Analysis method, peptide antioxidant for mass spectrometry, and kit for mass spectrometry

Abstract

An efficient method to suppress the oxidation of methionine in peptides during mass spectrometry is desirable. [Solution] The analytical method comprises preparing a sample containing the peptide to be analyzed and an antioxidant containing a protein to prevent oxidation of methionine, ionizing the peptide by laser desorption ionization, and detecting the ionized peptide by mass separation.

Description

[Technical field]

[0001] The present invention relates to an analytical method, a peptide antioxidant for use in mass spectrometry, and a kit for use in mass spectrometry. [Background technology]

[0002] Mass spectrometry using the matrix-assisted laser desorption / ionization (MALDI) method is a powerful tool for analyzing biomolecules such as peptides. However, when ionizing a sample using the MALDI method, peptides in the sample can be oxidized after the sample is placed on the sample plate.

[0003] Oxidation of peptides on a sample plate is likely to occur when the concentration of molecules in the sample is low, when the sample plate contains copper as an impurity that catalyzes oxidation, when the oxygen concentration in the sample is high, etc. When such oxidation of peptides on a sample plate occurs, the sample is erroneously analyzed as being more oxidized than it actually is.

[0004] In particular, methionine residues in the main chain of a peptide are more susceptible to oxidation than other amino acid residues, and in many cases, the sulfur atom in the side chain of the methionine residue is converted to a sulfoxide type by oxidation.

[0005] Patent Document 1 proposes a method aimed at converging methionine-containing chemical species in protein and peptide samples as methionine oxidized products. Such a method changes the oxidation state of the sample. Non-Patent Document 1 adds proteins such as bovine serum albumin (BSA) and transferrin to increase the sensitivity of mass spectrometry for high-mass peptides of 3000 Da or more such as insulin, but does not describe the purpose or effect of suppressing oxidation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2007-292523 [Non-patent literature]

[0007] [Non-Patent Document 1] Gustavsson N, Kokke BP, Harndahl U, Silow M, Bechtold U, Poghosyan Z, Murphy D, Boelens WC, Sundby C. “A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock protein,” The Plant Journal,(USA), Society for Experimental Biology, March 1, 2002, Volume 29, Issue 5, pp.545-553 Summary of the Invention [Problem to be solved by the invention]

[0008] An efficient method to suppress the oxidation of methionine in peptides during mass spectrometry is desirable. [Means for solving the problem]

[0009] According to a first aspect of the present invention, the method comprises the steps of preparing a sample comprising the peptide to be analyzed and an antioxidant comprising a protein to prevent the oxidation of methionine; The method includes ionizing the peptides by laser desorption ionization, and detecting the ionized peptides by mass separation.

[0010] According to a second aspect of the present invention, in the analysis method of the first aspect, the laser desorption / ionization method is preferably matrix-assisted laser desorption / ionization method.

[0011] According to the third aspect of the present invention, in the analysis method of the first or second aspect, the concentration of the protein is preferably 27.5 nM or more and less than 275 nM.

[0012] According to the fourth aspect of the present invention, in the analysis method of the third aspect, the concentration of the protein is preferably less than 260 nM.

[0013] According to a fifth aspect of the present invention, in the analysis method according to the fourth aspect, the concentration of the protein is preferably 45.2 nM or more and less than 151 nM.

[0014] According to a sixth aspect of the present invention, in the analysis method of any one of the first to fifth aspects, the antioxidant preferably further comprises an amino acid and / or a reducing agent in addition to the protein.

[0015] According to a seventh aspect of the present invention, in the analysis method of the sixth aspect, the amino acid and / or reducing agent is methionine, histidine, cysteine, tryptophan, dithiothreitol, tris(2-carboxyethyl)phosphine, 2-mercaptoethanol ... It is preferable that the antioxidant is at least one compound selected from the group consisting of n-butylphosphine, dithioerythritol, ascorbic acid, polyphenols, sodium pyrosulfite, citric acid, glucose, carotene, tocopherol, thioglycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, and thioglycerol.

[0016] According to an eighth aspect of the present invention, in the analysis method of the sixth or seventh aspect, the concentration of the amino acid and / or reducing agent is preferably 0.001 mM or more and 10 mM or less.

[0017] According to a ninth aspect of the present invention, in the analytical method of the first or second aspect, it is preferable that the peptide is purified using affinity purification, the protein is a ligand used in the affinity purification, and the affinity purification further comprises adding an amino acid and / or a reducing agent to an elution solution before or after eluting the peptide with an elution solution.

[0018] According to a tenth aspect of the present invention, in the analysis method of the ninth aspect, the amino acid and / or reducing agent is preferably at least one compound selected from the group consisting of methionine, histidine, cysteine, tryptophan, dithiothreitol, tris(2-carboxyethyl)phosphine, 2-mercaptoethanol, tri-n-butylphosphine, dithioerythritol, ascorbic acid, polyphenols, sodium pyrosulfite, citric acid, glucose, carotene, tocophenol, thioglycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, and thioglycerol.

[0019] According to an eleventh aspect of the present invention, in the analysis method of the ninth or tenth aspect, the concentration of the amino acid and / or reducing agent is preferably 0.0001 mM or more and 10 mM or less.

[0020] According to a twelfth aspect of the present invention, a peptide antioxidant for mass spectrometry is for preventing oxidation of methionine, and is used in the preparation of a sample containing a protein for mass spectrometry in which a peptide to be analyzed is ionized by laser desorption ionization.

[0021] According to a thirteenth aspect of the present invention, in the peptide antioxidant for mass spectrometry according to the twelfth aspect, the concentration of the protein is preferably 27.5 nM or more and less than 275 nM.

[0022] According to a fourteenth aspect of the present invention, the peptide antioxidant for mass spectrometry according to the twelfth or thirteenth aspect preferably further comprises an amino acid and / or a reducing agent in addition to the protein.

[0023] According to the fifteenth aspect of the present invention, in the peptide antioxidant for mass spectrometry of the fourteenth aspect, the amino acid and / or reducing agent is preferably at least one compound selected from the group consisting of methionine, histidine, cysteine, tryptophan, dithiothreitol, tris(2-carboxyethyl)phosphine, 2-mercaptoethanol, tri-n-butylphosphine, dithioerythritol, ascorbic acid, polyphenol, sodium pyrosulfite, citric acid, glucose, carotene, tocopherol, thioglycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, and thioglycerol.

