Isoaspartic acid analysis method and mass spectrometry device

Abstract

In a method for analyzing isoaspartic acid by dissociating precursor ions derived from sample components to generate product ions and analyzing them, the following steps are included: the precursor ions are irradiated with hydrogen radicals or radicals having oxidizing ability to dissociate the precursor ions to generate product ions, the product ions are separated and detected according to a mass-to-charge ratio, and the product ions derived from isoaspartic acid are determined based on the mass-to-charge ratio of the product ions.

Description

Technical Field

[0001] The present invention relates to an analysis method of isoaspartic acid and a mass spectrometry device for implementing the analysis method. Background Art

[0002] It has been reported that aspartic acid, one of the amino acids constituting proteins, isomerizes over time and transforms into isoaspartic acid, which is a major cause of cataracts (e.g., Non-Patent Document 1). Therefore, analyzing isoaspartic acid contained in peptides included in biological metabolites and inferring the extent of isoaspartic acid accumulation in the body based on the results may be effective in understanding the risk of cataracts or studying countermeasures for preventing cataracts.

[0003] In order to identify polymer compounds such as proteins, the following mass spectrometry is widely used: ions (precursor ions) derived from polymer compounds are dissociated once or multiple times to generate product ions (also called fragment ions), which are separated and detected according to the mass-to-charge ratio. As a representative method for dissociating ions in mass spectrometry, the collision-induced dissociation (CID: Collision-Induced Dissociation) method is known, in which molecules of inert gases such as nitrogen collide with ions. However, in the CID method, there is no difference between the product ions generated by the dissociation of the precursor ions derived from the peptide containing the isomer thereof, i.e., the aspartic acid residue. Therefore, even if the ions are dissociated by the CID method, isoaspartic acid and aspartic acid cannot be identified.

[0004] Non-patent document 1 states that: if negative ions collide with positive precursor ions derived from a peptide containing an isoaspartic acid residue and electron transfer dissociation (ETD) occurs, product ions characterizing isoaspartic acid named c+57 ions and z-57 ions are generated. The c+57 ion refers to an ion having a mass of 57 Da greater than the ion (c ion) on the N-terminal side generated by the cleavage of the N-Cα bond of the peptide main chain possessed by aspartic acid. In addition, the z-57 ion refers to an ion having a mass of 57 Da less than the ion (z ion) on the C-terminal side generated by the cleavage of the above-mentioned N-Cα bond. It should be noted that the product ions generated from the precursor ions derived from the peptide containing an isoaspartic acid residue are expressed based on the c ions and z ions of the peptide containing an isoaspartic acid residue because the bonds of the peptide main chains of the isoaspartic acid residue and the aspartic acid residue are different, and the definitions of the c ions and z ions are unclear.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2015 / 133259

[0008] Non-patent literature

[0009] Non-patent literature 1: Takumi Takata, “Studies on the mechanism of cataract caused by spontaneous chemical modification of amino acids in lens constituent proteins”, Journal of the Japanese Society of Cataract, 30, pp.7-12, 2018

[0010] Non-patent literature 2: Kempkes, LJ, Martens, J., Grzetic, J., Berden, G., & Oomens, J., "Deamidation Reactions of Asparagine-and Glutamine-Containing Dipeptides Investigated by Ion Spectroscopy", Journal of the American Society for MassSpectrometry, 27(11), pp.1855-1869, 2016

[0011] Non-patent document 3: Shimabukuro, Kasuya, and Wada, “Development of a small atomic source using microwave capacitively coupled plasma”, 77th Society of Applied Physics Lecture Series, September 2016, Society of Applied Physics

[0012] Non-patent document 4: Yuji Shimabukuro, Hidenori Takahashi, Shinichi Iwamoto, Koichi Tanaka, Motoi Wada, "Tandem Mass Spectrometry of Peptide Ions by Microwave Excited Hydrogen and Water Plasmas", Anal.Chem.2018,90(12)pp7239-7245 Summary of the invention

[0013] Problem that the invention aims to solve

[0014] In the ETD method, since negative ions are collided with positive precursor ions to generate product ions, only precursor ions with a valence of 2 or more can be dissociated. In addition, the ions generated in soft ionization methods such as the matrix-assisted laser desorption / ionization (MALDI) method used in the ionization of proteins and peptides are almost all univalent ions. Therefore, in the combination of the MALDI method and the ETD method, the dissociation efficiency of the precursor ions is poor, and the above-mentioned ions characterizing isoaspartic acid cannot be detected with sufficient intensity, so there is a problem of difficulty in distinguishing isoaspartic acid from aspartic acid.

