WO2011010649A1

WO2011010649A1 - Mass spectrometry method and ion dissociation device

Info

Publication number: WO2011010649A1
Application number: PCT/JP2010/062207
Authority: WO
Inventors: 直己万里; 宏之佐竹; 集平林
Original assignee: 株式会社日立製作所
Priority date: 2009-07-24
Filing date: 2010-07-21
Publication date: 2011-01-27
Also published as: JP5362009B2; JPWO2011010649A1

Abstract

Disclosed is a mass spectrometry method whereby information regarding to the structure of a substance can be acquired at an improved efficiency and the time required for measuring and identifying the substance can be shortened. Specifically disclosed is a mass spectrometry method characterized by comprising, in the course of ion dissociation in mass spectrometry, comparing the amount of ions measured before the reaction with the amount of ions measured after the dissociation and thus estimating the optimum ion dissociation strength.

Description

Mass spectrometry method and ion dissociation apparatus

The present invention relates to a mass spectrometry method and an ion dissociation apparatus suitable for use in mass spectrometry. The present invention can be used, for example, for dissociation analysis of biopolymers.

It is said that there are about 100,000 kinds of proteins in humans. Protein functions are clever by various post-translational modifications such as cleavage by protease, regulation of activity and interaction by addition of sugar chains and phosphate groups, localization to membranes by acylation such as myristylation and palmitylation Have undergone various adjustments. In eukaryotes in particular, it is rare that a protein synthesized based on a gene sequence exerts its function as it is. For example, after synthesis on a ribosome, the final synthesis in a cell or in a cell is performed. Undergo various modifications at various stages until specific localization is determined. The structures of these biopolymers that change in time and space cannot be determined solely by genome information, but proteins must be directly analyzed.

One of the structural analysis means is mass spectrometry (mass spectrometry: MS). By using mass spectrometry, it is possible to obtain sequence information and post-translational modification information of a polypeptide (peptide or protein) in which amino acids constituting a biopolymer are connected by peptide bonds. In particular, mass spectrometry using ion traps using a high-frequency electric field or quadrupole mass filter and time-of-flight mass spectrometry (Time-of-Flight mass spectrometry) (TOF-MS) are high-speed analytical methods. (Liquid-chromatography: LC) Good connectivity with a pretreatment means for separating a sample represented by an apparatus or the like. For this reason, these high-speed mass spectrometry methods are widely used because they meet the purpose of proteome analysis and the like that require continuous analysis of many types of samples.

In general, in mass spectrometry, a sample molecule is ionized and introduced into a vacuum (or ionized in a vacuum), and the mass-to-charge ratio (m / Z) is measured. Since the information to be obtained is a macroscopic quantity, that is, the ratio of mass to charge, it is not possible to obtain information on the internal structure of the mass analysis operation by simply executing it once. Therefore, a method called tandem mass spectrometry is used.

In tandem mass spectrometry, sample molecular ions are first identified or isolated in the first mass spectrometry operation. Specific or isolated ions are referred to as “precursor ions”. Subsequently, the precursor ions are dissociated by some method. The dissociated ions are called “fragment ions”. If this fragment ion is further subjected to mass analysis, information on the generation pattern of the fragment ion can be obtained.

It should be noted that the dissociation pattern has laws due to the dissociation method. For this reason, it becomes possible to infer the arrangement structure of the precursor ions. Especially in the field of analysis of biomolecules with amino acid skeletons, collision-induced-dissociation (CID) method and infrared multi-photon absorption (IRMPD) method are used as ion dissociation methods. Electron-capture-dissociation (ECD) method and electron-transfer-dissociation (ETD) method are used.

In the field of protein analysis, the most widely used method at present is the CID method in which a precursor ion is given kinetic energy and collides with a gas. In the CID method, molecular vibration of a precursor ion is excited by collision with a gas, and dissociation occurs at a portion where the molecular chain is easily broken.

A method that has recently been used is the IRMPD method in which a precursor ion is irradiated with infrared laser light to absorb a large number of photons. In the IRMPD method, molecular vibration of the precursor ion is excited, and dissociation occurs at a site where the molecular chain is easily broken. The dissociation site of the polypeptide is shown in FIG. In the figure, a, b, and c are molecules including the NH ₂ terminal side, and x, y, and z are molecules including the COOH terminal side.

The site that is easily cleaved by the CID method or IRMPD method is the site named by by in the main chain consisting of the amino acid sequence. Even the site of by may be difficult to cut depending on the amino acid sequence pattern. For this reason, it is known that structural analysis cannot be performed only by the CID method or the IRMPD method. Also, biopolymers that have undergone post-translational modification tend to break post-translational modifications that are present in the side chains of polypeptides when using the CID method or IRMPD method. Therefore, it is possible to determine whether or not the modified molecular species is modified from the lost mass. However, important information regarding the modification site indicating which amino acid moiety was modified is lost.

On the other hand, the ECD method and the ETD method do not depend on the amino acid sequence (except that the N-terminal side of the proline residue which is a cyclic structure is not cleaved), and one of the cz sites on the main chain of the amino acid sequence. Cut the point. For this reason, if ECD method or ETD method is used, a protein molecule can be analyzed only by mass spectrometry. In addition, since the ECD method and the ETD method have a feature that side chains are difficult to cleave, an amino acid sequence and a modified position can be identified even with a biopolymer subjected to post-translational modification. For this reason, dissociation techniques such as the ECD method and the ETD method have recently received particular attention as a means for research and analysis of post-translational modification.

Recently, an electron desorption-dissociation (EDD) method is attracting attention as a new dissociation method. The EDD method is implemented with an apparatus configuration similar to the ECD method. In the EDD method, a negatively charged precursor ion is reacted with an electron to cleave one of the ax sites on the main chain of the amino acid sequence. The EDD method has begun to be used for structural analysis of not only peptides but also oligonucleotides and sugar chains (Non-patent Document 1).

Note that the method of reacting positively charged precursor ions with electrons is called the ECD method. In addition, a technique that causes a positively charged precursor ion to react with a negatively charged ion and causes dissociation similar to the ECD method through transfer of electrons is called an ETD method. These reaction efficiencies are known to depend on the ion amount, valence, presence / absence of modification, type of modification and number of modifications (Non-Patent Documents 2 and 3), and the amount of electrons suitable for each precursor ion, It is necessary to set the amount of negative ions and the reaction time.

Here, FIG. 3 shows the ECD reaction time dependence of a divalent peptide ion called substance P (Non-patent Document 4). In the figure, the vertical axis represents relative ionic strength, and the horizontal axis represents ECD reaction time. While the amount of precursor ions decreases as the reaction time increases, fragment ions peak at around 7 ms. That is, if the precursor ion is reduced by the ECD reaction, the fragment ion is not produced accordingly. In substance P, when the amount of precursor ion at the reaction time of 0 is 1, fragment ions necessary for peptide identification are most produced when about 0.3 precursor ions remain. Here, the precursor ion residual ratio I can be expressed by I = 1 / e ^ (t / τ) using a reaction time t and a time constant τ determined by the properties of the sample.

FIG. 4 shows schematic diagrams of ECD spectra acquired with various amounts of electrons. In the figure, the vertical axis represents ionic strength, and the horizontal axis represents the mass-to-charge ratio (m / z) of molecular ions. If the amount of electrons is optimal, the precursor ion 200, the charge-reducing species 201, and the fragment ion 202 are detected, and the amino acid sequence, the kind of modified product, and the modification site can be identified from the m / z of the fragment ion 202 (FIG. 4). (A)). The charge reduce species 201 is a precursor ion 200 in which the precursor ion 200 has reacted with electrons but has not dissociated, and has a valence lower than that of the precursor ion. However, when the amount of electrons to be reacted is too small, a spectrum in which only the precursor ion 200 remains is obtained (FIG. 4B). When the amount of electrons to be reacted is too large, fragment ions produced by the ECD method receive electrons again, and fragment ions having a reduced valence are produced. Due to the occurrence of such a multistage reaction, the ions are finally lost and cannot be detected by the mass spectrometer (FIG. 4C).

Even when the reaction conditions are optimal, it is known that the dissociation efficiency is low when the valence is low, the number of modification sites is large, or the modification is large. In this case, charge-reducing species with high ionic strength are observed.

As a means for solving the problem, in the ECD method, the Hot) ECD (HECD) method and the Activated IonＥECD (AI-ECD) method are used. The HECD method is a dissociation method that uses electrons (2 eV or more) with higher energy than ordinary ECD (especially, the cold ECD (CECD) method is about 2 eV or less) and improves dissociation efficiency. Can be made. However, secondary dissociation other than cz may be induced, making subsequent identification difficult (Non-Patent Document 5).