[0024] According to a sixteenth aspect of the present invention, in the peptide antioxidant for mass spectrometry according to any one of the twelfth to fifteenth aspects, the laser desorption / ionization method is preferably matrix-assisted laser desorption / ionization method.

[0025] According to a 17th aspect of the present invention, a kit for mass spectrometry comprises a peptide antioxidant for mass spectrometry of any one of the 12th to 16th aspects, and at least one selected from the group consisting of a sample plate and a matrix. Effect of the Invention

[0026] According to the present invention, the oxidation of methionine in peptides in mass spectrometry can be effectively suppressed, and the oxidation state of methionine in a sample can be measured more accurately than ever before. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is a flowchart showing the flow of the analysis method according to the first embodiment. [Diagram 2] FIG. 2 is a schematic diagram showing the kit for mass spectrometry according to the first embodiment. [Diagram 3] FIG. 3 shows peaks corresponding to peptide Aβ1-40 in mass spectra obtained when the concentration of peptide Aβ1-40 is changed. [Figure 4] FIG. 4 shows peaks corresponding to each peptide in the mass spectrum when the concentration of BSA is changed. [Diagram 5] FIG. 5 is a graph showing the relationship between the amount of BSA added to the sample and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrometry. [Figure 6] FIG. 6 shows peaks corresponding to each peptide in the mass spectrum when the concentration of transferrin is changed. [Figure 7] FIG. 7 is a graph showing the relationship between the amount of transferrin added to a sample and the ratio of oxidized to non-oxidized form of each peptide based on mass spectrometry. [Figure 8] FIG. 8 shows peaks corresponding to each peptide in the mass spectrum when the concentration of fetuin is changed. [Figure 9] FIG. 9 is a graph showing the relationship between the amount of fetuin added to a sample and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrometry. [Figure 10] FIG. 10 is a flowchart showing the flow of the analysis method according to the second embodiment. [Figure 11] FIG. 11 is a schematic diagram showing a kit for mass spectrometry according to the second embodiment. [Figure 12] FIG. 12 shows peaks corresponding to each peptide in the mass spectrum when the concentration of glycine is changed. [Figure 13] FIG. 13 is a graph showing the relationship between the concentration of glycine added to a sample and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrum. [Figure 14]FIG. 14 shows peaks corresponding to each peptide in the mass spectrum when the concentration of L-methionine is changed. [Figure 15] FIG. 15 is a graph showing the relationship between the concentration of L-methionine added to a sample and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrometry. [Figure 16] FIG. 16 shows peaks corresponding to each peptide in the mass spectrum when the concentration of L-cysteine ​​is changed. [Figure 17] FIG. 17 is a graph showing the relationship between the concentration of L-cysteine ​​added to a sample and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrum. [Figure 18] FIG. 18 shows peaks corresponding to each peptide in the mass spectrum when the concentration of L-histidine is changed. [Figure 19] FIG. 19 is a graph showing the relationship between the concentration of L-histidine added to a sample and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrum. [Figure 20] FIG. 20 shows peaks corresponding to each peptide in the mass spectrum when the concentration of DTT is changed. [Figure 21] FIG. 21 is a graph showing the relationship between the concentration of DTT added to the sample and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrum. [Figure 22] FIG. 22 is a graph showing the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrometry in samples containing L-methionine or DTT with and without added BSA. [Figure 23] FIG. 23 is a flowchart showing the flow of an analysis method according to a modified example of the second embodiment. [Figure 24] FIG. 24 shows mass spectra of the immunoprecipitation eluates. [Diagram 25]FIG. 25 is a graph showing the relationship between the L-methionine concentration in the immunoprecipitation eluate and the ratio of oxidized to non-oxidized forms of each peptide based on mass spectrometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0029] (First embodiment) The analysis method of the first embodiment prevents oxidation of methionine in peptides in a sample to be used in mass spectrometry by adding a protein as an antioxidant to the sample.

[0030] FIG. 1 is a flow chart showing the flow of the analysis method of this embodiment. In step S1001, a sample is prepared containing a peptide to be analyzed and an antioxidant having a protein for preventing oxidation of methionine. The peptide to be analyzed (hereinafter referred to as target peptide) may be a peptide whose amino acid sequence has been fully or partially identified and contains methionine, or may be a peptide whose amino acid sequence is unknown. The number of residues in the amino acid sequence of the target peptide is not particularly limited, but a peptide having 100 or less residues, or 50 or less residues, such as each peptide used in the examples described below, is preferred.

[0031] In the examples described below, proteins such as bovine serum albumin (hereinafter referred to as BSA), transferrin, and fetuin are used as antioxidants, but the type of protein (hereinafter referred to as antioxidant protein) added to the sample as an antioxidant is not particularly limited. It is considered that the presence of easily oxidized molecules in addition to the target peptide in the sample disperses the target to be oxidized, suppressing the oxidation of the target peptide.

[0032] More specifically, in step S1001, an antioxidant protein is added to a solution containing a target peptide (hereinafter referred to as a sample solution) to a predetermined concentration. The concentration of the antioxidant protein is preferably 27.5 nM or more, more preferably 35.0 nM or more, even more preferably 39.9 nM or more, and even more preferably 45.2 nM or more. The amount of substance of the antioxidant protein per well is preferably 27.5 fmol / well or more, more preferably 35.0 fmol / well or more, even more preferably 39.9 fmol / well or more, and even more preferably 45.2 fmol / well or more. The amount of the antioxidant protein per well (in terms of mass) is preferably 1 ng / well or more, and even more preferably 3 ng / well or more. A higher concentration or a larger amount of the antioxidant protein is preferable because it has a higher effect of preventing oxidation of the target peptide.

[0033] The concentration of the antioxidant protein is preferably less than 275 nM, more preferably less than 260 nM, even more preferably less than 151 nM, and even more preferably less than 133 nM. The amount of substance of the antioxidant protein per well is preferably less than 275 fmol / well, more preferably less than 260 fmol / well, even more preferably less than 151 fmol / well, and even more preferably less than 133 fmol / well. The amount of the antioxidant protein per well (in terms of mass) is preferably less than 30 ng / well, and even more preferably 10 ng / well or less. If the concentration of the antioxidant protein is too high or the amount is too large, it is not preferable because the antioxidant protein is likely to suppress ionization or generate impurities.

