Peptide analysis method and peptide analysis device

Abstract

The present invention is provided with: a step for preparing a sample containing a peptide to be analyzed; a step for adding a labeling compound capable of specifically reacting with a thiol group to the sample to label a reduced cysteine residue contained in the peptide in the sample with the labeling compound; a separation step for separating the sample containing the peptide labeled with the labeling compound by chromatography; a measurement performance step for performing a product ion scanning measurement of the peptide labeled with the labeling compound using, as a precursor ion, a peptide ion generated as the result of the ionization under the conditions where the thiol group cannot be detached from the cysteine residue; and a determination step for determining whether or not there is a peak appearing at a specific (mass) / (charge number) ratio corresponding to the structure of the labeling compound in a mass spectrum produced on the basis of data obtained in the measurement performance step to determine whether or not the peptide to be analyzed contains a reduced cysteine residue.

Description

[Technical field]

[0001] The present invention relates to a peptide analysis method and a peptide analysis apparatus that use a chromatography mass spectrometer to determine whether or not an amino acid having a thiol group constituting a protein or peptide is in a reduced form. [Background technology]

[0002] Cysteine ​​(Cys) is a sulfur-containing amino acid with a thiol group (SH group) in the side chain, and is present in many proteins. The SH group is highly reactive, and can form disulfide (SS) bonds with the SH groups of other amino acids, including cysteine, within a protein molecule, or can break and return to the SH group. The number and positions of SS bonds within a protein molecule determine the three-dimensional structure of the protein. Cys residues have also been frequently used as targets for specific reactions by nucleophilic substitution reactions and nucleophilic addition reactions to thiol groups (Non-Patent Document 1).

[0003] Albumin, a type of protein contained in blood, contains 35 cysteine ​​residues (Cys residues). Usually, only the 34th Cys residue from the N-terminus (hereinafter referred to as "Cys34") has a free SH group, and the remaining 34 Cys residues all form SS bonds within the molecule. However, a free SH group of a certain albumin in blood may form an SS bond with a free SH group of another albumin, or may form an SS bond with a free SH group contained in a protein or peptide other than albumin. Albumin in which the SH group of Cys34 is free is called reduced albumin, and albumin that forms an SS bond is called oxidized albumin. In recent years, it has been reported that there is a correlation between the ratio of reduced albumin and oxidized albumin in blood and certain diseases, and the use of the ratio of reduced albumin and oxidized albumin as a marker for screening and diagnosing the specific disease is being considered.

[0004] Conventionally, oxidized and reduced albumin have been distinguished by liquid chromatography, but it is not clear whether the separation by liquid chromatography is due solely to the difference in the redox state of Cys residues.

[0005] In response to this, there is a method for detecting proteins and / or peptides containing Cys residues using DAABD-Cl (4-(dimethylaminoethyl aminosulfonyl)-7-chloro-2,1,3-benzoxadiazole), a fluorescent reagent that specifically reacts with SH groups (Patent Documents 1 and 2, Non-Patent Documents 2-4). In this method, in order to increase the detection sensitivity, a sample containing proteins and / or peptides is reduced, one or more SS bonds contained in the proteins and / or peptides are cleaved to convert them to SH groups, and then DAABD-Cl is added. As a result, the SH groups of the Cys residues of the proteins and / or peptides react with DAABD-Cl, and DAABD is bound to the Cys residues to obtain fluorescently labeled proteins and / or peptides, which are then separated by fluorescence detection, and if necessary, the proteins and / or peptides are fragmented by trypsin digestion before being introduced into a mass spectrometer. The data (MS spectrum, MS / MS spectrum) obtained by MS / MS analysis of the fluorescently labeled protein and / or peptide fragments is then compared with information stored in a database to determine the amino acid sequence of each fragment and identify the protein and / or peptide. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2005-017264 A [Patent Document 2] International Publication WO2005 / 056146 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] L. Dayon; Thiol‐targeted Microspray Mass Spectrometry of Peptides and Proteins through On‐line EC‐tagging. PhD Thesis, Ecole Polytechnique Federale de Lausanne, 2006. [Non-Patent Document 2] T. Santa, T. Fukushima, T. Ichibangase, and K. Imai; Biomed. Chromatogr. 2008, 22(4), pp.343-353 [Non-Patent Document 3] M. Masuda, C. Toriumi, T. Santa, and K. Imai; Anal. Chem. 2004, 76, pp.728-735 [Non-Patent Document 4] T. Ichibangase and K. Imai; J. Pharm. Biomed. Anal. 2014, 101, pp.31-39 Summary of the Invention [Problem to be solved by the invention]

[0008] In the above method, although it is possible to selectively introduce fluorescently labeled proteins and / or peptides, i.e., proteins and / or peptides having Cys residues, into a mass spectrometer, it is not possible to determine from the results of MS / MS analysis of protein and / or peptide fragments whether the fragments are DAABD-bound fragments (fluorescently labeled fragments). Therefore, it is necessary to determine the amino acid sequence of each fragment by collating the MS / MS analysis data obtained for all fragments with information stored in a database.