The AI-ECD method is a method in which precursor ions are excited with a high-frequency electric field during or before and after the ECD reaction to facilitate dissociation (Non-patent Document 6). In the AI-ECD method, dissociation other than cz is likely to occur as in the HECD method. On the other hand, the ETD method cannot increase the electron energy in principle. For this reason, the CID method is implemented for charge-reducing species to improve the dissociation efficiency (ETcaD method: Non-Patent Document 7). Fragment ions produced from Charge Reduce species are also mainly cz ions.

5A and 5B show spectra obtained as a result of analyzing a pentavalent neuropeptide Y consisting of 36 amino acids by the CECD (1.25 eV) method and the HECD (10 eV) method. In the figure, the vertical axis represents ionic strength, and the horizontal axis represents the mass-to-charge ratio (m / z) of molecular ions. The reaction time was set so that the precursor ion residual ratio I was 0.3.

In the CECD method, 21 of the 35 peptide bonds that can be dissociated were dissociated (sequence coverage: 60%). On the other hand, in the HECD method, 30 sites were dissociated (sequence coverage 86%). It can be seen that in the HECD method, the ionic strength of charge-reduced species ([M + 5H] ⁴⁺ and [M + 5H] ³⁺ ) is decreased and the types of fragment ions are increased. However, w ions due to secondary dissociation have been observed. Next, ECD method is performed on neuropeptide Y pentavalent and hexavalent ions with various electron energies, and the sequence coverage, charge-reduced species ratio of all ions, and fragment ion ratio depend on electron energy (Fig. 6). In addition, the vertical axis | shaft of FIG. 6 (A) is a sequence coverage, and a horizontal axis is electron energy. On the other hand, the vertical axis in FIG. 6B represents the relative ion intensity occupied by all ions, and the horizontal axis represents the electron energy.

As shown in FIG. 6, with hexavalent ions, a sequence coverage of 86% is obtained regardless of the electron energy, and the charge-reduced species ratio and the fragment ion ratio are almost unchanged. On the other hand, in the case of pentavalent ions, the sequence coverage, which was 60% in the CECD (1.25 eV) method, was improved as the electron energy was increased, and was 86% in the HECD (10 eV) method. Further, in the CECD method, the charge-reduced species ratio was 0.2. However, the charge-reduced species ratio decreased with increasing electron energy to a charge-reduced species ratio of about 0.1 at 10 eV. For the hexavalent ion of neuropeptide Y, the CECD method is optimal because it provides a high sequence coverage without causing secondary dissociation. On the other hand, in the case of pentavalent ions, the sequence coverage is low in the CECD method, and therefore it is desirable to use the HECD method that provides a high sequence coverage.

Also in the ETD method, it is necessary to optimize the amount of negative ions and reaction time. Non-Patent Document 2 describes that the amount of negative ions and reaction time are optimized using a typical glycopeptide as a preliminary study for attempting analysis of various glycopeptides.

It is necessary to set the dissociation energy specific to the precursor ion even in the CID method. According to Patent Document 1, it is necessary to set ion dissociation energy proportional to the mass-to-charge ratio (m / z) of molecular ions. In addition, since there is a gas pressure such as helium or an error inherent to a device such as an electronic device, the necessity of calibration is described.

The Thermo Scientific website discloses that the ACE (Automated Collision Energy) method is used as a function of the gas chromatograph mass spectrometer. It is described that, in order to obtain a fragment ion spectrum with a large amount of information with high sensitivity, the dissociation energy is changed in three stages in one scan, enabling the setting of optimal dissociation conditions and fragment ion generation. (Non-patent Document 8).

No.US6124591

Qualitative analysis of proteins and peptides requires not only the identification of amino acid sequences, but also the type of post-translational modification, the structure of the modified product, or the identification of the modification site. As described above, if the ECD method or the ETD method is used as the ion dissociation method, not only the amino acid sequence but also the modified site of the post-translationally modified product can be identified.

Also, when the CID method is used, the type of post-translational modification and the structure of the modified product can be identified. The ion dissociation efficiency when the ECD method or the ETD method is used depends on the valence of the precursor ion, the presence / absence of modification, the type of modification, and the number of modification, but information on the precursor ion is only the molecular weight and valence. Therefore, it is difficult to set the amount of electrons, the amount of negative ions, and the reaction time suitable for each precursor ion based only on this information.

Moreover, even if the amount of electrons, the amount of negative ions, and the reaction time are optimal, some precursor ions may not be sufficiently dissociated. In that case, the dissociation efficiency can be improved by applying an HECD method, an AI-ECD method, an ETcaD method, or the like to these ions.

However, in these dissociation methods, fragment ions that cannot be assigned due to secondary dissociation increase. This makes it difficult to identify amino acid sequences and modification sites. Furthermore, the AI-ECD method and the ETcaD method require excitation using a high-frequency electric field in addition to the ECD method and the ETD reaction. For this reason, the throughput decreases. Therefore, when it can be identified by the CECD method or the ETD method, it is desirable to use the CECD method or the ETD method and use the HECD method, the AI-ECD method, or the ETcaD method only for a precursor ion having a low dissociation efficiency. .

Patent Document 1 describes that the optimum dissociation energy in the CID method is proportional to the charge-to-mass ratio (m / z) of molecular ions. Therefore, the dissociation energy is set from the m / z value of the precursor ion.

However, as the m / z value of the precursor ion increases, there is an exception that the optimum value cannot be set with the estimated optimum dissociation energy, and the data is also described. That is, it is difficult to set an optimal dissociation energy only by the m / z value.

In the ACE method described above, the energy is changed in three stages, but it is not known whether the optimum dissociation energy has been set.

Accordingly, the present invention aims to solve the technical problems described above and provide a mass spectrometric method for improving the acquisition efficiency of information related to the structure of a substance and an apparatus suitable for ion dissociation during mass spectrometry. To do.

(Means 1)
The mass spectrometry method according to the present invention includes a step of separating a sample by a separation unit provided in a previous stage of an ion source for ionizing the sample, a step of ionizing the separated sample by an ion source, Obtaining a non-fragmented spectrum, selecting a precursor ion, measuring a precursor ion intensity, dissociating the generated sample ion at a desired intensity, and obtaining a fragmented spectrum of the sample ion And a step of calculating a ratio of the precursor ion intensity of the fragmentation spectrum to the precursor ion intensity of the non-fragmentation spectrum, and a desired ratio is obtained based on the calculated ratio and the set value of the ion dissociation intensity. The process of calculating the optimum value of ion dissociation intensity and ion dissociation of the generated sample ions A step of dissociating in degrees optimum value.

The ion dissociation apparatus according to the present invention includes an electron source, a ring-shaped first end electrode disposed on the far end side of the ion trap region with respect to the electron source, and a position near the ion trap region with respect to the electron source. An ion dissociation part having a ring-shaped second end electrode disposed on the end side and a ring-shaped electron amount control electrode disposed on the electron source side with respect to the second end electrode; and an electron amount control electrode And a control unit that variably controls the voltage applied to the.

(Means 2)
The mass spectrometry method according to the present invention includes a step of registering analysis conditions of desired ions in a database, a step of separating a sample by a separation unit provided in a previous stage of an ion source for ionizing the sample, and a separated sample. A step of ionizing with an ion source, a step of obtaining a non-fragmentation spectrum of the generated sample ions, a step of selecting a precursor ion, a step of measuring a precursor ion intensity, a step of measuring an electron current value, The step of calculating the ECD reaction time from the precursor ion intensity of the non-fragmented spectrum, the step of calculating the ECD reaction time from the electron current value, the step of updating the database, and the optimal value of the ion dissociation strength of the generated sample ions And a step of dissociating.

According to the present invention, it is possible to obtain a fragmentation spectrum in which a dissociation intensity suitable for each precursor ion is set. Thereby, a fragmentation spectrum with high identification accuracy can be acquired. Further, in the identification of a substance by mass spectrometry, the efficiency of acquiring information related to the structure of the substance is improved, and the time for measurement and substance identification is shortened.