[0034] Furthermore, a solution containing a matrix and a matrix additive (hereinafter referred to as an additive-containing matrix solution) is prepared. The type of matrix is ​​not particularly limited, and α-cyano-4-hydroxycinnamic acid (hereinafter referred to as CHCA), 2,5-dihydroxybenzoic acid (hereinafter referred to as DHB), sinapic acid, etc. can be used. The type of matrix additive is not particularly limited, and methylenediphosphonic acid (hereinafter referred to as MDPNA) etc. can be used. After step S1001 is completed, step S1003 is started.

[0035] In step S1003, the sample prepared in step S1001 is A matrix solution containing an additive is placed in each well of the sample plate and dried, and then a sample solution containing an antioxidant protein is added to the dried matrix and dried. A sample plate for MALDI is used as the sample plate, but when no matrix is ​​used, a sample plate for laser desorption ionization is used. After step S1003 is completed, step S1005 is started. It should be noted that the method of preparing samples for mass spectrometry is not limited to the above method. For example, a solution containing a target peptide, an antioxidant protein, a matrix and a matrix additive may be prepared, and then the solution may be placed on a sample plate and dried.

[0036] In step S1005, the sample placed on the sample plate is ionized by laser desorption ionization. The sample mixed with the matrix on the sample plate, which has been dried in step S1003, is irradiated with laser light emitted from a laser device of a mass spectrometer, causing ionization of the sample. The laser light used is ultraviolet light with a wavelength of 337 nm, but is not particularly limited as long as ionization is performed appropriately. In this embodiment, an example of the MALDI method in which a matrix and a sample are mixed and then ionized by irradiating a laser is shown as described above, but the present invention is also applicable to laser desorption ionization methods that do not use a matrix. When step S1005 is completed, step S1007 is started.

[0037] In step S1007, the sample ionized in step S1005 is mass-separated and detected. The type of mass spectrometer that performs mass separation of the ionized sample is not particularly limited as long as it can detect the target peptide with the desired accuracy, but a time-of-flight mass spectrometer is preferable from the viewpoint of accurate mass separation even when the peptide has a high mass of several thousand Da or more. A mass spectrum is created from a detection signal indicating the intensity of the mass-separated ions. For example, in a time-of-flight mass spectrometer, the flight time is converted to m / z based on previously acquired calibration data, and the intensity of the detected ions corresponding to m / z is shown in the mass spectrum. In addition, analysis of the data measured by mass analysis is performed as appropriate. When step S1007 is completed, the process is terminated.

[0038] 2 is a schematic diagram showing a mass spectrometry kit for providing reagents and the like used in the analysis method of the present embodiment. The mass spectrometry kit 1 includes an antioxidant protein 10, a matrix 20, and a sample plate 30.

[0039] The antioxidant protein 10 is a peptide antioxidant for mass spectrometry that is used in the preparation of a sample to be ionized by laser desorption ionization, particularly the MALDI method, and prevents the oxidation of methionine in a target peptide. The matrix 20 includes a matrix such as the above-mentioned CHCA, DHB, or sinapinic acid. The matrix 20 may be in a solid state or may be dissolved in any solvent including water and / or acetonitrile. In FIG. 2, the antioxidant protein 10 and the matrix 20 are stored in containers C1 and C2, respectively, but the type, shape, etc. of the containers C1 and C2 are not particularly limited. The sample plate 30 is a sample plate for laser desorption ionization, particularly for MALDI, and has a plurality of wells 31 formed therein, but the shape, number, arrangement position, etc. of the wells 31 are not particularly limited. The mass spectrometry kit according to this embodiment is required to include at least the antioxidant protein 10, and may not include the matrix 20 and the sample plate 30, or may include either one or both of them.

[0040] (Example of the first embodiment) Examples of the present embodiment will be described below, but the present invention is not limited to the following examples.

[0041] <1-1. Preparation of sample solution containing antioxidant protein> A mixture of three peptides, Aβ1-40 (Anaspec), Aβ1-42 (Anaspec), and APP669-711 (Peptide Institute), was added to the solution. Sample solutions containing either transferrin (Sigma-Aldrich) or bovine fetuin (Sigma-Aldrich) and no protein were prepared. The solvent for the sample solutions was 70% (v / v) acetonitrile containing 5 mM hydrochloric acid. The concentrations of the above proteins added to the sample solutions were 0.1, 0.3, 1, 3, 10, 30, and 100 ng / μL. Sample solutions containing none of the above three proteins but Aβ1-40 at concentrations of 10, 100, and 1000 fmol / μL (hereafter referred to as Aβ1-40 solutions) were also prepared.

[0042] The one-letter amino acid sequences of the peptides to be analyzed (target peptides) and the corresponding sequence numbers in the sequence table are shown in the following table. Aβ1-40 and Aβ1-42 contain one methionine (M) residue, so there is one site susceptible to oxidation corresponding to the sulfur atom in the side chain, while APP669-711 contains two methionine residues, so there are two sites susceptible to oxidation. [Table 1]

[0043] <1-2. Time-of-flight mass spectrometry> CHCA was used as a matrix for mass spectrometry. The matrix solution was prepared by dissolving 1 mg of CHCA in 1 mL of 70% (v / v) acetonitrile. A 0.4% (w / v) MDPNA solution containing MDPNA as a matrix additive was used as the matrix additive solution. After preparing an additive-containing matrix solution by mixing equal amounts of the matrix solution and the matrix additive solution, 0.5 μL of the additive-containing matrix solution was dropped into each well on a sample plate for MALDI (μFocus MALDI plate 900 μm, Hudson Surface Technology, Inc., Fort Lee, NJ) and allowed to dry. Then, 1 μL of the sample solution prepared in 1-1 was taken and dropped onto the matrix on the above sample plate and allowed to dry.

[0044] MALDI time-of-flight mass spectrometry (hereafter referred to as MALDI-TOF MS) was performed in linear mode using an AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) in positive ion mode. m / z values ​​were expressed as average masses of peaks. m / z values ​​were calibrated using human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, and bovine insulin as external standards.