[0009] It is conceivable to selectively detect fluorescently labeled fragments by multiple reaction monitoring (MRM) measurements using a mass spectrometer, but in this case, it is necessary to prepare standard samples in which DAABD is bound to Cys residues for peptides of various sequences, and to obtain MS / MS data for each standard sample by measuring them. Then, by analyzing the MS / MS data obtained for each standard sample, it is necessary to set control parameters such as the combination of mass-to-charge ratio (m / z) of precursor ion and product ion that is optimal for detecting fluorescently labeled fragments and the optimal collision energy for that combination, which is a tedious and complicated process.

[0010] The problem to be solved by the present invention is to provide a peptide analysis method and a peptide analysis device that can determine whether a Cys residue in a protein or peptide is reduced or oxidized without the need for tedious and complicated operations. [Means for solving the problem]

[0011] The present invention, which has been made to solve the above problems, is a peptide analysis method for analyzing reduced and oxidized cysteine ​​residues contained in a peptide using a chromatography mass spectrometer capable of MS / MS measurement, comprising the steps of: a) providing a sample containing a peptide to be analyzed; b) adding a labeling compound that specifically reacts with a thiol group to the sample to label reduced cysteine ​​residues contained in peptides in the sample with the labeling compound; c) a separation step of separating the sample containing the peptide labeled with the labeling compound by chromatography; d) a measurement execution step of performing a product ion scan measurement on the separated peptide labeled with the labeling compound, using peptide ions generated by ionization under conditions that do not cause thiol groups to be released from cysteine ​​residues as precursor ions; e) a determination step of determining whether or not the peptide to be analyzed contains a reduced cysteine ​​residue by determining whether or not a peak appears at a specific mass-to-charge ratio corresponding to the structure of the labeled compound in a mass spectrum created based on the data obtained in the measurement execution step. The present invention is characterized by comprising:

[0012] The present invention, which has been made to solve the above problems, is a peptide analysis device including a chromatography mass spectrometer capable of MS / MS measurement, for analyzing reduced and oxidized cysteine ​​residues contained in a peptide, comprising: a) an analysis control unit that operates the chromatographic mass spectrometer to separate a sample containing a peptide labeled with a labeling compound that specifically reacts with a thiol group by chromatography, and to perform a product ion scan measurement on the separated sample, using peptide ions generated by ionization under conditions that do not cause thiol groups to be dissociated from cysteine ​​residues as precursor ions; b) a mass spectrum generating unit for generating a mass spectrum based on data obtained by the product ion scan measurement; c) a determination unit for determining whether or not a peak appears at a specific mass-to-charge ratio corresponding to the structure of the labeled compound in the mass spectrum, thereby determining whether or not the peptide to be analyzed contains a reduced cysteine ​​residue; The present invention is characterized by comprising:

[0013] In the present invention, examples of chromatographs constituting a chromatograph mass spectrometer capable of MS / MS measurement include gas chromatographs, liquid chromatographs, and capillary electrophoresis, and examples of mass spectrometers include tandem quadrupole mass spectrometers (also called triple quadrupole mass spectrometers), quadrupole time-of-flight mass spectrometers (QTOF), ion trap mass spectrometers, and matrix-assisted laser desorption ionization mass spectrometers, and combinations of these can be used.

[0014] In the present invention, the term "peptide" includes both proteins and peptides. A reduced cysteine ​​residue (Cys residue) refers to a Cys residue in which the thiol group (SH group) is in a free state, and an oxidized Cys residue refers to a Cys residue that forms a disulfide bond (SS bond) with another Cys residue in the same peptide molecule, or a Cys residue in one peptide that forms an SS bond with a Cys residue in another peptide.