The figure explaining the ECD reaction control flow in Embodiment 1 of invention. The figure explaining the dissociation pattern of polypeptide. The experimental figure explaining the reaction time dependence of the fragment ion total amount by the ECD reaction using substance P, and the precursor ion amount. The schematic diagram of the ECD spectrum in the optimal amount of electrons (A), insufficient amount of electrons (B), and excess amount of electrons (C). The ECD spectrum experiment figure which dissociated neuropeptide Y (pentavalent) with the electron energy of 1.25 eV (A), and the ECD spectrum experiment figure which made it dissociate with the electron energy of 10 eV (B). The experimental figure of electron energy dependence of the sequence coverage (A) of neuropeptide Y (pentavalent and hexavalent), the total amount of fragments, and the total amount of charge-reducing species (B). The figure which shows the structural example of the mass spectrometry system containing the ECD cell based on Embodiment 1 of invention. The table | surface which shows the reaction time and control voltage value in each precursor ion valence when the amount control voltage of an electron is controlled and not controlling. The schematic diagram explaining an ion peak. The schematic diagram of a non-fragmentation spectrum (A) and a fragmentation spectrum (B). The lineblock diagram of a linear ion trap ECD cell and an electron source. The experiment figure which shows the dependence of the amount of electrons and the amount control voltage of electrons when the ion trap voltage is 20V (A) and when it is 28V (B). The figure explaining the ECD reaction control flow based on Embodiment 2 of invention. The figure explaining the ECD reaction control flow based on Embodiment 3 of invention. The figure explaining the ECD reaction control flow based on Embodiment 4 of invention. The figure which shows the structural example of the mass spectrometry system which uses ETD based on Embodiment 6 of invention as an ion dissociation means. The figure explaining the ECD reaction control flow based on Embodiment 6 of invention. The figure which shows the example of a data acquisition sequence based on Embodiment 1 of invention. The experimental figure which shows the relationship between the time constant (tau) by the ECD reaction using the substance P, and precursor ion intensity | strength. The schematic diagram which shows the reaction time in the predicted ion chromatogram and each ion intensity | strength. The schematic diagram of the ion chromatogram estimated by the actual measurement ion intensity and Gaussian function of a certain ion. The figure which shows the structural example of the mass spectrometry system which uses the CID method based on Embodiment 7 of invention as an ion dissociation means. The figure of the CID reaction control flow based on Embodiment 7 of invention. Experimental diagram showing the relationship between the precursor ionic strength using substance P, the relative strength of the total amount of fragment ions, and the ionic dissociation strength The schematic diagram which shows the actual value of the precursor ion residual rate dissociated by various ion dissociation intensity | strength, and optimal ion dissociation intensity | strength. The figure which shows the example of a data acquisition sequence based on Embodiment 7 of invention. The figure which shows the structural example of the mass spectrometry system containing the ECD cell based on Embodiment 5 of invention. The figure which shows the operation screen of the data dependence analysis mode based on Embodiment 5 of invention. The figure which shows the operation screen of the database mode based on Embodiment 5 of invention. The figure of the database mode control flow based on Embodiment 5 of invention. The figure which shows the example of a database which sets ECD reaction time for every ionic strength. The figure of a control flow at the time of using the database which sets ECD reaction time for every ionic strength. The block diagram of a linear ion trap ECD cell and electron current measurement. The figure of the flow which correct | amends ECD reaction time and an electron amount control voltage dependently on an electronic current. The figure of the database mode control flow which correct | amends an ECD reaction time and an electron amount control voltage depending on an electron current value and ion intensity. The figure of the control flow which correct | amends an ECD reaction time and an electron amount control voltage dependently on an electron current value and ion intensity at the time of using the database which sets ECD reaction time for every ion intensity.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The embodiment of the present invention to be described later is merely an example, and other embodiments can be realized by a combination or replacement with a known or well-known technique.

(A) Embodiment 1: When ECD method is used as an ion dissociation method (part 1)
(Configuration of mass spectrometry system)
FIG. 7 shows a configuration example of a mass spectrometry system that uses the ECD method for ion dissociation. The sample 1 to be analyzed is separated by pretreatment by a sample separation device 2 (in the figure, liquid chromatography (LC)). Note that a gas chromatogram (GC) can also be used for the sample separation device 2.

The separated sample is ionized in the ion source 3 and introduced into the mass spectrometer. In the case of non-fragmented spectrum acquisition, the introduced ions are accumulated in a linear ion trap (LIT) 4 and discharged in a state where all ions (or a specific ion) are isolated. The discharged ions are detected by the TOF detector 8. A TOF 7 having a high resolution is present in front of the TOF detector 8.

In the case of fragmentation spectrum acquisition, ions introduced into the mass spectrometry system are accumulated in LIT4, and certain ions are discharged in an isolated state. The discharged ions enter the ECD cell 6 via the Q deflector 5. Here, the ECD reaction is performed at a specific ion dissociation intensity, and the ions are ejected. The discharged ions again pass through the Q deflector 5 and are separated by the TOF 7 according to the mass-to-charge ratio m / z of the ions.

The ion source 3 is selected from an electrospray ion source, an ion source based on atmospheric pressure chemical ionization, a matrix-assisted laser desorption ion source, an electric impact ion source, an ion source based on chemical ionization, and an ion source based on field ionization.

LIT4 can accumulate a large number of ions and can be detected with high sensitivity. However, instead of LIT4, a three-dimensional quadrupole ion trap, a quadrupole mass filter, a collision cell, or a Fourier transform ion cyclotron resonance mass separation unit may be used.

The ECD cell 6 is selected from LIT, a three-dimensional quadrupole ion trap, a collision cell, a quadrupole mass filter, and a Fourier transform ion cyclotron resonance mass spectrometer. In this apparatus configuration, the ECD method is used as the ion dissociation method, but the EDD method may be used. In this case, ionization is performed in the negative ion mode.

TOF7 is preferable because of its high resolution and high mass accuracy, but it may be a quadrupole mass filter, ion trap, magnetic field type mass analyzer, Fourier transform ion cyclotron resonance mass analyzer, or orbitrap type analyzer. The electron source for supplying electrons may be a filament or a dispenser cathode.

Each ion detected by the TOF detector 8 is given to the overall processing unit 10 together with the m / z value. The overall processing unit 10 performs data organization and / or data processing as will be described later. The overall processing unit 10 can be realized not only when it has a hardware circuit structure but also through a processing function of a program that operates on a computer system. The ion dissociation parameter determination unit 12, which is one of the processing functions of the overall processing unit 10, includes an ion dissociation intensity determination unit 13, a precursor ion intensity measurement unit 15, a precursor ion residual rate calculation unit 16, and a reaction time calculation unit 1000. The The entire processing unit 10 corresponds to a “calculation processing unit” in the claims.

The precursor ion intensity measuring unit 15 measures the intensity of each precursor ion in the non-fragmented spectrum and the fragmented spectrum. The precursor ion residual ratio calculation unit 16 calculates a precursor ion residual ratio based on the measured precursor ion intensity. The precursor ion residual ratio is a ratio of the precursor ion intensity of the fragmentation spectrum to the precursor ion intensity of the non-fragmentation spectrum.

The reaction time calculation unit 1000 calculates the optimum reaction time based on the precursor ion residual ratio and the user desired setting value. The ion dissociation strength determination unit 13 determines the ion dissociation strength according to the calculated optimum reaction time. A signal giving the determined intensity is given from the ion dissociation intensity determining unit 13 to the ECD cell 6 via the control unit 9. That is, the ECD cell 6 is controlled to the determined strength.

Also, as indicated by a broken line in the figure, the amount of precursor ions can be adjusted by controlling the ion accumulation time in LIT4. Similarly, as shown by a broken line in the figure, it is possible to adjust the ion elution profile by controlling the sample separation device 2 to change the flow rate of the sample 1.

Mass spectrometry spectrum, precursor ion intensity in unfragmented spectrum and fragmented spectrum, precursor ion residual ratio, ECD reaction time, electron energy, parameters controlling ECD cell 6, control parameters of LIT4, solvent mixing of sample separation apparatus 2 The ratio, flow rate, and the like are displayed on the data display unit 18. The residual ratio of the desired precursor ion intensity with respect to the overall processing unit 10 is input through the parameter input unit 19.

(Outline of processing operation)
FIG. 1 shows an ECD reaction control flow based on Embodiment 1 of the present invention. A sample 1 which is a mixture of a plurality of substances is introduced into a sample separation device 2 (liquid chromatography in the figure). Chromatography is equipped with a separation column for separation according to the nature of the substance, and the sample that has passed through the separation column elutes at different times for each component. Each component of the eluted sample is ionized by the ion source 3 (302). The ions are introduced into the LIT 4 in the mass spectrometer and accumulated (303) and discharged from the outlet. The ejected ions are detected by the TOF detector 8 to obtain a non-fragmented spectrum with the horizontal axis representing m / z and the vertical axis representing ion intensity (304).

