising:
3,5-dihydroxy-2-nitrobenzoic acid represented by the following structural formula (1) and at least one of 3,5-dihydroxy-4-nitrobenzoic acid represented by the following structural formula (2), Specific cleaving reagent.

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