[0015] A peptide has a structure in which a plurality of amino acids are peptide-bonded, and when a peptide in a sample contains a reduced Cys residue, the SH group of the Cys residue reacts with a labeling compound to obtain a peptide in which the Cys residue is labeled with the labeling compound. Here, "labeled with a labeling compound" refers to a state in which, for example, H (hydrogen) of the SH group and a part of the labeling compound are separated, and S (sulfur) of the SH group and the remaining part of the labeling compound are bonded to form a new functional group (hereinafter also referred to as "labeling functional group"). In this case, H separated from the SH group and a part of the labeling compound are also bonded to form a molecule.

[0016] In the present invention, a sample containing peptides labeled with a labeling compound is separated by chromatography and then introduced into a mass spectrometer. At this time, if the molecular weight of the peptide to be analyzed is relatively small, the sample is introduced directly into the chromatograph, but if the molecular weight is relatively large, the peptides labeled with the labeling compound are digested and fragmented with a protein digestive enzyme to obtain peptides with small molecular weights, and then introduced into the chromatograph. For the sake of convenience, in the following, it is assumed that the peptides labeled with the labeling compound are introduced into the mass spectrometer.

[0017] In the present invention, conditions are set so that when peptides in a sample are introduced into a mass spectrometer and ionized, thiol groups are not dissociated from cysteine ​​residues. Therefore, it is preferable to use an ion source that performs so-called soft ionization, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), as the ion source for the mass spectrometer. When ionized under the above conditions, if the peptide in the sample contains a reduced Cys residue, ions of the peptide in a state in which the Cys residue is labeled with a labeling compound (i.e., in a state in which the labeling functional group is present) are generated by ionization. When such peptide ions are cleaved by collision-induced dissociation or the like, the labeling functional group is released, and when a product ion scan measurement is performed using the peptide ions as precursor ions, a peak appears in the MS / MS spectrum of the product ions at a specific mass-to-charge ratio corresponding to the structure of the labeling functional group, i.e., the structure of the labeling compound. Therefore, if the presence of such a peak is confirmed, it can be determined that the Cys residue contained in the peptide in the sample is highly likely to be in a reduced form.

[0018] On the other hand, if no peak is detected at the specific mass-to-charge ratio, it can be determined that the Cys residue contained in the peptide in the sample is oxidized, or that the peptide in the sample is highly likely to not contain a Cys residue. In this case, whether or not the peptide in the sample contains a Cys residue can be determined by comparing the MS / MS spectrum with information stored in a database to identify the peptide. Even if a peak is detected at the specific mass-to-charge ratio, the peptide can be identified by comparing the pattern obtained by removing the peak from the MS / MS spectrum with information stored in a database.

[0019] In the present invention, a compound represented by the following formula (1) can be used as the labeling compound. [ka] This compound is a DAABD-Cl reagent, which is known as a reagent that specifically binds to SH groups and fluorescently labels peptides. In this case, examples of specific mass-to-charge ratios corresponding to the structure of the labeled compound include "303.06" and "344.08."

[0020] The peak with a mass-to-charge ratio of 303.06 corresponds to the ion represented by the following formula (2), and the peak with a mass-to-charge ratio (m / z) of 344.08 corresponds to the ion represented by the following formula (3). Both of these correspond to ions derived from DAABD-Cl. [ka] [ka] Effect of the Invention

[0021] According to the present invention, a labeled compound is added to a sample containing a peptide to be analyzed, and the sample is then analyzed using a chromatographic mass spectrometer. The mass spectrum is generated based on the data obtained, and a peak with a specific mass-to-charge ratio corresponding to the structure of the labeled compound that specifically reacts with an SH group is determined to determine whether the peptide contains a reduced Cys residue. This makes it possible to eliminate the need for tedious and complicated tasks such as identifying the peptide by comparing data obtained about the sample with information stored in a database, or measuring standard samples to set conditions for measuring the sample. [Brief description of the drawings]