Next, the overall processing unit 10 selects a precursor ion from the total ion spectrum (non-fragmented spectrum) (305). The total ion spectrum is generally called the MS1 spectrum. In general, the precursor ions are selected in the order of ionic strength. However, the precursor ion may be selected in consideration of the valence and molecular weight. Thereafter, the overall processing unit 10 specifies the valence and m / z value of the selected precursor ion, and calculates the molecular weight from the valence and m / z value (306). Details of this calculation operation will be described later. In parallel with this calculation operation, the overall processing unit 10 also measures the ion intensity of the precursor ions (308).

For example, for the precursor ion analyzed for the first time, the overall processing unit 10 sets the electron energy, ECD reaction time, and electron amount control voltage that are estimated to be suitable for each precursor ion based on the m / z value, valence, and molecular weight. (307). Of course, a predetermined specific value may be given as a set value for the precursor ion analyzed for the first time. For example, the electron energy is set to 1 eV, the reaction time is set to 10 ms, and the electron amount control voltage is set to 20V. The electron voltage control voltage will be described later.

Next, a precursor ion is isolated with LIT4 (309), an ECD reaction (310) is performed on the isolated precursor ion, and then an ECD spectrum is acquired in the overall processing unit 10 (311). Next, the overall processing unit 10 measures the precursor ion intensity existing in the ECD spectrum (308).

The overall processing unit 10 compares the precursor ion intensity in the non-fragmented spectrum with the precursor ion intensity in the ECD spectrum, and obtains the residual ratio of precursor ions (312). Details of this operation will be described later. When the remaining value of the precursor ion is outside the desired range (314), the overall processing unit 10 calculates and sets the optimum ion dissociation intensity (313), and acquires the ECD spectrum again. On the other hand, when the remaining rate of the precursor ions is within a desired range, the overall processing unit 10 determines that the ion dissociation strength is optimal.

In this case (315), the overall processing unit 10 returns to the acquisition of the non-fragmented spectrum. But you may continue acquisition of the ECD spectrum of the same ion. Note that the precursor ion residual ratio calculation, reaction time calculation, electron quantity control voltage calculation, and parameter setting by the overall processing unit 10 need to be performed in real time so as not to disturb the analysis.

(Calculation of valence and molecular weight)
Here, a method for calculating the valence and molecular weight of ions will be described. FIG. 9 shows a schematic diagram of a protonated [H ⁺ ] ion peak observed by mass spectrometry. The vertical axis represents ionic strength, and the horizontal axis represents m / z. The constituent atoms of proteins are mainly carbon, hydrogen, oxygen, and nitrogen. In addition to ¹² C with a mass number of 12 in natural carbon, ¹³ C with a mass number of about 1% ¹³ exists. Therefore, the protein is a mixture containing ¹² C and ¹³ C at a constant ratio. In mass spectrometry, there is an isotope mixed with ¹³ C in addition to the monoisotopic peak 400 composed only of ¹² C. Isotope ¹³ C containing ^{one, 13} containing two isotopes ^{C, 13} isotope respectively ¹² C molecule weight +1 C including ^{three, 12} C molecule weight ^{+2, 12} C molecule weight +3, and the isotope peaks 401 is called.

In the case of protonated ions, the proton addition number corresponds to the valence z. In mass spectrometry, it is displayed in m / z. Therefore, the m / z value of the monoisotopic peak 400 is (M + z) / z, and the m / z value of the isotope peak 401 is (M + 1 + z) / z and (M + 2 + z), respectively, where the molecular weight of the ¹² C isomer is M. / Z, (M + 3 + z) / z. The difference between these peaks is 1 / z. That is, the peak interval in the case of monovalent is 0.5 in the case of 1 and 2. Therefore, the valence can be determined from the peak interval. If the valence is known, the molecular weight can be calculated from the valence and the m / z value.

(Calculation of optimal reaction time)
In the case of the apparatus configuration of FIG. 7, the amount of electrons introduced into the ECD cell 6 is proportional to the reaction time. For this reason, the amount of electrons reacting with the precursor ion can be adjusted by manipulating the reaction time. A method for calculating the optimum reaction time will be described below. The calculation of the optimal reaction time is executed by the overall processing unit 10. 10A and 10B are schematic diagrams of a non-fragmentation spectrum and a fragmentation spectrum. In addition, a vertical axis | shaft is an ionic strength and a horizontal axis is m / z.

Here, a precursor ion 200 (FIG. 10A) having an ionic strength x in an unfragmented spectrum was isolated and subjected to an ECD reaction for t [ms]. As a result, the precursor ion 201 having an ionic strength Ix (FIG. 10 ( Assume that an ECD spectrum in which B)) remains is obtained. In this case, the precursor ion residual ratio I can be expressed by the following equation using the time constant τ determined by the properties of the sample and the reaction time t [ms].

I = 1 / e ^ (t / τ)
If the time constant τ is obtained from this equation, the reaction time t required to obtain a desired precursor ion residual rate (for example, 0.3) is obtained from the relationship between the obtained time constant τ and the precursor ion residual rate. Can do. When the ECD reaction is carried out with this reaction time, an ECD spectrum of a desired precursor ion residual ratio can be obtained. It is also possible to control the control voltage of the amount of electrons instead of controlling the reaction time.

(ECD cell equipment configuration)
FIG. 11 shows an apparatus configuration example of the ECD cell 6 using a linear ion trap. The ECD cell 6 corresponds to the ion dissociation apparatus in the claims.

The ECD cell 6 has a configuration in which a first end electrode 100 and a second end electrode 101 are arranged at both ends of a quadrupole LIT4. Here, the 1st end electrode 100 and the 2nd end electrode 101 are comprised by the flat electrode which has a ring shape. That is, a pore is formed in the center of the substrate of each end electrode.

As shown in the figure, the precursor ion 102 is held along the central axis of the LIT4. In addition, a plate-shaped electron quantity control electrode 103 having a ring shape is disposed between the electron source 104 and the second end electrode 101. The electrons irradiated from the electron source 104 sequentially pass through the pores of the electron control electrode 103 and the second end electrode 101 and reach the LIT 4 to generate an ECD reaction. As will be described later, the control unit 9 adjusts the amount of electrons introduced by controlling the voltage applied to the electron amount control electrode 103.

11 shows that a voltage of 20V is applied as the electron source voltage 105, and the ion trap voltage 106 is adjusted in the range of 20V to 28V. By raising the ion trap voltage 106, high-energy electrons can be irradiated (corresponding to HECD).

Here, the current value obtained when the ion trap voltage 106 is fixed at 20 V or 28 V and the electron quantity control electrode 103 is changed from 10 V to 23 V is shown in FIG. 12A shows an example in which the ion trap voltage 106 is 20V, and FIG. 12B shows an example in which the ion trap voltage 106 is 28V. In both graphs, electrons are not incident on the ion trap while the applied voltage to the electron amount control electrode 103 is up to 14V. When the ion trap voltage is 20V, the maximum current flows when the electron amount control voltage is around 20V. On the other hand, when the ion trap voltage is 28V, the maximum current flows when the electron amount control voltage is 23V. That is, the amount of electrons incident on the LIT 4 can be adjusted by controlling the electron amount control electrode 103. Thus, the amount of electrons used for the reaction can be controlled not only by the reaction time t but also by the voltage value applied to the electron amount control electrode 103.

In this embodiment, the DC voltage applied to the electron quantity control electrode 103 is varied, but the control method differs depending on the ion dissociation part. For example, the ion dissociation intensity can be adjusted by variable control of the frequency and amplitude of the high-frequency voltage.

FIG. 8 shows examples of ion dissociation strength parameters for two examples of the invention that incorporates the conventional method and the electron quantity control electrode 103. When ECD analysis of divalent to 12-valent ions is performed, in the conventional method, the electron amount control voltage 103 is fixed at 23 V, and the amount of electrons is adjusted only by the reaction time. However, it is difficult to control the reaction time at the first decimal place, and the amount of electrons cannot be accurately controlled for ions from 9 to 12 valences. On the other hand, in the case of the invention, the reaction time is fixed at 3 ms, and the voltage applied to the electron quantity control electrode 103 may be variably set according to the required quantity of electrons. Thereby, in the ECD cell 6 used in the embodiment, it is possible to control the amount of electrons more accurately than in the conventional method.