[0045] <1-3. Peptide concentration and oxidation state> Figure 3 shows the mass spectrometry results of a sample (10-1000 fmol / well Aβ1-40) obtained by dropping 1 μL of Aβ1-40 solution (10-1000 fmol / μL) onto a sample plate and drying it. This is a diagram showing the peaks in the spectrum corresponding to Aβ1-40. As the peptide concentration decreased, the oxidized form was detected more strongly than the non-oxidized form, and at 10 fmol / well the peaks had the same height.

[0046] <1-4. Evaluation of oxidation inhibition by proteins> Figures 4, 6, and 8 show the peaks corresponding to each peptide in the mass spectrum when the concentrations of BSA, transferrin, and fetuin proteins were changed, respectively. When each peptide (each peptide concentration 500 amol / μL) was analyzed by MALDI-TOF MS without adding protein ("No protein added" in the figures), prominent peaks were detected for oxidized forms of Aβ1-40 and Aβ1-42, and for dioxide ("Oxidized form (2 places)" in the figures) in APP669-711 (Figures 4, 6, and 8).

[0047] In samples where each protein was added at a concentration of 0.1, 0.3, 1, 3, 10, 30, and 100 ng / μL, the non-oxidized form became the main peak as the amount of protein added was increased, and the oxidation of each peptide was suppressed. Here, when each protein was added at a concentration of 0.1, 0.3, 1, 3, 10, 30, and 100 ng / μL, 0.1, 0.3, 1, 3, 10, 30, and 100 ng / well of protein was present on the sample plate. The effect of suppressing oxidation was observed for all proteins used, namely BSA, transferrin, and fetuin. However, when the amount of protein added was large, the peak of the non-oxidized form became weaker. It is thought that ion suppression occurred due to the amount of protein added being too high. Furthermore, when the amount of protein added was too high, the peak corresponding to the protein-derived impurities (impurity peak) was detected strongly. In this example, the m / z of the impurity peak and the peak corresponding to the target peptide were different, but if the m / z values ​​of these two peaks were close and overlapped, the specificity of the target peptide would decrease. In view of this, it is preferable to appropriately select the type of protein to be added depending on the m / z of the peak corresponding to the target peptide.

[0048] The protein amount range in which the peaks corresponding to non-oxidized forms were more intense than those corresponding to oxidized forms and had a clear peak shape was 3-10ng / well (45.2-151 fmol / well) for BSA, and the concentration of the sample solution when dropped onto the sample plate was 3-10ng / μL (45.2-151 nM). The range for transferrin was 3-10 ng / well (39.9-133 fmol / well), and the concentration of the sample solution when dropped onto the sample plate was 3-10 ng / μL (39.9-133 nM). The range for fetuin was 1-10 ng / well (27.5-275 fmol / well), and the concentration of the sample solution when dropped onto the sample plate was 1-10 ng / μL (27.5-275 nM). When comparing weight (g), fetuin was confirmed to have an oxidation inhibitory effect even at an amount of 1 ng / well, which is three times lower than the others. When comparing the amount of substance (mol), the lower limit of the amount of substance (mol) that has an oxidation inhibitory effect was similar, 45.2 fmol / well for BSA, 39.9 fmol / well for transferrin, and 27.5 fmol / well for fetuin. In other words, it is thought that the oxidation inhibitory effect depends on the amount of substance (mol) rather than on the weight (g). On the other hand, the range of protein amounts at which the peak shape becomes unclear is 30 ng / well or more for BSA, transferrin, and fetuin, so it is thought that ion suppression depends on weight (g). From the above data, the appropriate amount of protein to be added for oxidation inhibition is 27.5-275 fmol / well (27.5-275 nM).

[0049] Figures 5, 7, and 9 are graphs showing the relationship between the amount of BSA, transferrin, and fetuin added to the sample, respectively, and the ratio of oxidized to non-oxidized peptides based on mass spectra. In samples with no added protein, the peaks of oxidized peptides relative to non-oxidized peptides were 8.5 times higher for Aβ1-40 and 6.1 times higher for Aβ1-42, and in APP669-711, the combined peak intensity of oxidized peptides (monoxidized peptide (one site) and dioxide (oxidized peptide (two sites)) was 11.6 times higher than that of non-oxidized peptides. On the other hand, the ratio of oxidized peptides relative to non-oxidized peptides decreased with the addition of protein. The results showed that adding protein to the sample was effective in suppressing oxidation, and that the effect was the same when using a variety of proteins.

[0050] Second embodiment The analytical method of the second embodiment prevents oxidation of methionine in peptides in a sample by adding, in addition to proteins, amino acids and / or reducing agents as antioxidants to the sample used in mass spectrometry.

[0051] FIG. 10 is a flow chart showing the flow of the analysis method of this embodiment. In step S2001, a sample containing a peptide to be analyzed (target peptide) and an antioxidant is prepared to prepare a sample containing a protein and an antioxidant comprising amino acids other than the protein (free amino acids) and / or a reducing agent to prevent oxidation of methionine. The target peptide may be a peptide whose amino acid sequence has been identified in whole or in part and contains methionine, or may be a peptide whose amino acid sequence is unknown. The number of residues in the amino acid sequence of the target peptide is not particularly limited, but a peptide having 100 or less residues or 50 or less residues such as each peptide used in the examples is preferable.

[0052] The type of protein (antioxidant protein) contained in the antioxidant is not particularly limited. As in the first embodiment, the presence of easily oxidized molecules in addition to the target peptide in the sample is thought to disperse the target to be oxidized, thereby suppressing the oxidation of the target peptide.

[0053] More specifically, in step S2001, an antioxidant protein is added to a solution (sample solution) containing a target peptide to a predetermined concentration. The concentration of the antioxidant protein is not particularly limited, but from the viewpoint of promoting the prevention of oxidation by the antioxidant protein, it is desirable that the concentration, amount of substance, or mass be within the range of the preferred concentration, amount of substance, or mass in the first embodiment described above.

[0054] The antioxidant according to this embodiment includes an amino acid and / or a reducing agent in addition to an antioxidant protein. The amino acid and / or the reducing agent is preferably a low molecular weight compound, and the molecular weight of the low molecular weight compound is preferably less than 10,000, more preferably less than 5,000. The molecular weight of the low molecular weight compound is preferably smaller than the molecular weight of the antioxidant protein. It is considered that the presence of a low molecular weight compound that captures oxygen in the sample solution suppresses the oxidation of the target peptide, which cannot be prevented by the antioxidant protein alone. Hereinafter, the free amino acid and / or the reducing agent contained in the antioxidant according to this embodiment is referred to as a low molecular weight oxygen capture substance.