[0022] [Figure 1] 1 is a schematic diagram of an embodiment of a peptide analysis device for carrying out a peptide analysis method according to the present invention. [Diagram 2] 1 is a flowchart showing the procedure of a peptide analysis process. [Diagram 3] FIG. 1 shows an example of the results of an MS / MS ion search performed on the results of LC-MS / MS analysis of a sample containing human albumin. [Figure 4] FIG. 1 shows the MS / MS spectrum of sequence number 571 and the amino acid sequence identified from the spectrum. [Diagram 5] FIG. 1 shows the MS / MS spectrum of sequence number 589 and the amino acid sequence identified from the spectrum. [Figure 6] FIG. 1 shows the MS / MS spectrum of sequence number 627 and the amino acid sequence identified from the spectrum. [Figure 7] FIG. 1 shows an example of the MS / MS spectrum of sequence number 655 and the amino acid sequence identified from the spectrum. [Figure 8] FIG. 1 shows the results of an MS / MS ion search for a sample containing a peptide in which no peak was observed at the mass-to-charge ratio corresponding to the DAABD-Cl structure. [Figure 9] An example of the MS spectrum and MS / MS spectrum of a sample containing glutathione. [Figure 10] Examples of MS and MS / MS spectra of a sample containing oxytocin. [Figure 11] FIG. 1 shows peaks with common mass-to-charge ratios that appeared in the MS / MS spectra of samples containing albumin, glutathione, and oxytocin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The analytical method according to the present invention will be described below with reference to the drawings. Fig. 1 is a schematic diagram of a peptide analyzer for carrying out the peptide analysis method according to the present invention. This peptide analyzer is a liquid chromatograph system (LC-MS / MS system) including a liquid chromatograph (LC) section 1 and a mass spectrometry (MS / MS) section 2.

[0024] The LC section 1 comprises a mobile phase container 10 in which a mobile phase is stored, a pump 11 that draws in the mobile phase and delivers it at a constant flow rate, an injector 12 that injects a predetermined amount of sample liquid into the mobile phase, and a column 13 that separates various compounds contained in the sample liquid in the time direction.

[0025] The MS / MS section 2 has a multi-stage differential pumping system configuration with a first intermediate chamber 21, a second intermediate chamber 22, and a third intermediate chamber 23, the degree of vacuum of which is increased stepwise, between an ionization chamber 20, which is at approximately atmospheric pressure, and an analysis chamber 24, which is at a high vacuum and evacuated by a vacuum pump (not shown). An electrospray ionization probe (ESI probe) 201 is installed in the ionization chamber 20, which sprays the sample liquid eluted from the column 13 of the LC section 1 while imparting an electric charge to the sample liquid.

[0026] The ionization chamber 20 and the first intermediate chamber 21 communicate with each other through a thin-diameter heated capillary 202. The first intermediate chamber 21 and the second intermediate chamber 22 are separated by a skimmer 212 having a small hole at the top, and the first intermediate chamber 21 and the second intermediate chamber 22 are provided with ion guides 211 and 221 for converging ions and transporting them to the rear stage, respectively. The third intermediate chamber 23 is provided with a quadrupole mass filter 231 for separating ions according to their mass-to-charge ratio, a collision cell 232 having a multipole ion guide 233 therein, and an ion guide 234 for transporting ions emitted from the collision cell 232. A CID gas such as argon or nitrogen is continuously or intermittently supplied to the inside of the collision cell 232.

[0027] The analysis chamber 24 is equipped with an ion transport electrode 241 for transporting ions incident from the third intermediate chamber 23 to the orthogonal acceleration section, an orthogonal acceleration electrode 242 consisting of two electrodes 242A and 242B arranged opposite each other across the ion incident optical axis (orthogonal acceleration region), an acceleration electrode 243 for accelerating ions sent out into the flight space by the orthogonal acceleration electrode 242, reflectron electrodes 244 (244A, 244B) that form a return trajectory of the ions in the flight space, a detector 245, and a flight tube 246 located at the outer edge of the flight space.

[0028] In the MS / MS section 2, MS scan measurement, MS / MS scan measurement, or MS nScan measurements (n is an integer of 3 or more) can be performed. For example, in the case of MS / MS scan measurements (product ion scan measurements), only ions set as precursor ions are passed through the quadrupole mass filter 231. Also, CID gas is supplied to the inside of the collision cell 232 to fragment the precursor ions and generate product ions. The product ions are then introduced into the flight space, and the mass-to-charge ratio is calculated based on their flight times.

[0029] The analysis control unit 3 has a function of controlling the operations of the LC unit 1 and the MS / MS unit 2 under the instructions of the central control unit 31. The central control unit 31, to which an input unit 32 and a display unit 33 are connected, is responsible for the overall control of the entire system as well as the user interface through these. A control program 35 for peptide structure estimation that performs characteristic control for estimating the structure and amino acid sequence of a specific amino acid (specifically, cysteine) residue contained in a protein or peptide that is the subject of analysis is stored in a storage device included in the central control unit 31, and the CPU or the like controls each unit through the analysis control unit 3 in accordance with this program 35, thereby performing measurements and data processing required for estimating the peptide structure.