(Sequence example when ECD is executed multiple times)
18A to 18G show sequence examples according to Embodiment 1 of the present invention. In the case of the sequence example shown in FIG. 18 (A), a precursor ion is selected after acquisition of a non-fragmented spectrum (indicated as “MS1” in FIG. 18), and the pre-reaction is performed with a reaction time corresponding to the valence and molecular weight. Perform ECD. Thereafter, the optimum reaction time is calculated by comparing the precursor ion intensities contained in the non-fragmented spectrum and the pre-ECD spectrum, respectively, and the ECD spectrum is obtained at the optimum reaction time. Here, pre-ECD is an ECD spectrum used for determining conditions in order to obtain a real ECD spectrum under optimum ion dissociation conditions.

The sequence example shown in FIG. 18B is used when the liquid chromatography is arranged in the front stage of the mass spectrometer. In the case of this apparatus configuration, the precursor ion intensity varies with time. Therefore, after the pre-ECD reaction, the MS1 spectrum is obtained again, and the average value of the precursor ion intensity in the MS1 spectrum before and after the pre-ECD is used for calculating the optimum reaction time. In addition, since the spectrum after the ECD reaction needs only the precursor ion intensity, it is not necessary to acquire the spectrum of all ions. Thereby, shortening of the measurement time is expected. That is, only the precursor ion is isolated from the spectrum after the ECD reaction. In this case, losses that occur during the isolation process and ion transport are also taken into account. Therefore, more accurate ionic strength can be measured. As in the examples of FIGS. 18A and 18B, the time constant τ can be calculated by executing pre-ECD only once, that is, by obtaining data of one reaction time. However, in order to further improve the accuracy, it is preferable to acquire a plurality of points of data while changing the reaction time.

FIG. 18C shows a sequence in which independent pre-ECD is executed a plurality of times, the time constant τ is calculated each time, and the optimum reaction time is calculated from the averaged value. This makes it possible to calculate a reaction time that is more reliable than a single pre-ECD measurement. In addition, it is possible to calculate a highly reliable precursor ion residual ratio by acquiring the MS1 spectrum (or only isolation of precursor ions) without fail (that is, every time) before each pre-ECD.

FIG. 18D shows a sequence in which the pre-ECD is executed a plurality of times with a plurality of predetermined reaction times, and an actual ECD spectrum is acquired under the condition closest to the desired precursor ion residual rate.

FIG. 18 (E) obtains a pre-ECD spectrum of each ion in the first liquid chromatography analysis, calculates an optimal reaction time for each ion, and creates a database. The second liquid chromatography analysis shows a sequence example in which only pre-ECD is not performed and only the ECD spectrum is acquired.

FIG. 19 is a graph showing the results of pre-ECD performed at various ionic strengths using substance P and the time constant τ obtained. In the case of FIG. 19, a proportional relationship is recognized between the amount of precursor ions and a plurality of time constants τ (R ² = 0.9949). That is, a function (linear equation) can be obtained by using ions having different ionic strengths and obtaining pre-ECD spectra and calculating the time constant τ. Thus, even when the ion amount of the precursor ion changes with time as in liquid chromatography, the optimal reaction time can be calculated if the ion amount can be predicted.

In the sequence of FIG. 18 (F), in the first chromatographic analysis, pre-ECD is performed a plurality of times with different ion intensities for each ion, and a function (primary equation) of ion amount and time constant τ. Following the calculation, the relationship between the ion intensity and the optimum reaction time for each ion is compiled into a database. In the second chromatographic analysis, the profile of each ion can be predicted to be approximately the same, so ECD is performed with the optimal reaction time corresponding to each ion intensity.

Fig. 20 shows the processing image. The vertical axis represents ionic strength and the horizontal axis represents retention time. Moreover, each peak is symmetric in many cases. Therefore, even in the first chromatographic analysis, if several points of ion intensity can be measured in the first half of the ion chromatograph, the subsequent ion intensity can be predicted by a Gaussian function or the like (FIG. 21). In addition, the vertical axis | shaft of FIG. 21 is ionic strength, and a horizontal axis is holding time.

The sequence example shown in FIG. 18 (G) is an example in which the MS1 spectrum is acquired before acquiring the ECD spectrum when the optimum time is calculated with the ion amount for which pre-ECD has been acquired. Here, if the amount of precursor ions per unit accumulation time in LIT4 is calculated and the accumulation time in LIT4 is adjusted, the precursor ions that undergo ECD reaction can be made constant. Thereby, even when the ion amount of the precursor ion changes with time as in liquid chromatography, ECD analysis can be performed with an optimum reaction time for each ion. However, the storage time may be adjusted by the ECD cell 6 instead of the LIT4.

(Effect of embodiment)
By applying the mass spectrometry method according to the above-described embodiment, optimum ion dissociation intensity data can be accumulated for each precursor ion. These data can be stored as data for each mass, valence, and electron energy. This is an index of the ion dissociation intensity of ions to be analyzed for the first time. Thereby, the time required to derive the optimum ion dissociation strength can be shortened.

Also, when these data are plotted in a chart with the date on the horizontal axis and the reaction time on the vertical axis, the reaction time is expected to increase over time due to the exhaustion of the electron source filament. Since the throughput decreases when the reaction time is extended, a threshold value for the reaction time may be set, and when the threshold value is exceeded, the operator may be prompted to replace the electron source filament using an alarm sound or other notification technology.

(B) Embodiment 2: When using an ECD method as an ion dissociation method (part 2)
FIG. 13 shows an ECD reaction control flow based on the second embodiment of the present invention. In FIG. 13, the same reference numerals are given to the portions corresponding to those in FIG. 1.

In the first embodiment described above, it is assumed that the electron energy is not changed during the measurement. However, as shown in FIG. 6, depending on the nature of the substance, it may not be dissociated unless high-energy electrons are used. For example, for neuropeptide Y (pentavalent), only 60% sequence coverage can be obtained even if CECD is performed with the reaction time set to a desired precursor ion residual rate of 0.3. Similarly, when the reaction time was set so that the remaining amount of precursor ion remaining 0.3 for the divalent phosphorylated peptide VNQIGTLSESIK (S is the phosphorylated modification site) and CECD was performed, only 55% sequence coverage was obtained. I couldn't.

In both samples, the charge-reduced species ratio was about 0.2. However, when the reaction time is set so that the precursor ion residual rate becomes 0.3 and HECD (10 eV) is performed, the charge-reducing species ratio is reduced to about 0.1 for the pentavalent ion of neuropeptide Y. Coverage increased to 86%. On the other hand, with the phosphorylated peptide, the charge-reduced species ratio was reduced to about 0.01 and the sequence coverage was 100%.

As described above, in the CECD, there are cases where a large amount of charge-reducing species remains and cannot be sufficiently dissociated. Therefore, data may be acquired by increasing the electron energy using the charge-reducing species ratio as an index.

Here, when the remaining ratio of the precursor ions is within a desired range (315), the overall processing unit 10 has a desired charge-reduced species ratio (a ratio of charge-reduced species occupying all ions in the ECD spectrum). It is determined whether or not. Here, the m / z value of the charge-reducing species is [M + z] ^{(z−1) +} , [M + z] ^(z− , where M is the molecular weight of the precursor ion and z is the valence of protonation [H ⁺ ]. ²⁾ Continue with ⁺ . Therefore, the charge-reduced species ratio can be calculated by dividing the sum of the amounts of ions present at this position by the total amount of ions.

When the charge reduce species ratio is equal to or less than a specific value (316), the overall processing unit 10 returns to the acquisition of the non-fragmented spectrum. If the charge-reduced species ratio is not less than or equal to a specific value (317), the overall processing unit 10 raises a specific value ion trap voltage and executes an HECD analysis (318). Further, AI-ECD may be used instead of HECD. Other control flows are the same as those in the first embodiment.

(C) Embodiment 3: When ECD method is used as an ion dissociation method (part 3)
FIG. 14 shows an ECD reaction control flow based on Embodiment 3 of the present invention. In FIG. 14, the same reference numerals are given to the portions corresponding to FIG. 1.

In the second embodiment described above, the case where the electron energy is increased using the charge-reduced species ratio as an index has been described. However, the electron energy may be increased by using one or both of the fragment ion ratio and the fragment type as an index.