[0015] An object of the present invention is to provide a technique capable of easily distinguishing isoaspartic acid and aspartic acid contained in a sample.

[0016] Solutions for solving problems

[0017] One embodiment of the present invention made to solve the above-mentioned problems is a method for analyzing isoaspartic acid by dissociating precursor ions derived from a peptide component to generate product ions and analyzing the product ions, which comprises the following steps:

[0018] The precursor ions are irradiated with hydrogen radicals or radicals having oxidizing ability to dissociate the precursor ions to generate product ions.

[0019] The aforementioned product ions are separated and detected according to their mass-to-charge ratio.

[0020] The product ions derived from isoaspartic acid were determined based on the mass-to-charge ratio of the aforementioned product ions.

[0021] Another aspect of the present invention made to solve the above-mentioned problems is a mass spectrometer for analyzing isoaspartic acid by generating product ions from precursor ions derived from sample components and analyzing the product ions, comprising:

[0022] A reaction chamber, which is used to introduce the aforementioned precursor ions;

[0023] A radical generating unit for generating hydrogen radicals or radicals having oxidizing ability;

[0024] a radical irradiation unit for irradiating the interior of the reaction chamber with the radicals generated in the radical generation unit;

[0025] a separation and detection unit for separating and detecting product ions generated by dissociation of the precursor ions through the reaction with the free radicals according to a mass-to-charge ratio; and

[0026] A product ion determination unit is used to determine the product ion derived from isoaspartic acid based on the mass-to-charge ratio of the product ion.

[0027] Effects of the Invention

[0028] In the isoaspartic acid analysis method and mass spectrometry apparatus of the present invention, hydrogen radicals or radicals with oxidizing ability are irradiated to precursor ions derived from sample components to generate product ions. Radicals with oxidizing ability are, for example, oxygen radicals and hydroxyl radicals. The ion dissociation method of irradiating these radicals to precursor ions is the hydrogen attachment dissociation (HAD: Hydrogen-Attachment Dissociation) method and the oxygen attachment dissociation (OAD: Oxygen-Attachment Dissociation) method proposed by the inventors in Patent Document 1.

[0029] The present inventors have found that if the precursor ions derived from isoaspartic acid are dissociated by the HAD method, c+57 ions and z-57 ions that characterize isoaspartic acid (not generated by aspartic acid) are generated in the same manner as the ETD method. In addition, it has been found that if the precursor ions derived from isoaspartic acid are dissociated by the OAD method, a+16 ions and / or x ions are generated. The a+16 ion refers to an ion that has a mass 16 Da greater than the ion (a ion) on the N-terminal side generated by the cleavage of the Cα-C bond of the peptide main chain possessed by aspartic acid. The a+16 ion and the x ion generated by the isoaspartic acid residue have different masses from the ions derived from the aspartic acid residue, and therefore, the present invention can be used to identify these residues.

[0030] In other words, it was found that the product ions generated by dissociating the precursor ions from the sample components were separated and detected according to the mass-to-charge ratio, and the C+57 ion, Z-57 ion, A+16 ion or X ion were determined from them, thereby being able to identify isoaspartic acid and aspartic acid and analyze them. The HAD method and the OAD method are methods for dissociating the precursor ions by free radical addition, and can be used regardless of the polarity and valence of the ions. Therefore, the precursor ions from the sample components can be efficiently dissociated, and isoaspartic acid and aspartic acid as its isomer can be easily identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a diagram showing the main parts of an embodiment of the mass spectrometer described in the present invention.

[0032] Figure 2 It is a schematic diagram of the structure of the radical irradiation unit in the mass spectrometer of this embodiment.

[0033] Figure 3 This is the mass spectrum of product ions generated by dissociating isoaspartic acid and aspartic acid by the CID method.

[0034] Figure 4 It is a diagram illustrating the molecular structure and dissociation position of aspartic acid and isoaspartic acid.

[0035] Figure 5 This is a diagram illustrating the results of quantum chemical calculations related to the dissociation of isoaspartic acid.