[0030] At this time, the detection signal (ion intensity signal) from the detector 245 is input to the data processing unit 4. The data processing unit 4 includes a data collecting unit 41, a data storage unit 42, a graph creating unit 43, and a peptide structure estimation processing unit 44 as functional blocks, and performs data processing using information stored in a database 45 to estimate the structure of cysteine ​​contained in a peptide and the amino acid sequence, and from these results, judges whether or not a protein or peptide in a sample has a reduced Cys residue, and identifies the protein or peptide. As will be described later, whether or not a Cys residue is reduced is judged based on whether or not a peak with a specific mass-to-charge ratio appears in an MS / MS spectrum. Since the mass-to-charge ratio of such a peak is predetermined, it is preferable to store it in the peptide structure estimation control program 35.

[0031] The database 45 stores time-of-flight-mass-to-charge ratio information and applied voltage information. The time-of-flight-mass-to-charge ratio information is information describing the time required for ions having various mass-to-charge ratios to fly through the flight space of the mass analysis unit 2. The applied voltage information is information relating to values ​​of voltages applied to the ion transport electrode 241, the orthogonal acceleration electrode 242, the acceleration electrode 243, the reflectron electrode 244, and the flight tube 246, and in this embodiment, for the orthogonal acceleration electrode 242, applied voltages of different magnitudes are associated with the ion transmission period.

[0032] The central control unit 31 and the data processing unit 4 can be realized by using a personal computer as hardware and executing dedicated control and processing software installed in the computer. In this case, the input unit 32 is a keyboard and a pointing device (mouse), and the display unit 33 is a display monitor.

[0033] When analyzing the structure of Cys residues contained in peptides using the above-mentioned LC-MS / MS system, first, a sample containing the peptide to be analyzed is prepared. At this time, a labeling compound that specifically reacts with thiol groups is added to the sample to react with the thiol groups of the Cys residues contained in the peptides in the sample. After such pretreatment is completed, the operator sets the sample in the LC-MS / MS system and operates the input unit 32 to set conditions such as separation conditions in the LC unit 1, ionization conditions in the MS / MS unit 2, and MS / MS measurement conditions (step S1).

[0034] When measurement is started according to an instruction from the operator, the analysis control unit 3 controls the LC unit 1 and the MS / MS unit 2 based on instructions from the central control unit 31. As a result, the sample separated in the LC unit 1 is introduced into the MS / MS unit 2, where it is ionized and then a product ion scan measurement is performed (step S2). At this time, the ionization is performed under conditions that do not cause the labeled compound to leave the peptide. The data obtained by the product ion scan measurement is stored in the data storage unit 42 in the data processing unit 4.

[0035] Then, when the measurement is completed, the graph creation unit 43 processes the data to create a mass spectrum (step S3). Next, peaks are extracted from the mass spectrum, and it is determined whether or not a peak with a specific mass-to-charge ratio is included in the extracted peaks (step S4). Furthermore, the peak information of the mass spectrum is collated with information stored in a protein / peptide database to estimate the amino acid sequence of the peptide (step S5). When the above processing is completed, the determination result of step S4 and the estimated amino acid sequence are output to the display unit 33 (step S6). The determination result of step S4 is, for example, the number of reduced Cys residues contained in the peptide in the sample, the ratio of reduced Cys residues to oxidized Cys residues, which Cys residues in the amino acid sequence are reduced, etc.

[0036] Next, an example of actual measurement using the LC-MS / MS system according to the above-mentioned embodiment will be described, and it will be shown that the system is useful for determining whether Cys residues contained in proteins or peptides are in the reduced or oxidized form.

[0037] 1. Example of measurement of human albumin (1) DAABD modification of human albumin The following solutions and buffer solutions were added to 10 μL of human albumin (1 mM), and finally water was added to make the total volume 100 μL, and then the mixture was gently stirred at 500 rpm at 40° C. for 10 minutes to react human albumin with DAABD-Cl. Then, 3 μL of 20% TFA aqueous solution was added to the stirred mixture to terminate the reaction. ·3.3mM EDTA / 17mM CHAPS aqueous solution: 58.8μL ·TCEP (42mM) aqueous solution: 1.2μL 6M Guanidine hydrochloride (GdnHCl) buffer (pH 8.7): 25 μL 0.14M DAABD-Cl acetonitrile solution: 5μL Note that EDTA is the abbreviation of "ethylenediaminetetraacetic acid", CHAPS is the abbreviation of "3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate)", TCEP is the abbreviation of "tris(2-carboxyethyl)phosphine hydrochloride", and TFA is the abbreviation of "trifluoroacetic acid". The structural formula of DAABD-Cl is shown below. [Chemical formula]