When the fragment ion ratio or the fragment type is a certain value or more (319), the overall processing unit 10 returns to the acquisition of the non-fragmented spectrum. If the fragment ion ratio or the fragment type is not greater than a certain value (320), the overall processing unit 10 increases the ion trap voltage at a certain value and performs analysis by the HECD method (318). Further, the AI-ECD method may be used instead of the HECD method. If post-translational modification is not envisaged, the type of fragment is proportional to the number of peptide bonds. For example, a peptide consisting of 10 amino acids has 9 peptide bonds. Therefore, when all the peptide bonds are dissociated, a total of 18 fragment ions with 9 c fragments and 9 z fragments are assumed. Since the average amino acid molecular weight is 110, it is desirable to set the desired fragment type for each precursor ion molecular weight by calculating the approximate number of amino acids from the molecular weight of the precursor ion. In the case of the HECD method or the AI-ECD method, w or u ions may be detected, so it is desirable to take that possibility into consideration. Other control flows are the same as those in the first embodiment.

(D) Embodiment 4: When ECD method is used as an ion dissociation method (part 4)
FIG. 15 shows an ECD reaction control flow based on the fourth embodiment of the present invention. In FIG. 15, parts corresponding to those in FIG.

In the case of the above-described third embodiment, the case where the electron energy is increased using one or both of the fragment ion ratio and the fragment type as an index has been described. However, amino acid sequence analysis (de novo analysis) may be performed in real time, and the electron energy may be increased using as an index whether or not an amino acid sequence greater than a specific value can be analyzed.

When the amino acid sequence analysis of a certain specific value or more has been completed (321), the overall processing unit 10 returns to the acquisition of the non-fragmented spectrum. On the other hand, when the amino acid sequence analysis of a certain specific value or more has not been completed (322), the overall processing unit 10 increases the ion trap voltage to a certain specific value, and executes analysis by the HECD method (318). Further, the AI-ECD method may be used instead of the HECD method. Other control flows are the same as those in the first embodiment.

(E) Embodiment 5: ECD method is used as an ion dissociation method (part 5)
In the first to fourth embodiments described above, the embodiments are described in which each ion is dissociated by ECD, and the reaction time, the electron amount control voltage, and the electron energy are controlled based on the dissociation state. In this embodiment, these analysis conditions are determined in advance, and mass spectrometry is executed based on these analysis conditions.

(Configuration of mass spectrometry system)
FIG. 27 shows a configuration example of a mass spectrometry system that uses the ECD method for ion dissociation. The sample 1 to be analyzed is separated by a pretreatment of a sample separation device 2 (in the figure, liquid chromatography (LC)). Note that a gas chromatogram (GC) can also be used for the sample separation device 2.

The separated sample is ionized in the ion source 3 and introduced into the mass spectrometer. In the case of non-fragmented spectrum acquisition, the introduced ions are accumulated in a linear ion trap (LIT) 4 and all ions (or certain ions) are isolated and ejected. The discharged ions are detected by the TOF detector 8. A TOF 7 having a high resolution is present in front of the TOF detector 8.

In the case of fragmentation spectrum acquisition, ions introduced into the mass spectrometry system are accumulated in LIT4, and certain ions are discharged in an isolated state. The discharged ions enter the ECD cell 6 via the Q deflector 5. Here, the ECD reaction is performed at a specific ion dissociation intensity by the electrons irradiated from the electron source 104, and the ions are ejected. The discharged ions again pass through the Q deflector 5 and are separated by the TOF 7 according to the mass-to-charge ratio m / z of the ions. The ion guide 2001 measures the irradiated electron current value. Further, the amount of electrons incident on the ECD cell 6 is adjusted by controlling the electron amount control voltage 103.

TOF7 is preferable because it has high resolution and high mass accuracy, but a quadrupole mass filter, ion trap, magnetic field type mass analyzer, Fourier transform ion cyclotron resonance mass analyzer, or orbitrap type analyzer may be used. The electron source for supplying electrons may be a filament or a dispenser cathode.

Each ion detected by the TOF detector 8 is given to the overall processing unit 10 together with the m / z value. The overall processing unit 10 performs data organization and / or data processing as will be described later. The overall processing unit 10 can be realized not only when it has a hardware circuit structure but also through a processing function of a program that operates on a computer system. The ion dissociation parameter determination unit 12 that is one of the processing functions of the overall processing unit 10 includes a database 2102, a database update unit 2101, a precursor ion intensity measurement unit 15, an electron current measurement unit 2100, and a reaction time calculation unit 1000. . The entire processing unit 10 corresponds to a “calculation processing unit” in the claims.

The precursor ion intensity measuring unit 15 measures each precursor ion intensity in the non-fragmented spectrum or the isolated precursor ion intensity.

The electron current measuring unit 2100 measures the value of the electron current irradiated from the electron source 104.

The reaction time calculation unit 1000 calculates the optimum reaction time based on the information on the electron current value and the precursor ion intensity.

The database update unit 2101 rewrites (updates) the reaction time described in the database 2102 to the optimum reaction time calculated by the reaction time calculation unit 1000. When ions to be analyzed are described in the database 2102, ECD reaction conditions associated with the ions are given to the ECD cell 6 from the database 2102 via the control unit 9 prior to the start of mass analysis. That is, the ECD cell 6 is controlled to a predetermined analysis condition.

Mass spectrometry spectrum, non-fragmentation spectrum and precursor ion intensity in fragmentation spectrum, ECD reaction time, electron energy, parameters for controlling ECD cell 6, control parameters for LIT4, database, etc. are displayed on data display section 18. Desired parameters for the overall processing unit 10 are input through the parameter input unit 19.

(Outline of processing operation)
Here, an operation method when ions to be analyzed are not determined before analysis will be described with reference to FIG. 28 which is a display example of an operation screen. In this case, the operator selects the data dependence analysis mode through an input device (not shown). Next, the operator selects the valence (z) to be analyzed (selects from 2 to 4 in FIG. 28), and sets the ECD reaction time, electron energy, and electron quantity control voltage for each valence. . After the set parameters are stored in the database 2102, analysis by the mass spectrometer is started. When ions of the selected valence appear during the analysis, the ECD reaction is performed according to the set ECD analysis conditions.

Next, an operation method when ions to be analyzed are determined before analysis will be described with reference to FIG. 29 which is a display example of an operation screen. In this case, the operator selects a database (DB) mode through an input device (not shown). Next, the operator selects a DB to be used (selects mouse_plasma_Trp_f3 in FIG. 29). At this time, the contents of the parameter of the selected DB are displayed on the data display unit 18. In DB, the retention time in chromatography of each ion, m / z value, valence (z), ionic strength, ECD reaction time, electron energy, electron amount control voltage, and electron current reference value are described. Among these, ECD reaction time, electron energy, and electron quantity control voltage are items that define ECD reaction conditions. That is, DB describes the ECD reaction conditions that are applied when a certain ion has a previously described ionic strength. The electron current reference value will be described later. The operator inputs a desired value for the ECD reaction condition displayed on the data display unit 18. When the changed value is stored, an ion analysis process is performed by the mass spectrometer.

Incidentally, when the ionic strength changes as shown in FIG. 19, it is necessary to change the reaction time and the electron amount control voltage in accordance with the change. FIG. 30 shows a processing flow when the database shown in FIG. 29 is used.

First, the database to be used is selected on the screen of the data display unit 18, and after changing the value, it is stored in the database 2102 (3000). Thereafter, analysis of ions by the mass spectrometer is started (3001). Simultaneously with the start of analysis, a total ion spectrum is acquired (304). Based on the acquired information, the overall processing unit 10 creates a peak list including the retention time, m / z, valence, and ion intensity (3003).

Next, the overall processing unit 10 executes an inclusion DB search (3005). The overall processing unit 10 collates the created peak list with the database 2102 and searches whether or not there is an ion whose retention time, m / z, and valence match. If there is no matching ion (3007), the overall processing unit 10 returns to the acquisition process (304) of the total ion spectrum. If there is a matching ion (3006), the overall processing unit 10 compares the ion intensity in the peak list with the ion intensity described in the database 2102 and compares whether or not a certain value has deviated. The constant value here is determined by the operator before the analysis is executed.

Here, when the value does not deviate more than a certain value (3008), the overall processing unit 10 gives the ECD reaction conditions described in the database 2102 to the control unit 9. The control unit 9 controls the ECD cell based on the ECD reaction conditions. The overall processing unit 10 acquires an ECD spectrum through the TOF detector 8 (311). Thereafter, the overall processing unit 10 returns to the acquisition process (304) of the total ion spectrum. The ECD spectrum may be acquired multiple times.

On the other hand, when the value has deviated by a certain value or more (3009), the overall processing unit 10 corrects the ECD reaction time using the graph of FIG. 19 as an example, or uses the graph of FIG. 12 as an example. The electron quantity control voltage is corrected (3002). The overall processing unit 10 gives the corrected ECD reaction condition to the control unit 9. The control unit 9 controls the ECD cell based on the ECD reaction conditions. The overall processing unit 10 acquires an ECD spectrum through the TOF detector 8 (311). Thereafter, the overall processing unit 10 returns to the acquisition process (304) of the total ion spectrum.