[0036] Figure 6 This is a mass spectrum of product ions generated by dissociating a peptide having an isoaspartic acid residue and an aspartic acid residue by the HAD method.

[0037] Figure 7 This is a mass spectrum of product ions generated by dissociating other peptides having an isoaspartic acid residue and an aspartic acid residue by the HAD method. DETAILED DESCRIPTION

[0038] An embodiment of the mass spectrometer of the present invention is described below with reference to the accompanying drawings. The mass spectrometer of this embodiment is an ion trap-time of flight (IT-TOF) mass spectrometer.

[0039] Figure 1 The schematic structure of the ion trap-time-of-flight mass spectrometer (hereinafter also referred to as "mass spectrometer") of the present embodiment is shown in FIG. The mass spectrometer of the present embodiment is provided inside a vacuum chamber (not shown) which maintains a vacuum atmosphere: an ion source 1 for ionizing components in a sample; an ion trap 2 for capturing ions generated by the ion source 1 by the action of a high-frequency electric field; a time-of-flight mass separation unit 3 for separating ions ejected from the ion trap 2 according to a mass-to-charge ratio; and an ion detector 4 for detecting the separated ions. The ion trap mass spectrometer of the present embodiment further comprises: a radical irradiation unit 5 for irradiating the precursor ions captured in the ion trap 2 with radicals to dissociate the ions captured in the ion trap 2; an inert gas supply unit 6 for supplying a specified inert gas into the ion trap 2; a trap voltage generating unit 7; a control unit 8 and a data processing unit 9.

[0040] The ion source 1 of the mass spectrometer of this embodiment is a MALDI ion source. In the MALDI ion source, a substance (matrix substance) that easily absorbs laser light and is easily ionized is pre-coated on the surface of the sample. Thereafter, the matrix substance mixed with the sample molecules is microcrystallized and irradiated with laser light to ionize the sample molecules. The ion trap 2 is a three-dimensional ion trap including a circular ring electrode 21 and a pair of end cap electrodes (an inlet-side end cap electrode 22 and an outlet-side end cap electrode 24) arranged opposite to each other with the ring electrode 21 sandwiched therebetween. A radical particle introduction port 26 and a radical particle discharge port 27 are formed in the ring electrode 21, an ion introduction hole 23 is formed in the inlet-side end cap electrode 22, and an ion ejection hole 25 is formed in the outlet-side end cap electrode 24. The trap voltage generating unit 7 applies either a high-frequency voltage or a direct current voltage or a voltage synthesized from them to the ring electrode 21, the inlet-side end cap electrode 22, and the outlet-side end cap electrode 24 at a predetermined timing, according to instructions from the control unit 8.

[0041] The radical irradiation unit 5 includes: a nozzle 54, which has a radical generation chamber 51 formed therein; a raw material gas supply unit (raw material gas supply source) 52, which is used to introduce a raw material gas into the radical generation chamber 51; a vacuum pump (vacuum exhaust unit) 57, which is used to exhaust the radical generation chamber 51; an inductively coupled high-frequency plasma source 53, which is used to supply microwaves for generating vacuum discharge in the radical generation chamber 51; a skimmer 55, which has an opening on the central axis of the jet flow from the nozzle 54, and separates the diffused raw material gas molecules and the like to take out a small diameter radical flow; and a valve 56, which is provided in the flow path from the raw material gas supply source 52 to the radical generation chamber 51. As the raw material gas, for example, hydrogen, oxygen, water vapor (water), air, etc. can be used. If hydrogen is used as the raw material gas, hydrogen radicals are generated, and if oxygen is used as the raw material gas, oxygen radicals are generated. When water vapor is used as the raw material gas, hydroxyl radicals, oxygen radicals, and hydrogen radicals are generated, and when air is used as the raw material gas, oxygen radicals and nitrogen radicals are mainly generated.

[0042] The radical irradiation unit 5 may be, for example, a radical irradiation unit described in Non-Patent Document 3. Its schematic structure is shown in FIG. Figure 2. The radical irradiation section 5 is generally composed of a raw gas supply source 52, a high-frequency plasma source 53, a nozzle 54 and a vacuum pump 57. The high-frequency plasma source 53 includes a microwave supply source 531 and a three-stub tuner 532. The nozzle 54 includes a ground electrode 541 constituting the outer periphery and a torch 542 made of Pyrex (registered trademark) glass located inside the ground electrode 541, and the interior of the torch 542 constitutes a radical generation chamber 51. Inside the radical generation chamber 51, a needle electrode 543 connected to the high-frequency plasma source 53 by means of a connector 544 passes through the length direction of the radical generation chamber 51. In addition, a flow path for supplying the raw gas from the raw gas supply source 52 to the radical generation chamber 51 is provided, and a valve 56 for adjusting the flow rate of the raw gas is provided on the flow path.