[0055] The concentration of the low molecular weight oxygen scavenging substance is preferably 0.001 mM or more, more preferably 0.01 mM or more, and even more preferably 0.1 mM or more. The amount of the low molecular weight oxygen scavenging substance per well is preferably 1 pmol / well or more, more preferably 10 pmol / well or more, and even more preferably 100 pmol / well or more. The higher the concentration or the larger the amount of the low molecular weight oxygen scavenging substance, the higher the effect of preventing oxidation of the target peptide, and therefore it is preferable.

[0056] The concentration of the low molecular weight oxygen scavenging substance is preferably less than 10 mM, more preferably less than 1 mM. The amount of the low molecular weight oxygen scavenging substance per well is preferably less than 10 nmol / well, more preferably less than 1 nmol / well. If the concentration of the low molecular weight oxygen scavenging substance is too high or the amount is too large, it is not preferable because it may cause ionization suppression and reduce the sensitivity in mass spectrometry.

[0057] The amino acid in the low-molecular-weight oxygen scavenging substance is preferably at least one of methionine, histidine, cysteine, and tryptophan, which have a side chain that is easily oxidized. When the side chain of the acid receives oxygen, the target peptide to be oxidized in the sample solution is dispersed, making it difficult for the target peptide to be oxidized.

[0058] The reducing agent in the low molecular weight oxygen scavenger is preferably a low molecular weight compound as described above, but is not particularly limited thereto, and is preferably at least one compound selected from the group consisting of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethanol (2-ME), tri-n-butylphosphine (TBP), dithioerythritol (DTE), ascorbic acid, polyphenol, sodium pyrosulfite, citric acid, glucose, carotene, tocopherol, thioglycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, and thioglycerol. Of the above, the polyphenol is particularly preferably catechin, isoflavone, ellagic acid, or tannin.

[0059] Furthermore, a solution containing a matrix and a matrix additive (additive-containing matrix solution) is prepared. The type of matrix is ​​not particularly limited, and CHCA, DHB, sinapic acid, etc. can be used. The type of matrix additive is not particularly limited, and MDPNA, etc. can be used. After step S2001 is completed, step S2003 is started.

[0060] In step S2003, the sample prepared in step S2001 is placed on a sample plate of a mass spectrometer. After an additive-containing matrix solution is placed in each well of the sample plate and dried, a sample solution to which an antioxidant protein and an amino acid and / or a reducing agent have been added is added to the dried matrix and dried. The sample plate is for laser desorption ionization, and a sample plate for MALDI is particularly preferred. After step S2003 is completed, step S2005 is started. It should be noted that the method of preparing samples for mass spectrometry is not limited to the above method. For example, a solution containing a target peptide, an antioxidant protein, amino acids and / or a reducing agent, a matrix and a matrix additive may be prepared, and then the solution may be placed on a sample plate and dried.

[0061] Steps S2005 and S2007 are similar to steps S1005 and S1007, respectively, and therefore will not be described. When step S2007 ends, the process ends.

[0062] 11 is a schematic diagram showing a mass spectrometry kit for providing reagents and the like used in the analysis method of this embodiment. The mass spectrometry kit 2 according to this embodiment includes an antioxidant protein 10, a matrix 20, a sample plate 30, and a low-molecular-weight oxygen-scavenging substance 40 containing an amino acid and / or a reducing agent.

[0063] The antioxidant protein 10 and the low molecular weight oxygen scavenging substance 40 are peptide antioxidants for mass spectrometry that are used in the preparation of a sample to be ionized by laser desorption ionization, particularly the MALDI method, and prevent the oxidation of methionine in a target peptide. The matrix 20 includes a matrix such as the above-mentioned CHCA, DHB, or sinapinic acid. The matrix 20 may be in a solid state or may be dissolved in any solvent including water and / or acetonitrile. In FIG. 11, the antioxidant protein 10, the matrix 20, and the low molecular weight oxygen scavenging substance 40 are stored in containers C1, C2, and C3, respectively, but the types, shapes, etc. of the containers C1, C2, and C3 are not particularly limited. The sample plate 30 is a sample plate for laser desorption ionization, and has a plurality of wells 31 formed therein, but the shapes, numbers, positions, etc. of the wells 31 are not particularly limited. The mass spectrometry kit according to this embodiment includes at least a low molecular weight oxygen scavenger substance 40. It is sufficient that the antioxidant protein 10, the matrix 20 and the sample plate 30 are included, and the antioxidant protein 10, the matrix 20 and the sample plate 30 may not be included, or one, two or all of them may be included.

[0064] (Example of the second embodiment) Examples according to this embodiment are shown below, but the present invention is not limited to the following examples. In the examples of the first embodiment, a sample solution was prepared by adding 0.1, 0.3, 1, 3, 10, 30, or 100 ng / μL of BSA to a mixture of Aβ1-40, Aβ1-42, and APP669-711, and mass spectrometry was performed to examine the inhibition of peptide oxidation (see FIG. 4). In the following, amino acids and / or reducing agents were further added to this sample solution to evaluate the inhibition of peptide oxidation.

[0065] <2-1. Evaluation of peptide oxidation inhibition by antioxidant proteins and amino acids> A sample solution was prepared by adding amino acids together with 10 ng / μL of BSA to a mixture of three types of synthetic peptides Aβ1-40, Aβ1-42 and APP669-711 (see the table of the examples of the first embodiment), and a sample solution without adding amino acids. The solvent used was 70% (v / v) acetonitrile containing 5 mM hydrochloric acid. The amino acid-containing sample solution was added with any of glycine (Nacalai Tesque), L-methionine (Nacalai Tesque), L-cysteine ​​(Thermo Fisher Scientific) and L-histidine (Fluka) as the amino acid. As described above, methionine, cysteine ​​and histidine have a site that is easily oxidized in the side chain, but glycine does not have a site that is easily oxidized in the side chain. Therefore, glycine was used as a comparative example. The concentrations of the added amino acids were 0.0001, 0.001, 0.01, 0.1, 1 and 10 mM for each amino acid. These samples were measured by MALDI-TOF MS under the same conditions as in the examples of the first embodiment.