In the case of FIG. 29, the case where the database 2102 in which one ion intensity is registered for a certain ion and one ECD reaction condition is registered for the combination has been described. However, it is also possible to use a database 2102 in which a plurality of ion intensities are registered for a certain ion and unique ECD reaction conditions are registered for each ion intensity. In this case, it is not necessary to correct the reaction time and the electron amount control voltage in accordance with the change in ionic strength.

An example of this type of database 2102 is shown in FIG. In the case of FIG. 31, three types of ion intensity and three types of ECD reaction conditions (here, ECD reaction time) corresponding to each of ions having an m / z value of 1110.3 are registered.

In this example, 3 ms is registered as the ECD reaction time applied when the ionic strength is 1000 or less. Further, 5 ms is registered as an ECD reaction time to be applied when the ionic strength is 1000 to 3000. Further, 8 ms is registered as an ECD reaction time applied when the ion intensity is 3000 or more.

In the case of FIG. 31, the electron control voltage is set to a fixed value, and different values of ECD reaction time are set according to the ion intensity. However, the ECD reaction time is set to a fixed value and varies depending on the ion intensity. A value electron quantity control voltage may be set.

FIG. 32 shows a processing flow when the database 2102 having the data structure shown in FIG. 31 is used.

Next, the overall processing unit 10 executes an inclusion DB search (3005). The overall processing unit 10 collates the created peak list with the database 2102 and searches whether or not there is an ion whose retention time, m / z, and valence match. If there is no matching ion (3007), the overall processing unit 10 returns to the acquisition process (304) of the total ion spectrum. If there is a matching ion (3006), the overall processing unit 10 determines which ion intensity range in which the ion intensity in the peak list corresponds to the database 2102 as well as the ECD reaction time to be applied to the control unit 9. give. The control unit 9 controls the ECD cell based on the ECD reaction conditions. The overall processing unit 10 acquires an ECD spectrum through the TOF detector 8 (3006). Thereafter, the overall processing unit 10 returns to the acquisition process (304) of the total ion spectrum.

(Description and flow of electronic current reference value)
As described with reference to FIG. 11, electrons are irradiated from the electron source 104 in the ECD. If the supply of the amount of electrons is not always constant, highly reproducible ECD reaction cannot be performed. For example, when the amount of electrons irradiated for each analysis varies, even if the conditions of the database 2102 used for the previous analysis are applied, excessive or insufficient reaction time occurs, making it difficult to obtain the same data as before. Therefore, the monitoring of the amount of electrons irradiated is an essential condition in the embodiment using the database.

FIG. 33 shows a device configuration example of an electron current measuring unit 2100 (FIG. 27) that monitors the amount of electrons (electron current value). In the case of this embodiment, LIT2001 having an ion guide function is used as the electron current measuring unit 2100. As shown in FIG. 33, the LIT 2001 is installed outside the end electrode 100 when viewed from the electron source 104. Some of the electrons that have passed through LIT4 change their trajectory from the central axis due to the influence of the high-frequency electric field formed in LIT2001, and collide with one of the electrodes. The voltage of the electrodes constituting the LIT 2001 varies due to the collision of electrons. By measuring the fluctuation of the electrode voltage, the amount of electron current irradiated from the electron source 104 can be estimated (2002). However, other methods may be used for measuring the electron current.

電子 It is possible to determine whether or not a certain amount of electrons is being supplied by constantly monitoring the electron current during analysis. As a method of keeping the amount of electrons irradiated from the electron source 104 constant, there is a method of adjusting a voltage applied to the electron source 104. However, when the voltage applied to the electron source 104 is changed, it may take 10 minutes or more for the electron current to stabilize. On the other hand, in chromatography, ions eluted in several tens of seconds are switched. Therefore, when chromatography is used for the sample separation device 2, the reproducibility cannot be improved even if a method of changing the applied voltage of the electron source 104 is applied.

Therefore, in the case of this embodiment, in order to realize a highly reproducible ECD reaction, the ECD reaction time and the voltage value of the electron amount control electrode 103 are changed according to the electron current value during the ECD reaction.

FIG. 34 shows a processing flow for correcting the ECD reaction time based on the monitored electron current value and realizing a highly reproducible ECD reaction.

First, the database to be used is selected on the screen of the data display unit 18, and after changing the value, it is stored in the database 2102 (3000). Thereafter, analysis of ions by the mass spectrometer is started (3001).

Simultaneously with the start of the analysis, the LIT 2001 measures the value of the electron current irradiated from the electron source 104 (3010).

The overall processing unit 10 determines whether or not the measured value is different from the electronic current reference value (for example, 0.6 μA) described in the database by a certain value (for example, ± 0.02 μA) or more. When not deviating more than a certain value (3011), the overall processing unit 10 returns to the measurement 3010 of the electronic current value. When it deviates more than a certain value (3011), the overall processing unit 10 automatically corrects the ECD reaction time (3013). For example, when 0.05 μA is higher than the electronic current reference value, the overall processing unit 10 corrects the value by subtracting a predetermined value (for example, 1 ms) from the ECD reaction time described in the database 2102. On the other hand, when the value is 0.05 μA lower than the electronic current reference value, the overall processing unit 10 corrects the ECD reaction time described in the database 2102 to a value obtained by adding a predetermined value (for example, 1 ms).

In this processing flow, the ECD reaction time is corrected based on the measured electron current value, but the electron quantity control voltage 103 may be corrected instead of the ECD reaction time.

Next, when using the database shown in FIG. 29 (when one ion intensity is set for each precursor ion), the ECD reaction time is corrected based on the measured electron current value and the precursor ion intensity. The processing flow to be executed is shown in FIG.

In this case as well, the operator selects the database to be used on the screen of the data display unit 18, changes the value, and saves it in the database 2102 (3000). Thereafter, analysis of ions by the mass spectrometer is started (3001).

The overall processing unit 10 determines whether or not the measured value is different from the electronic current reference value (for example, 0.6 μA) described in the database by a certain value (for example, ± 0.02 μA) or more. If there is no deviation beyond a certain value (3011), the overall processing unit 10 proceeds to the acquisition process of the total ion spectrum (304). On the other hand, when there is a deviation from a certain value (3012), the overall processing unit 10 automatically corrects the ECD reaction time or the electron quantity control voltage described in the database 2102 (3013). The process proceeds to the spectrum acquisition process (304).

When all the ion spectra are acquired, the overall processing unit 10 creates a peak list including the retention time, m / z, valence, and ion intensity (3003).

Next, the overall processing unit 10 executes an inclusion DB search (3005). The overall processing unit 10 collates the created peak list with the database 2102 and searches whether or not there is an ion whose retention time, m / z, and valence match. If there is no matching ion (3007), the overall processing unit 10 returns to the electron current value measurement process (3010). If there is a matching ion (3006), the overall processing unit 10 compares the ion intensity in the peak list with the ion intensity described in the database 2102 and compares whether or not a certain value has deviated. The constant value here is determined by the operator before the analysis is executed.

Here, when the value does not deviate more than a certain value (3008), the overall processing unit 10 acquires an ECD spectrum (311). Then, the whole process part 10 returns to the acquisition process (304) of a total ion spectrum. The ECD spectrum may be acquired multiple times.

Next, in the case of using the database shown in FIG. 31 (when a plurality of ECD reaction times are set for each ion intensity for each precursor ion), an ECD reaction is performed based on the measured electron current value and the precursor ion intensity. FIG. 36 shows a processing flow executed when correcting the time.

Next, the overall processing unit 10 executes an inclusion DB search (3005). The overall processing unit 10 collates the created peak list with the database 2102 and searches whether or not there is an ion whose retention time, m / z, and valence match. If there is no matching ion (3007), the overall processing unit 10 returns to the electron current value measurement process (3010). When there is a matching ion (3006), the overall processing unit 10 determines which ion intensity range described in the database 2102 the ion intensity in the peak list is, and the corresponding ion intensity range is set. An ECD spectrum is acquired based on the ECD reaction time (311), and the process returns to the measurement 3010 of the electron current value. The ECD spectrum may be acquired multiple times.

(F) Embodiment 6: When ETD method is used as ion dissociation method (configuration of mass spectrometry system)
FIG. 16 shows a mass spectrometry system using the ETD method for ion dissociation. In FIG. 16, the same reference numerals are given to portions corresponding to FIG. 7. Also in the case of this mass spectrometry system, the sample 1 as an analysis target is separated by the pretreatment of the sample separation device 2 (in the figure, liquid chromatography (LC)). Note that a gas chromatogram (GC) can also be used for the sample separation device 2.