[0043] The inert gas supply unit 6 includes a gas supply source 61 storing helium, argon or the like used as a buffer gas or a cooling gas, and a gas introduction pipe 63. The gas introduction pipe 63 is provided with a valve 62 for adjusting the flow rate of the gas supplied from the gas supply source 61 to the ion trap 2.

[0044] Next, the analysis in the mass spectrometer of this embodiment is described. Before the analysis begins, the interior of the vacuum chamber that accommodates the ion trap 2 and the like is evacuated to a predetermined vacuum level using a vacuum pump (not shown). In addition, the interior of the radical generation chamber 51 is evacuated to a predetermined vacuum level using a vacuum pump 57. Next, a raw gas is supplied from a raw gas supply source 52 to the radical generation chamber 51 of the radical irradiation section 5, and microwaves are supplied from a high-frequency plasma source 53, thereby generating free radicals inside the radical generation chamber 51. As described later, in this embodiment, hydrogen, oxygen, water vapor, etc. are used as raw gas to generate hydrogen radicals, or oxygen radicals and / or hydroxyl radicals.

[0045] Various ions (mainly monovalent ions) generated by the sample in the ion source 1 are ejected from the ion source 1 in a packet shape and introduced into the interior of the ion trap 2 through the ion introduction hole 23 formed in the entrance side end cover electrode 22. The ions introduced into the ion trap 2 are captured by the high-frequency electric field formed in the ion trap 2 by the voltage applied to the ring electrode 21 by the trap voltage generator 7. Thereafter, a predetermined voltage is applied to the ring electrode 21 and the like by the trap voltage generator 7, thereby exciting ions having a mass-to-charge ratio other than ions having a specific mass-to-charge ratio as a target and ejecting them from the ion trap 2. In this way, only precursor ions having a specific mass-to-charge ratio are captured in the ion trap 2.

[0046] Next, the valve 62 of the inert gas supply unit 6 is opened, and an inert gas such as helium is introduced into the ion trap 2 to cool the precursor ions. As a result, the precursor ions are gathered near the center of the ion trap 2. Thereafter, the valve 56 of the radical irradiation unit 5 is opened, and the gas containing the radicals generated in the radical generation chamber 51 is ejected from the nozzle 54. Furthermore, the gas containing the radicals is formed into a thin-diameter beam by the separator 55, and ejected into the ion trap 2 from the radical particle inlet 26 penetrated in the ring electrode 21, and irradiated to the precursor ions captured in the ion trap 2.

[0047] At this time, the opening degree and opening time of the valve 56 (i.e., the time for irradiating the precursor ions with radicals) are adjusted in advance so that the amount of radicals irradiating the precursor ions becomes a predetermined amount or more. These can be determined in advance based on the results of preliminary experiments.

[0048] When the precursor ions in the ion trap 2 are irradiated with free radicals, the precursor ions undergo lone pair electron induced dissociation to generate product ions derived from the peptide. The various product ions generated are captured in the ion trap 2 and cooled by helium gas or the like from the inert gas supply unit 6. Thereafter, a high DC voltage is applied to the inlet end cap electrode 22 and the outlet end cap electrode 24 by the trap voltage generator 7 at a predetermined timing, whereby the ions captured in the ion trap 2 receive acceleration energy and are ejected together through the ion ejection hole 25. As described above, the product ions generated here may include both fragment ions and adduct ions.

[0049] In this way, ions with a certain acceleration energy are introduced into the flight space of the time-of-flight mass separation unit 3, and are separated according to the mass-to-charge ratio during the flight in the flight space. The ion detector 4 detects the separated ions in sequence, and the data processing unit 9 that receives the detection signal produces a flight time spectrum, for example, with the time when the ions are ejected from the ion trap 2 as the zero time. In addition, by using the mass correction information obtained in advance, the flight time is converted into a mass-to-charge ratio, thereby producing a product ion spectrum. In the data processing unit 9, as described below, by detecting the mass peak of the product ion of a predetermined type, the product ion derived from isoaspartic acid is determined, thereby identifying isoaspartic acid and aspartic acid. That is, the data processing unit 9 of this embodiment has the function of a product ion determination unit as the mass spectrometry analysis device described in the present invention.