[0066] Fig. 12 shows the peaks corresponding to each peptide in the mass spectrum when the concentration of glycine is changed. Fig. 13 is a graph showing the relationship between the concentration of glycine added to the sample and the ratio of oxidized to non-oxidized peptides based on the mass spectrum. Even when glycine was added to the sample solution at various concentrations in addition to BSA, there was no change in the peak of the oxidized peptide. In other words, the effect of suppressing the oxidation of peptides by adding glycine was not confirmed.

[0067] Figures 14, 16, and 18 are diagrams showing the peaks corresponding to each peptide in the mass spectrum when the concentrations of L-methionine, L-cysteine, and L-histidine are changed, respectively. Figures 15, 17, and 19 are graphs showing the relationship between the concentrations of L-methionine, L-cysteine, and L-histidine added to the sample, respectively, and the ratio of oxidized to non-oxidized peptides based on the mass spectrum. When L-methionine, L-cysteine, and L-histidine were added to the sample solution, the peak of the oxidized peptide decreased, and the ratio of the peak of the oxidized peptide to the non-oxidized peptide decreased.

[0068] The inhibition of oxidation was evaluated by Dunnett's test with a significance level of p<0.05. The amino acid concentrations at which the ratio of oxidized products was statistically significantly decreased in at least one of the three peptides compared to the control without amino acid addition were 0.001-10 mM for L-methionine, 10 mM for L-cysteine, and 0.01-10 mM for L-histidine. No statistically significant decrease in oxidized products was observed in the case of glycine. The concentrations with the greatest inhibitory effect on oxidation were 100 μM or 1 mM for L-methionine, 1 mM or 10 mM for L-cysteine, and 1 mM for L-histidine. At a high concentration of 10 mM, ion suppression was strong and no peaks could be detected for L-histidine. In the case of L-methionine and L-cysteine, the sensitivity tended to decrease at a concentration of 10 mM. The amino acids that showed inhibition of oxidation have one thing in common: they have side chains that are easily oxidized. In other words, it is believed that the side chains capture oxygen and thus suppress the oxidation of the target peptide. do.

[0069] <2-2. Evaluation of peptide oxidation inhibition by antioxidant proteins and reducing agents> A sample solution was prepared by adding the reducing agent DTT together with 10 ng / μL BSA to a mixture of three synthetic peptides, Aβ1-40, Aβ1-42, and APP669-711, and a sample solution without DTT added. The solvent used was 70% (v / v) acetonitrile containing 5 mM hydrochloric acid. The concentrations of DTT added were 0.0001, 0.001, 0.01, 0.1, 1, and 10 mM. These samples were measured by MALDI-TOF MS under the same conditions as in the case of amino acids (2-1) above.

[0070] Fig. 20 shows the peaks corresponding to each peptide in the mass spectrum when the concentration of DTT is changed. Fig. 21 is a graph showing the relationship between the concentration of DTT added to the sample and the ratio of oxidized to non-oxidized peptides based on the mass spectrum. When DTT was added to the sample solution at various concentrations in addition to BSA, the oxidized peak decreased at 10 μM or more, and the ratio of the oxidized to non-oxidized peaks decreased.

[0071] The inhibition of oxidation was evaluated by Dunnett's test with a significance level of p<0.05. Compared to the control without the addition of a reducing agent, the concentration of DTT at which the proportion of oxidized products was statistically significantly reduced for at least one of the three peptides was 10 mM. This indicates that oxidation on the sample plate is inhibited even when a reducing agent is added. The most effective concentration was 10 mM. Note that there was a tendency for sensitivity to decrease at a concentration of 10 mM.

[0072] <2-3. Evaluation of inhibition of peptide oxidation by amino acids or reducing agents when the sample solution does not contain antioxidant proteins> A mixture of three synthetic peptides, Aβ1-40, Aβ1-42 and APP669-711, was used to prepare a sample solution containing 1 mM L-methionine and no antioxidant protein, a sample solution containing 10 mM DTT and no antioxidant protein, and a sample solution in which 10 ng / μL BSA was added to each of these sample solutions. The solvent used was 70% (v / v) acetonitrile containing 5 mM hydrochloric acid. These samples were measured by MALDI-TOF MS under the same conditions as in the case of amino acids (2-1) above.

[0073] FIG. 22 is a graph showing the ratio of oxidized to non-oxidized peptides based on mass spectra when BSA was added to a sample containing L-methionine or DTT and when it was not added. Even when 1 mM L-methionine or 10 mM DTT was added to the sample solution alone, the ratio of the oxidized to non-oxidized peaks decreased, and the effect of inhibiting oxidation was observed. The ratio of the oxidized to non-oxidized peaks was further decreased by adding BSA in addition to these. This result indicates that the addition of antioxidant proteins to a sample solution containing amino acids and reducing agents but not antioxidant proteins further inhibits peptide oxidation.

[0074] Third embodiment The analysis method of the third embodiment, like the analysis method of the second embodiment, prevents oxidation of methionine in peptides in a sample by adding amino acids and / or reducing agents as antioxidants in addition to proteins (antioxidant proteins) to a sample used for mass spectrometry. However, in the analysis method of this embodiment, a peptide to be analyzed (target peptide) is purified by affinity purification, and a ligand contained in the eluate in affinity purification is used as an antioxidant protein. This makes it possible to effectively suppress oxidation of the target peptide without the need for an operation of adding an antioxidant protein to the sample solution.

[0075] 23 is a flow chart showing the flow of the analysis method of this embodiment. In step S3001, a target peptide is purified using affinity purification. The method is not particularly limited as long as a protein can be used as a ligand bound to a support, and immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), pull-down assay, affinity chromatography, etc. can be used. When an antibody is used as a ligand, the antibody may be immobilized on any column including a spin column, or on a pipette tip or a microchannel.

[0076] In affinity purification, a peptide solution containing the target peptide is contacted with a ligand bound to a support, and the target peptide in the peptide solution is bound to the ligand. The type of ligand is not particularly limited as long as it can be bound to the support and can bind to the target peptide, and an antibody or the like that can bind specifically or nonspecifically to the target peptide can be used.