[0038] (2) Fractionation by high performance liquid chromatography (HPLC) 10 μL (about 1 nmol of human albumin) was collected from 103 μL of the reaction solution obtained in (1) above, and this was injected into a preparative column to fractionate an albumin fraction (about 2 - 5 mL). The fractionated albumin fraction was dried to dryness using a centrifugal dryer (or freeze dryer). The LC conditions are shown below. <LC conditions> HPLC: Nexera XR manufactured by Shimadzu Corporation Column: Aeris WIDEPORE 3.6u XB-C8 manufactured by Phenomenex (HPLC column filled with ODS group-modified silica gel (inner diameter 4.6 mm, length 250 mm, particle size of packing material 3.6 μm)) Column temperature: 60 °C Elution method: Gradient elution with two solutions, mobile phase A: 0.1% TFA, 1% IPA, 9% ACN aqueous solution, mobile phase B: 0.1% TFA, 1% IPA, 74% ACN aqueous solution Mobile phase flow rate: 0.8 mL / min

[0039] (3) Reduction treatment The dried albumin fraction obtained in (2) above was dissolved in 75 μL of a 500 mM TrisHCl (pH 8.0) aqueous solution containing urea (8M). 1 μL of a TCEP (42 mM) aqueous solution was added thereto and mixed, and this mixed solution was heated at 37 °C for 30 minutes. After the reduction treatment, an alkylation treatment is usually performed, but this treatment was not performed in the actual measurement example presented here. The reason is that the protein was first reduced and then modified with DAABD, so all Cys residues reduced to free SH groups were modified with DAABD. However, if necessary depending on the purpose or analysis method, an alkylation treatment should be performed appropriately. The alkylation treatment method involves adding 1 μL of IAA (iodoacetamide) (100 mM) to the above mixture, mixing, and heating the mixture at 37°C for 30 minutes.

[0040] (4) Trypsin digestion In (3), 225 μL of 50 mM NH4CO3 aqueous solution was added to the mixed solution heated to 37°C, and mixed to obtain 300 μL of mixed sample solution, after which trypsin digestion was performed. Trypsin digestion was performed using a trypsin column (MonoSpin (registered trademark) Trypsin column (GL Sciences Inc.)). Specifically, first, the trypsin column was equilibrated with 50 mM NH4CO3 aqueous solution, and the mixed sample solution was passed through the column slowly twice to perform trypsin digestion. The mixed sample solution after trypsin digestion is called trypsin-digested sample solution.

[0041] (5) LCMS / MS measurement Of the 300 μL of the trypsin digested sample solution obtained in (4) above, 3 μL (corresponding to 10 pmol of human albumin) was introduced into an HPLC (Shimadzu Corporation, Nexera X2) for LC separation, and then LCMS analysis and LCMS / MS analysis were performed using an LCMS-IT-TOF (Shimadzu Corporation). Note that here, the three peaks with the highest intensity were selected from the peaks detected in the mass spectrum obtained by the LCMS analysis, and MS / MS (automatic) analysis was performed. LC: Shimadzu Nexera XR Column: Shimadzu Corporation XR-ODS III (ODS group-modified silica gel packed HPLC column (inner diameter 2.0 mm, length 150 mm, packing particle size 2.2 μm)) Column temperature: 40℃ Elution method: Gradient elution with two solutions: mobile phase A: 5 mM ammonium formate / 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile solution. Mobile phase flow rate: 0.3mL / min Ionization: ESI Ion accumulation: 30 msec Mass range: MS m / z 150 - 1,500, MS / MS m / z 50 - 2,000

[0042] (6) Database search The peak information extracted from the MS / MS spectrum obtained in the MS / MS analysis in (5) above was subjected to the MS / MS ion search implemented in Mascot (Matrix Science), to identify the peptides corresponding to each peak in the mass spectrum, and also to identify the assignment of the fragments corresponding to each peak in the MS / MS spectrum.

[0043] Figure 3 shows some of the results of the MS / MS ion search. This shows information on the peptide identification of each MS / MS spectrum, and from the left, it shows the serial number of the MS / MS spectrum data (Query), the experimental value of the m / z value of the precursor ion (Observed), the experimental value of the mass of the precursor molecule (Mr(expt)), the theoretical value of the mass of the precursor molecule (Mr(calc)), the difference between the experimental value and the theoretical value of the mass of the precursor molecule (Delta), the number of uncleaved sites (Miss), the ion score (Score), the expected value (Expect), the score rank (Rank), whether it is a unique peptide that exists only in a certain protein (Unique, if it is a unique peptide, it is written as "U"), and the amino acid sequence of the peptide (Peptide). "+DAABD(C)" displayed to the right of the symbol representing the amino acid sequence indicates that the Cys residue of the peptide is labeled with DAABD.