[0050] In the mass spectrometer of this embodiment, isoaspartic acid and aspartic acid are identified by dissociating precursor ions using the HAD method and the OAD method. The details are described below.

[0051] First, the spectrum of product ions generated by dissociation of a peptide containing an isoaspartic acid residue and a peptide containing an aspartic acid residue by the widely used collision-induced dissociation (CID) method will be described. As an example, Figure 3 : shows the mass spectra (product ion spectra) of product ions generated by dissociating the following precursor ions by the CID method, the precursor ions being derived from peptides obtained by digesting and fragmenting the proteins constituting the eye lens, i.e., crystallin (protein containing aspartic acid residues) and crystallin denatured product (protein containing isoaspartic acid residues obtained by isomerization of aspartic acid) with trypsin. The upper part is the mass spectrum of the product ions derived from the peptide containing isoaspartic acid residues, and the lower part is the mass spectrum of the product ions derived from the peptide containing aspartic acid residues.

[0052] If Figure 3 By comparing the mass spectra above and below, it can be seen that the mass peaks of the two appear at the same position. In other words, even if the peptide containing aspartic acid residues and the peptide containing isoaspartic acid residues are dissociated by the CID method, there is no difference in the generated product ions. In the precursor ion dissociation based on the CID method, it is impossible to distinguish between the peptide containing aspartic acid residues and the peptide containing isoaspartic acid residues as their isomers.

[0053] like Figure 4 As shown in the figure, even if the aspartic acid residue and the isoaspartic acid residue are dissociated at the position of the N-Cα bond, product ions with different masses will not be generated. On the other hand, if the two are dissociated at the position of the Cα-C bond, product ions with different masses can be generated. It should be noted that the bonds of the peptide backbone of the isoaspartic acid residue and the aspartic acid residue are different, so Figure 4 The positions corresponding to these bonds in aspartic acid residues are indicated in brackets.

[0054] The inventor Figure 5 The results of quantum chemical calculations for the dissociation of isoaspartic acid residues based on the HAD method are shown in FIG. When hydrogen radicals are irradiated to isoaspartic acid residues, the hydrogen radicals attach to the carbon atom of the carboxyl group contained in the amide bond, and then the precursor ion dissociates in two ways. The first is a method in which dissociation occurs at the position of the N-Cα bond as in the CID method ( Figure 5 The first is the method where the N-Cα bond is cleaved, and the second is the method where the dissociation occurs at the Cα-C bond position ( Figure 5(The method described as "pathway specific to isoaspartic acid" in the method). The activation energy from the attachment of hydrogen radicals to the dissociation is 64 kJ / mol and 66 kJ / mol, respectively, with almost no difference. Therefore, if the precursor ions derived from a peptide containing an isoaspartic acid residue are dissociated by the HAD method, product ions obtained by dissociation at the Cα-C bond are generated from about half of them. On the other hand, aspartic acid dissociates only at the position of the N-Cα bond. Therefore, by detecting the product ions generated by dissociation at the position of the Cα-C bond, isoaspartic acid and aspartic acid can be distinguished.

[0055] Based on the above calculation results, the measurement results of the product ions generated by dissociating the peptide containing the isoaspartic acid residue and the aspartic acid residue by the HAD method are shown in Figure 6 .exist Figure 6 and Figure 7 In the measurement of , hydrogen gas was used as the raw material gas. Figure 6 The upper part is the product ion spectrum of the peptide with isoaspartic acid residues, Figure 6 The lower part of is the product ion spectrum of the peptide with aspartic acid residues. Figure 6 As shown in the upper part of FIG. , the z7+57 ion is detected in the product ion spectrum of the peptide having the isoaspartic acid residue, while on the other hand, Figure 6 The z7+57 ion was not detected in the product ion spectrum of the peptide having an aspartic acid residue shown in the lower part of . It should be noted that z7 refers to an ion (z ion) on the C-terminal side generated by dissociation at the position of the N-Cα bond of the aspartic acid residue, and has 7 amino acids on the C-terminal side of its bonding site. In addition, z7+57 refers to an ion with a mass 57 Da greater than that of the z7 ion.