[0077] Once the target peptide has bound to the ligand, the support is washed with a buffer solution while maintaining the bond, and components of the peptide solution that have not bound to the ligand are removed. The target peptide is then eluted by contacting the support with an elution solution containing a low or high pH buffer solution or the like. After step S3001 is completed, step S3003 is started.

[0078] In step S3003, amino acids and / or reducing agents are added to the eluate from affinity purification containing the target peptide to prepare a sample solution. The amino acids and reducing agents used are preferably those of the low molecular weight oxygen scavenger in the second embodiment. The sample solution contains the ligand used in affinity purification. As in the above-mentioned embodiment, the presence of easily oxidized molecules in addition to the target peptide in the sample is thought to disperse the target to be oxidized and inhibit oxidation of the target peptide. In the affinity purification, the amino acid of the low molecular weight oxygen scavenger and a reducing agent may be added to the elution solution in advance, which makes it possible to prepare a sample solution in which oxidation of the target peptide is suppressed without the need to add a low molecular weight oxygen scavenger after elution.

[0079] The concentration of the low molecular weight oxygen scavenging substance in this embodiment is preferably 0.0001 mM or more, more preferably 0.00067 mM or more, even more preferably 0.0067 mM or more, and even more preferably 0.067 mM or more. The higher the concentration of the low molecular weight oxygen scavenging substance, the higher the effect of preventing oxidation of the target peptide, which is preferable. The concentration of the low molecular weight oxygen scavenging substance in this embodiment is preferably less than 10 mM, more preferably less than 1 mM. If the concentration of the low molecular weight oxygen scavenging substance is too high, ionization suppression or the like may occur, which may reduce the sensitivity in mass spectrometry, and this is not preferable.

[0080] Also, a solution containing a matrix and a matrix additive (additive-containing matrix solution) is prepared. The type of matrix is ​​not particularly limited, and CHCA, DHB, sinapic acid, etc. can be used. The type of matrix additive is not particularly limited, and MDPNA, etc. can be used. After step S3003 is completed, step S3005 is started.

[0081] In step S3005, the sample prepared in step S3003 is placed on a sample plate of a mass spectrometer. After an additive-containing matrix solution is placed in each well of the sample plate and dried, a sample solution to which the ligand as the antioxidant protein, and an amino acid and / or a reducing agent have been added is added to the dried matrix and dried. After step S3005 is completed, step S3007 is started. It should be noted that the method of preparing samples for mass spectrometry is not limited to the above method. For example, a solution containing the target peptide, the above-mentioned ligand, amino acids and / or a reducing agent, a matrix and a matrix additive may be prepared, and then the solution may be placed on a sample plate and dried.

[0082] Steps S3007 and S3009 are similar to steps S1005 and S1007, respectively, and therefore will not be described. When step S3009 is completed, the process ends.

[0083] (Example of the third embodiment) Examples of the present embodiment will be described below, but the present invention is not limited to the following examples.

[0084] <3-1. Immunoprecipitation (IP)> Anti-Aβ antibody (clone 6E10) was bound to Epoxy magnetic beads (Thermo Fisher Scientific) to prepare anti-Aβ antibody beads for immunoprecipitation. Human plasma was spiked with synthetic peptides Aβ1-40 (40 pM), Aβ1-42 (10 pM), and APP669-711 (10 pM) to prepare samples. 250 μL of plasma spiked with the synthetic peptide (target peptide) was mixed with 250 μL of Tris buffer containing DDM and NTM, and then left to stand on ice for 5 minutes. The plasma was mixed with anti-Aβ antibody beads and shaken on ice for 1 hour. The antibody beads were then washed three times with 100 μL of Tris buffer containing DDM and NTM, and twice with 50 μL of 50 mM ammonium acetate buffer, after which the target peptide bound to the antibody beads was eluted with glycine buffer containing DDM. The eluate was mixed with Tris buffer containing DDM, mixed with the antibody beads, and shaken on ice for 1 hour. Thereafter, the antibody beads were washed five times with 50 μL of Tris buffer containing DDM, twice with 50 μL of 50 mM ammonium acetate buffer, and once with 30 μL of H2O, after which the peptides bound to the antibody beads were eluted with 5 μL of elution solution (70% (v / v) acetonitrile containing 5 mM hydrochloric acid). The elution solutions used were either containing L-methionine or not. The L-methionine concentration was 0.0067 to 67 μM. The target peptides eluted by immunoprecipitation were measured by MALDI-TOF MS under the same conditions as in Example (2-1) of the second embodiment.

[0085] FIG. 24 is a diagram showing the mass spectrum of the eluate of immunoprecipitation. In FIG. 24, the range of m / z 4000 to 20000 is shown, and the peak corresponding to Aβ1-40 is shown on the left side of the figure. As a peak corresponding to IgG is observed in the mass spectrum of the eluate in FIG. 24, the sample in which human plasma is treated by immunoprecipitation is contaminated with IgG derived from antibody beads. Therefore, it is not necessary to add a protein such as BSA for the purpose of preventing oxidation, but it is possible that oxidation can be further suppressed by adding a low molecular weight oxygen scavenger such as an amino acid. Therefore, it was evaluated whether the oxidation of the target peptide can be further suppressed by adding methionine.

[0086] Figure 25 is a graph showing the relationship between the concentration of L-methionine in the immunoprecipitation eluate and the ratio of oxidized to non-oxidized peptides based on mass spectrometry. The ratio of oxidized to non-oxidized peaks was reduced in the eluate to which L-methionine was added, compared to the eluate to which L-methionine was not added. The inhibition of oxidation was evaluated by Dunnett's test with a significance level of p<0.05. When the concentration of L-methionine was 0.67 μM or higher, the ratio of oxidized peptides was statistically significantly reduced for at least one of the three target peptides, compared to the control without added amino acids. This result indicates that the presence of a low-molecular-weight oxygen scavenger such as L-methionine in the post-immunoprecipitation sample inhibits oxidation.

[0087] The present invention is not limited to the above-described embodiment, and other aspects that are conceivable within the scope of the technical concept of the present invention are also included within the scope of the present invention.