[0044] The search results shown in Figure 3 showed that many of the peaks were identified with peptides with high ion score values, and all of these were identified as human albumin (trypsin digestion fragments). In addition, 20 of the peptides identified as belonging to human albumin were labeled with DAABD, and it was shown that a peak appeared at a mass-to-charge ratio (m / z) of 303.06 in 18 of these 20 peptides. The ion (C) shown in the following structural formula (2) that can be generated by cleavage of the chemical bond from the structure in which DAABD-Cl is modified to a Cys residue was 10 H 15 NO3S2 + ) is 303.06, the peak that appeared at the mass-to-charge ratio (m / z) position of 303.06 can be said to be derived from a structure in which DAABD-Cl is modified with a Cys residue.

[0045] [ka]

[0046] Figures 4 to 7 show the MS / MS spectra of the four peptides enclosed in square frames in Figure 3, and the amino acid sequences of the peptides estimated from these spectra. Figure 4 shows the MS / MS spectrum of the peptide with the serial number "571" (amino acid sequence: C*C*TESLVNR), Figure 5 shows the MS / MS spectrum of the peptide with the serial number "589" (amino acid sequence: YIC*ENQDSISSK), Figure 6 shows the MS / MS spectrum of the peptide with the serial number (Query) 627 (amino acid sequence: SLHTLFGDKLC*TVATLR), and Figure 7 shows the MS / MS spectrum of the peptide with the serial number (Query) 655 (amino acid sequence: ALVLIAFAQYLQQC*PFEDHVK) (C* indicates DAABD-labeled Cys). As can be seen from Figures 4 to 7, the MS / MS spectra of these four peptides all show a peak at a mass-to-charge ratio (m / z) of 303.06 (peaks enclosed in square frames in Figures 4 to 7), and all of the peptides contain a Cys residue. The peptide shown in Figure 7 is a peptide that contains Cys34 of human albumin. These results suggest that the appearance of a peak at the mass-to-charge ratio (m / z) of 303.06 on an MS / MS spectrum may be an indicator for determining whether a peptide estimated from the MS / MS spectrum contains a reduced Cys residue in the original sample.

[0047] In addition, even though the reduced Cys residue of human albumin was labeled with DAABD, some peptides did not show a peak at the mass-to-charge ratio (m / z) of 303.06 in the MS / MS spectrum. It is presumed that such peptides had low ion scores or the mass-to-charge ratio of the precursor ion was close to or below the cutoff value of the ion trap, and therefore were not trapped. Figure 8 shows an example of the results of the MS / MS ion search for such a peptide (peptide with serial number (Query): 628 enclosed in a dashed rectangular frame in Figure 8).

[0048] 2. Measurement example of a sample containing peptides other than albumin DAABD-Cl was added to the sample containing glutathione and the sample containing oxytocin in the same manner as in the above-mentioned measurement example of the sample containing human albumin (except for the trypsin digestion step), and DAABD was bound to the Cys residues contained in each of glutathione and oxytocin. Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine (Glu-Cys-Gly). Oxytocin is a peptide consisting of nine amino acids (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly). Figures 9 and 10 show the results of LC-MS / MS analysis of a sample containing glutathione and a sample containing oxytocin. The upper parts of Figures 9 and 10 are MS spectra, and the lower parts are MS / MS spectra of the precursor ions enclosed in ellipses in the MS spectra in the upper parts.

[0049] As shown in the MS / MS spectra of FIGS. 9 and 10, it was confirmed that a peak was observed at a mass-to-charge ratio (m / z) of 303.06 in the sample containing glutathione and oxytocin.

[0050] 3.Other As shown in FIG. 11, in the MS / MS spectra obtained for the sample containing human albumin, the sample containing glutathione, and the sample containing oxytocin, in addition to the peak with a mass-to-charge ratio (m / z) of 303.06, a peak with a mass-to-charge ratio (m / z) of 344.08 appeared in common. When the structure of the ion derived from the structure in which DAABD-Cl is modified with a Cys residue, which has a mass of 344.08, was examined, the structure shown in the following formula (3) was found. This suggests that the peak with a mass-to-charge ratio (m / z) of 344.08, like the peak with a mass-to-charge ratio (m / z) of 303.06, may be an indicator for determining whether the Cys residue is in a reduced form as a peak corresponding to the structure of DAABD-Cl.