[0056] In addition, for other peptides, the HAD method was used to dissociate peptides containing an isoaspartic acid residue and an aspartic acid residue in the same manner as above, and the measurement results of the generated product ions are shown in FIG. Figure 7 . Figure 7 The upper part of the figure shows the product ion spectrum of the peptide with isoaspartic acid residues. Figure 7 The lower part of the figure is the product ion spectrum of the peptide with aspartic acid residues. Figure 7 As shown in the upper part of FIG. , c7-57 ions are detected in the product ion spectrum of the peptide having an isoaspartic acid residue, while on the other hand, Figure 7 The c7-57 ion was not detected in the product ion spectrum of the peptide with aspartic acid residues shown in the lower part of . It should be noted that c7 refers to the ion (c ion) on the N-terminal side generated by dissociation at the position of the N-Cα bond of the aspartic acid residue, and has 7 amino acids on the N-terminal side of its bonding site. In addition, c7-57 refers to an ion with a mass 57Da smaller than that of the c7 ion.

[0057] Depend on Figure 6 and Figure 7 The results shown show that isoaspartic acid and aspartic acid can be identified by detecting z+57 ions or c-57 ions. Figure 6 The z7+57 ion was detected in Figure 7 The c-57 ion is detected in the peptide, but which of the z ion and the c ion is detected depends on the composition and position of the amino acids contained in the peptide. Specifically, Figure 6 In the peptide shown, the basic arginine (R) to which hydrogen ions are easily attached is located at the C-terminal side of aspartic acid, so z ions are generated. Figure 7 In the peptide shown, basic arginine (R) to which hydrogen ions are easily attached is located on the N-terminal side of aspartic acid, so that c ions are generated.

[0058] In addition, as reported by the present inventors in non-patent document 4, if the precursor ion is irradiated with oxygen free radicals, the precursor ion derived from the peptide is cleaved at the position of the Cα-C bond of the peptide bond to generate an a+16 ion. The a+16 ion refers to an ion on the N-terminal side generated by dissociation of the peptide bond at the position of the Cα-C bond, that is, an ion with an oxygen atom attached to the a ion and a mass increased by 16Da. In addition, by cleaving at the position of the Cα-C bond of the peptide bond, an x ​​ion can also be generated on the C-terminal side. As shown in reference Figure 4 As described above, if cleavage occurs at the position of the Cα-C bond, product ions (a+16 ions and / or x ions) of different masses are generated from aspartic acid and isoaspartic acid. Therefore, it can be considered that: instead of the hydrogen used in the above measurement example, water vapor or oxygen is used as the raw material gas, and the precursor ions are irradiated with oxygen radicals or hydroxyl radicals, thereby isoaspartic acid and aspartic acid can also be identified. In addition, it is not limited to oxygen radicals and hydroxyl radicals, and free radicals having oxidizing ability like these free radicals can also be used.

[0059] The above measurement examples all involve the identification of isoaspartic acid and aspartic acid contained in a peptide with a known structure, but the structure of the peptide does not necessarily need to be known. For example, by storing the names and masses of various substances predicted to be contained in the peptide as a sample component, such as amino acids, in advance in the data processing unit 9, and by detecting whether the difference in mass-to-charge ratio between mass peaks of the product ion spectrum is consistent with the mass of any amino acid, the structure of the peptide can be inferred.

[0060] In the above-mentioned embodiment, a mass spectrometer having a three-dimensional ion trap-time-of-flight mass separation unit is described, but a mass spectrometer having other structures can also be used. For example, a mass spectrometer having the following structure can be used, which is composed of: a front-stage mass separation unit for screening precursor ions, a collision cell for irradiating free radicals to dissociate the precursor ions, and a rear-stage mass separation unit for separating the product ions generated by dissociating the precursor ions in the collision cell according to the mass-to-charge ratio. An example of such a mass spectrometer is a triple quadrupole mass spectrometer. In this case, it is sufficient to spray free radicals from the nozzle into the interior of the collision cell and irradiate the precursor ions passing through the collision cell.

[0061] [Way]

[0062] It should be understood by those skilled in the art that the above-described multiple exemplary embodiments are specific examples of the following aspects.