[0088] The disclosures of the following priority applications are incorporated herein by reference: Japanese Patent Application No. 2018-013052 (filed January 29, 2018) [Explanation of symbols]

[0089] 1,2…Mass spectrometry kit 10. Antioxidant Protein 20. Matrix 30...Sample plate 31…Well 40...Low molecular weight oxygen scavenger C1,C2,C3…Container

Claims

1. An analytical method for analyzing a peptide containing methionine, comprising the steps of: In order to measure the oxidation state of methionine contained in the peptide to be analyzed in the sample, a mixed sample is prepared containing the peptide to be analyzed and an antioxidant having a protein for preventing oxidation of methionine on a sample plate of a laser desorption / ionization mass spectrometer; placing the mixed sample containing the antioxidant in a well of a sample plate of a laser desorption ionization mass spectrometer; Ionizing a mixed sample containing an antioxidant placed in a well of the sample plate by laser desorption ionization; and detecting at least one of the oxidized methionine and the non-oxidized methionine of the peptide in the mixed sample containing the ionized antioxidant by mass separation, The analytical method, wherein the protein prevents oxidation of methionine in a peptide in a mixed sample containing the antioxidant by oxidizing the protein itself in the mixed sample.

2. The method of claim 1 , The analytical method, wherein the laser desorption / ionization method is matrix-assisted laser desorption / ionization method.

3. The method of claim 1 , The method of analysis, wherein the antioxidant comprises a reducing agent, the reducing agent comprising an organic compound.

4. The method of claim 1 , The analysis method, wherein the concentration of the protein is equal to or greater than 27.5 nM and less than 275 nM.

5. The method of claim 4, The method of analysis, wherein the concentration of the protein is less than 260 nM.

6. The method of claim 5, The method for analysis, wherein the concentration of the protein is equal to or greater than 45.2 nM and less than 151 nM.

7. The method according to any one of claims 1 to 6, The antioxidant further comprises, in addition to the protein, an amino acid having an oxidizable site in a side chain, The analytical method, wherein the amino acid suppresses oxidation of methionine in a peptide in the mixed sample by capturing oxygen through the side chain in the mixed sample.

8. The method of claim 7, The antioxidant contains a reducing agent in addition to the protein and the amino acid, the amino acid is at least one compound selected from the group consisting of methionine, histidine, cysteine, and tryptophan; The analytical method, wherein the reducing agent is at least one compound selected from the group consisting of dithiothreitol, tris(2-carboxyethyl)phosphine, 2-mercaptoethanol, tri-n-butylphosphine, dithioerythritol, ascorbic acid, polyphenols, citric acid, glucose, carotene, tocopherol, thio-glycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, and thioglycerol.

9. The method of claim 1 , the antioxidant comprises a reducing agent; The analysis method, wherein the concentration of the reducing agent in the mixed sample is 0.001 mM or more and 10 mM or less.

10. The method of claim 1 , the antioxidant comprises a reducing agent; The amount of the reducing agent per well is 1 pmol / well or more and 10 nmol / well or less.

11. The method of claim 1 , the antioxidant comprises a reducing agent; The amount of the reducing agent per well is 10 pmol / well or more and 1 nmol / well or less.

12. In the analysis method according to claim 1 or 2, the antioxidant comprises a reducing agent; the peptide has been purified using affinity purification; the protein is a ligand used in the affinity purification, The analytical method comprises adding the reducing agent to an elution solution before or after elution of the peptide with an elution solution during the affinity purification.

13. The method of claim 12, The analytical method, wherein the reducing agent is at least one compound selected from the group consisting of dithiothreitol, tris(2-carboxyethyl)phosphine, 2-mercaptoethanol, tri-n-butylphosphine, dithioerythritol, ascorbic acid, polyphenols, citric acid, glucose, carotene, tocopherol, thio-glycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, and thioglycerol.

14. The method of claim 12, The method for analysis, wherein the concentration of the reducing agent is 0.0001 mM or more and 10 mM or less.

15. The method of any one of claims 1 to 14, The analytical method, wherein the protein is at least one selected from the group consisting of bovine serum albumin, transferrin, and fetuin.

16. A peptide antioxidant for use in the analysis method according to any one of claims 1 to 3, A method for preparing a sample for mass spectrometry in which a peptide to be analyzed is ionized by laser desorption / ionization, the method comprising the steps of: The peptide antioxidant for mass spectrometry, wherein the protein is at least one selected from the group consisting of bovine serum albumin, transferrin, and fetuin, and the protein itself is oxidized in the mixed sample, thereby preventing oxidation of methionine in the peptide in the mixed sample.

17. 17. The peptide antioxidant for mass spectrometry according to claim 16, the antioxidant comprises a reducing agent; A peptide antioxidant for mass spectrometry, wherein the reducing agent comprises an organic compound.

18. 17. The peptide antioxidant for mass spectrometry according to claim 16, A peptide antioxidant for mass spectrometry, wherein the protein has a concentration of 27.5 nM or more and less than 275 nM.

19. 17. The peptide antioxidant for mass spectrometry according to claim 16, In addition to the protein, the method further comprises providing an amino acid having an oxidizable site in a side chain, A peptide antioxidant for mass spectrometry, wherein the amino acid suppresses oxidation of methionine in the peptide in the mixed sample by capturing oxygen through the side chain in the mixed sample.

20. 20. The peptide antioxidant for mass spectrometry according to claim 19, In addition to the protein and the amino acid, a reducing agent is included, the amino acid is at least one compound selected from the group consisting of methionine, histidine, cysteine, and tryptophan; The reducing agent is a peptide antioxidant for mass spectrometry, which is at least one compound selected from the group consisting of dithiothreitol, tris(2-carboxyethyl)phosphine, 2-mercaptoethanol, tri-n-butylphosphine, dithioerythritol, ascorbic acid, polyphenol, citric acid, glucose, carotene, tocopherol, thio-glycolic acid, N-acetylcysteine, hydroxylamine, reduced glutathione, 2-aminoethylisothiouronium bromide, and thioglycerol.

21. 21. The peptide antioxidant for mass spectrometry according to claim 16, The peptide antioxidant for mass spectrometry, wherein the laser desorption / ionization method is matrix-assisted laser desorption / ionization method.

22. A peptide antioxidant for mass spectrometry according to any one of claims 16 to 21; A kit for mass spectrometry comprising at least one selected from the group consisting of a sample plate and a matrix.

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