[0051] [ka]

[0052] The present invention is not limited to the above-described embodiment, and various modifications are possible. For example, SH groups are not limited to those possessed by Cys residues, and for proteins or peptides containing SH groups, it is possible to determine whether the SH groups are in a free state or form an S-S bond.

[0053] The experiments were carried out using DAABD-Cl as an example of a labeling compound that specifically reacts with the SH group of a Cys residue contained in a protein or peptide. However, other compounds that can be used include those represented by the following general formula (4) or (5), or isotope compounds thereof, which are described in, for example, Patent Documents 1 and 2. [ka] In formula (4), X is halogen, Y is O, Se or S, and R is -NH2, -NHR' (wherein R' is alkyl-substituted N-alkyl, dialkyl-substituted N-alkyl or trialkyl-substituted N-alkyl), or -NR''R''' (wherein R'' is alkyl and R''' is alkyl-substituted N-alkyl, dialkyl-substituted N-alkyl or trialkyl-substituted N-alkyl).

[0054] [ka] In the formula (5), X represents a halogen, and Y represents O, Se or S.

[0055] In the present invention, instead of the product ion scan measurement, a precursor ion scan measurement may be performed in which an ion derived from a functional group formed by a reaction between the thiol group released from the Cys residue and the labeling compound is used as the product ion, or an MRM measurement may be performed in which multiple types of ions derived from a functional group formed by a reaction between the thiol group released from the Cys residue and the labeling compound are used as product ions for the precursor ion used in the product ion scan measurement. [Explanation of symbols]

[0056] 1...LC section 2…MS / MS section 3. Analysis and control section 31...Central control unit 32...Input section 33...Display section 34…Cys 35…Program 35…Control program for peptide structure prediction 4. Data processing section 41…Data collection section 42...Data storage unit 43…Graph Creation Section 44...Peptide structure prediction processing unit 45…Database

Claims

1. A peptide analysis method for analyzing reduced and oxidized cysteine ​​residues contained in a peptide using a chromatography mass spectrometer capable of MS / MS measurement, comprising: a) providing a sample containing a peptide to be analyzed; b) adding a labeling compound that specifically reacts with a thiol group to the sample to label reduced cysteine ​​residues contained in peptides in the sample with the labeling compound; c) a separation step of separating the sample containing the peptide labeled with the labeling compound by chromatography; d) a measurement execution step of performing a product ion scan measurement on the separated peptide labeled with the labeling compound, using peptide ions generated by ionization under conditions that do not cause thiol groups to be released from cysteine ​​residues as precursor ions; e) a determination step of determining whether or not the peptide to be analyzed contains a reduced cysteine ​​residue by determining whether or not a peak appears at a specific mass-to-charge ratio corresponding to the structure of the labeled compound in a mass spectrum created based on the data obtained in the measurement execution step. Equipped with The peptide analysis method, wherein the labeled compound is a compound represented by the following formula (1): 【Chemistry 1】

2. The peptide analysis method according to claim 1, A peptide analysis method, characterized in that in the separation step, a sample in which the peptide labeled with the labeling compound is fragmented into peptides by a proteolytic enzyme is separated over time by chromatography.

3. The peptide analysis method according to claim 1, The peptide analysis method, wherein the mass-to-charge ratio of the peak derived from the structure of the labeled compound is 303.

06.

4. The peptide analysis method according to claim 1, The method for peptide analysis, wherein the mass-to-charge ratio of the peak derived from the structure of the labeled compound is 344.

08.

5. A peptide analysis device for analyzing reduced and oxidized cysteine ​​residues contained in a peptide, the peptide analysis device including a chromatography mass spectrometer capable of MS / MS measurement, a) an analysis control unit that operates the chromatographic mass spectrometer to separate a sample containing a peptide labeled with a labeling compound that specifically reacts with a thiol group by chromatography, and to perform a product ion scan measurement on the separated sample, using peptide ions generated by ionization under conditions that do not cause thiol groups to be dissociated from cysteine ​​residues as precursor ions; b) a mass spectrum generating unit for generating a mass spectrum based on data obtained by the product ion scan measurement; c) a determination unit for determining whether or not a peak appears at a specific mass-to-charge ratio corresponding to the structure of the labeled compound in the mass spectrum, thereby determining whether or not the peptide to be analyzed contains a reduced cysteine ​​residue; Equipped with The peptide analyzer, wherein the labeled compound is a compound represented by the following formula (1): 【Chemistry 1】

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