[0063] (First method)

[0064] A first aspect of the present invention is a method for analyzing isoaspartic acid by dissociating precursor ions derived from sample components to generate product ions and analyzing the product ions. The method comprises the following steps:

[0065] The precursor ions are irradiated with hydrogen radicals or radicals having oxidizing ability to dissociate the precursor ions to generate product ions.

[0066] The aforementioned product ions are separated and detected according to their mass-to-charge ratio.

[0067] The product ions derived from isoaspartic acid were determined based on the mass-to-charge ratio of the aforementioned product ions.

[0068] (Fifth Method)

[0069] A fifth aspect of the present invention is a mass spectrometer for analyzing isoaspartic acid by dissociating precursor ions derived from sample components to generate product ions and analyzing the product ions, comprising:

[0070] A reaction chamber, which is used to introduce the aforementioned precursor ions;

[0071] A radical generating unit for generating hydrogen radicals or radicals having oxidizing ability;

[0072] a radical irradiation unit for irradiating the interior of the reaction chamber with the radicals generated in the radical generation unit;

[0073] a separation and detection unit for separating and detecting product ions generated by dissociation of the precursor ions through the reaction with the free radicals according to a mass-to-charge ratio; and

[0074] A product ion determination unit is used to determine the product ion derived from isoaspartic acid based on the mass-to-charge ratio of the product ion.

[0075] In the above-mentioned first mode of isoaspartic acid analysis method and fifth mode of mass spectrometry, hydrogen radicals or radicals with oxidizing ability are irradiated to precursor ions derived from sample components to generate product ions. Radicals with oxidizing ability are, for example, oxygen radicals and hydroxyl radicals. Ion dissociation methods for irradiating precursor ions with these radicals are hydrogen attachment dissociation (HAD: Hydrogen-Attachment Dissociation) method and oxygen attachment dissociation (OAD: Oxygen-Attachment Dissociation) method proposed by the inventors in Patent Document 1.

[0076] The present inventors have found that when the precursor ions derived from isoaspartic acid are dissociated by the HAD method, c+57 ions and z-57 ions representing isoaspartic acid (not generated from aspartic acid) are generated in the same manner as the ETD method. In addition, it has been found that when the precursor ions derived from isoaspartic acid are dissociated by the OAD method in which oxygen free radicals and hydroxyl free radicals are attached to the precursor ions to dissociate the precursor ions, a+16 ions representing isoaspartic acid (not generated from aspartic acid) are generated. The a+16 ion refers to an ion having a mass 57 greater than the ion (c ion) on the N-terminal side generated by the cleavage of the Cα-C bond of the peptide main chain possessed by aspartic acid.

[0077] In other words, it was found that the product ions generated by dissociating the precursor ions from the sample components were separated and detected according to the mass-to-charge ratio, and the ions corresponding to them were determined, thereby being able to identify and analyze isoaspartic acid and aspartic acid. The HAD method and the OAD method are methods for dissociating precursor ions by free radical addition, and can be used regardless of the polarity and valence of the ions. Therefore, the precursor ions from the sample components can be efficiently dissociated, and isoaspartic acid and aspartic acid, which are its isomers, can be easily identified and analyzed.

[0078] (Second method)

[0079] In the isoaspartic acid analysis method according to the second aspect of the present invention, in the analysis method of the first aspect,

[0080] The aforementioned product ions derived from isoaspartic acid are determined by probing for c+57 ions or z-57 ions.

[0081] (Sixth Method)

[0082] A mass spectrometer according to a sixth aspect of the present invention is, in the mass spectrometer according to the fifth aspect,

[0083] The product ion determination unit detects c+57 ions or z-57 ions.

[0084] In the second aspect of the isoaspartic acid analysis method and the sixth aspect of the mass spectrometry apparatus, isoaspartic acid and aspartic acid can be distinguished by detecting c+57 ions or z-57 ions unique to isoaspartic acid generated by the HAD method using hydrogen radical irradiation.

[0085] (Third Method)

[0086] In the isoaspartic acid analysis method according to the third aspect of the present invention, in the isoaspartic acid analysis method according to the first aspect or the second aspect,

[0087] The aforementioned product ions derived from isoaspartic acid were determined by probing for a+16 ions.

[0088] (Seventh Method)

[0089] A mass spectrometer according to a seventh aspect of the present invention is, in the mass spectrometer according to the fifth aspect or the sixth aspect,

[0090] The product ion determination unit detects a+16 ions.

[0091] In the isoaspartic acid analysis method of the third aspect and the mass spectrometer of the seventh aspect, isoaspartic acid and aspartic acid can be distinguished by detecting a+16 ions unique to isoaspartic acid generated by the OAD method or the like of irradiating free radicals having oxidizing ability.

[0092] (Fourth Method)

[0093] In the isoaspartic acid analysis method according to a fourth aspect of the present invention, in the isoaspartic acid analysis method according to the third aspect,

[0094] The precursor ions are irradiated with oxygen radicals and / or hydroxyl radicals.

[0095] (Eighth Method)

[0096] In the mass spectrometer according to an eighth aspect of the present invention, in the mass spectrometer according to the seventh aspect,

[0097] The radical generating unit generates oxygen radicals and / or hydroxyl radicals.

[0098] In the mass spectrometer of the seventh aspect, the ratio of the component having a cis-type unsaturated bond to the component having a trans-type unsaturated bond contained in the sample component can be estimated without requiring any operation by the user.

[0099] Description of Reference Numerals

[0100] 1…Ion source

[0101] 2…Ion trap

[0102] 21…Ring electrode

[0103] 22…Inlet side end cap electrode

[0104] 23…Ion introduction hole

[0105] 24…Outlet side end cap electrode

[0106] 25…Ion ejection hole

[0107] 26…Free radical particle inlet

[0108] 27…Free radical particle discharge port

[0109] 3…Time-of-flight mass separation unit

[0110] 4…Ion detector

[0111] 5…Free radical irradiation

[0112] 51…Free Radical Generation Chamber

[0113] 52…Raw material gas supply source

[0114] 53…High frequency plasma source

[0115] 531…Microwave supply source

[0116] 532…Three-stub tuner

[0117] 54…Nozzle

[0118] 541…Ground electrode

[0119] 542…Torch

[0120] 543…Needle Electrode

[0121] 544…Connector

[0122] 55…Separator

[0123] 56…Valve

[0124] 57…Vacuum pump

[0125] 6…Inert gas supply unit

[0126] 61…Gas supply source

[0127] 62…Valve

[0128] 63…Gas inlet pipe

[0129] 7… Well voltage generating unit

[0130] 8…Control Unit

[0131] 9…Data Processing Department

Claims

1. A method for analyzing isoaspartic acid, comprising dissociating precursor ions derived from sample components to generate product ions and analyzing the product ions, the method comprising the following steps: The precursor ions are irradiated with hydrogen radicals or radicals having oxidizing ability to dissociate the precursor ions to generate product ions, The product ions are separated and detected according to their mass-to-charge ratio, determining a product ion derived from isoaspartic acid based on a mass-to-charge ratio of the product ion, The radical irradiated to the precursor ion is a hydrogen radical, and the product ion derived from isoaspartic acid is determined by detecting c+57 ion or z-57 ion, or the product ion derived from isoaspartic acid is determined by detecting a+16 ion.

2. The method for analyzing isoaspartic acid according to claim 1, wherein: In the case where the product ions derived from isoaspartic acid are determined by probing for a+16 ions, the precursor ions are irradiated with oxygen radicals and / or hydroxyl radicals.

3. A mass spectrometer for analyzing isoaspartic acid by dissociating precursor ions derived from sample components to generate product ions and analyzing the product ions, the mass spectrometer comprising: a reaction chamber for introducing the precursor ions; A radical generating unit for generating hydrogen radicals or radicals having oxidizing ability; a radical irradiation unit for irradiating the interior of the reaction chamber with the radicals generated in the radical generation unit; a separation and detection unit for separating and detecting product ions generated by dissociating the precursor ions through the reaction with the free radicals according to a mass-to-charge ratio; as well as a product ion determination unit, configured to determine the product ions derived from isoaspartic acid according to the mass-to-charge ratio of the product ions, The radical generating unit generates hydrogen radicals, and the product ion determining unit detects c+57 ions or z-57 ions, or the product ion determining unit detects a+16 ions.

4. The mass spectrometer according to claim 3, wherein: When the product ion determination unit detects a+16 ions, the radical generation unit generates oxygen radicals and / or hydroxyl radicals.

Images