The separated sample is ionized in the ion source 3 and then introduced into the mass spectrometer.

When acquiring a non-fragmented spectrum, the introduced positive ions are accumulated in the middle stage 601 of the LIT or the front stage 600 of the LIT, and are discharged in a state where all ions (or a specific ion) are isolated. The discharged positive ions are separated by the LIT detector 606 according to the mass-to-charge ratio m / z of the ions. In addition, when acquiring a fragmentation spectrum, the introduced positive ions are accumulated in the middle stage 601 of LIT or the front stage 600 of LIT, and are held in the front stage 600 of LIT in an isolated state.

On the other hand, negative ions used for the ETD reaction are generated by ionization of the negative ion reagent 604 by the ion source 603 and introduced into the mass spectrometer. The introduced negative ions are accumulated and isolated in the middle stage 601 of LIT (or the latter stage 602 of LIT) for a certain period of time.

The precursor ions accumulated in the front stage 600 of the LIT and the negative ions accumulated in the middle stage 601 of the LIT undergo an ETD reaction in the entire LIT for a certain period of time, and after removing excess negative ions, the LIT detector 606 Is separated according to the mass-to-charge ratio m / z of ions. The ion source is selected from an electrospray ion source, an ion source by atmospheric pressure chemical ionization, a matrix-assisted laser desorption ion source, an electric impact ion source, an ion source by chemical ionization, and an ion source by field ionization.

A lot of ions can be stored in LIT. For this reason, highly sensitive detection is possible. However, a three-dimensional quadrupole ion trap, a quadrupole mass filter, a collision cell, or a Fourier transform ion cyclotron resonance mass separation unit may be used. Further, since improvement in sensitivity can be expected, a plurality of LIT detectors 606 may be installed. In addition, isolation of precursor ions and negative ions may be performed by installing a Q mass filter in the front stage of the front stage 600 of the LIT or the rear stage of the rear stage 602 of the LIT. Further, in the case of the embodiment, the detection is performed by the LIT detector 606, but the TOF, the three-dimensional quadrupole ion trap, the quadrupole mass filter, the magnetic field type mass analyzer, the Fourier transform ion cyclotron resonance mass spectrometry. Or an orbit trap type.

Each ion detected by the LIT detector 606 is arranged and / or processed by the overall processing unit 10 together with the m / z value. The ion dissociation parameter determination unit 12 of the overall processing unit 10 includes an ion dissociation intensity determination unit 13, a precursor ion intensity measurement unit 15, a precursor ion residual ratio calculation unit 16, and a reaction time calculation unit 1000.

The precursor ion intensity measurement unit 15 measures the non-fragmented spectrum and the precursor ion intensity in the fragmented spectrum. Precursor ion residual ratio calculation unit 16 calculates a precursor ion residual ratio. The reaction time calculation unit 1000 calculates the optimal reaction time based on the precursor ion residual ratio and the set value desired by the user. The method for calculating the reaction time is the same as the method described in the first embodiment. The signal giving the determined intensity calculated by the ion dissociation intensity determining unit 13 controls the middle stage 601 of the LIT via the control unit 9.

The mass display spectrum, the non-fragmented spectrum, the precursor ion intensity in the fragmented spectrum, the precursor ion residual rate, and the like are displayed on the data display unit 18. Further, the remaining rate of the precursor ion intensity is input from the parameter input unit 19 to the overall processing unit 10.

(Outline of processing operation)
FIG. 17 shows an ETD reaction control flow based on the sixth embodiment of the present invention. A sample 1 which is a mixture of a plurality of substances is introduced into a sample separation device 2 (liquid chromatography in the figure). Chromatography is equipped with a separation column for separation according to the nature of the substance. The sample that has passed through the separation column elutes at different times for each component. Each component of the eluted sample is ionized by an ion source (302). The ions are introduced and accumulated in the middle stage 601 of the LIT in the mass spectrometer, and then discharged (702). The ejected ions are detected by the LIT detector 606, and a non-fragmented spectrum is obtained with the horizontal axis being m / z and the vertical axis being the ion intensity (304).

Next, a precursor ion is selected from the unfragmented spectrum (305). Precursor ions are generally selected in order of ionic strength. However, depending on the nature of the ion dissociation technique, the valence and molecular weight may be taken into consideration. The valence and m / z value of the selected precursor ion are specified, and the molecular weight is calculated from the valence and m / z value (306). In parallel with this calculation operation, the ion intensity of the precursor ion is also measured (308).

For precursor ions analyzed for the first time, it is desirable to set the amount of negative ions and ETD reaction time estimated to be suitable for each precursor ion from the valence and molecular weight (703). Next, a precursor ion is isolated in the front stage 600 of LIT (704). On the other hand, for the negative ions necessary for the ETD reaction, the negative ion reagent 604 is ionized by the ion source (603) and accumulated in the middle stage 601 of the LIT for a desired time (706). The precursor ion isolated in the front stage 600 of the LIT performs an ETD reaction for a desired time with the negative ions accumulated in the middle stage 601 of the LIT in the LIT (705), and acquires an ETD spectrum (708). .

Next, the precursor ion intensity present in the ETD spectrum is measured (308). The precursor ion intensity in the non-fragmented spectrum and the precursor ion intensity in the ETD spectrum are compared to determine the remaining ratio of precursor ions (312). If this value is outside the desired range, the optimal ETD reaction time is calculated and set (313), and the ETD spectrum is acquired again. At this time, the amount of negative ions may be adjusted. It can be said that the ion dissociation strength was optimal when the remaining proportion of precursor ions was within a desired range. In this case (315), the process returns to the acquisition of the non-fragmented spectrum.

In the case of the sixth embodiment, as in the case of the second, third and fourth embodiments described above, ETcaD may be performed using the charge-reducing species ratio, the fragment ion ratio, the fragment type, and the real-time amino acid sequence analysis as indices. . In this case, after performing the ETD reaction, CID is performed by exciting only the charge-reducing species. Further, IRMPD may be used instead of CID.

Also in the case of the sixth embodiment, the sequence example to be used (FIG. 18) and the precursor ion examples (FIGS. 20 and 21) in which the ion amount changes are the same as those in the first embodiment.

Also in the case of the sixth embodiment, calculation of precursor ion residual rate, calculation of reaction time, charge reduction species ratio, calculation of fragment ion ratio and fragment type, de novo analysis, and setting of various parameters are all performed in real time. Need not interfere with the analysis.

(Effect of embodiment)
By applying the mass spectrometry method according to the above-described embodiment, optimum ion dissociation intensity data can be accumulated for each precursor ion. These data can be stored for each mass and valence. By using these data, it is possible to shorten the time until the optimum ion dissociation intensity is derived for the ions analyzed for the first time.

In addition, when these data are plotted on a chart with the date on the horizontal axis and the reaction time on the vertical axis, changes in the reaction time may be observed over time. In this case, the negative ion amount may not be supplied stably. Accordingly, a system may be adopted in which a range for a preset reaction time is determined, and when the range is exceeded, an alarm or the like is issued and the negative ion reagent 604 or the ion source 603 is observed.

(G) Embodiment 7: When CID method is used as an ion dissociation method (configuration of mass spectrometry system)
FIG. 22 shows a mass spectrometry system using the CID method for ion dissociation. In FIG. 22, the same reference numerals are given to the portions corresponding to FIG. 7.

Sample 1 as an analysis target is separated by a pretreatment of a sample separation device 2 (in the figure, liquid chromatography (LC)). Note that a gas chromatogram (GC) can also be used for the sample separation device 2.

The separated sample is ionized in the ion source 3 and introduced into the mass spectrometer. In the case of non-fragmented spectrum acquisition, the introduced ions are accumulated in a linear ion trap (LIT) 4 and all ions (or a specific ion) are discharged in an isolated state. The discharged ions are detected by the TOF detector 8. A TOF 7 having a high resolution is present in front of the TOF detector 8.

In the case of fragmentation spectrum acquisition, the introduced ions are accumulated in LIT4, and are discharged after isolation of certain specific ions and execution of the CID method. The discharged ions are separated by the TOF detector 8 according to the mass-to-charge ratio m / z of the ions. The ion source is selected from an electrospray ion source, an ion source by atmospheric pressure chemical ionization, a matrix-assisted laser desorption ion source, an electric impact ion source, an ion source by chemical ionization, and an ion source by field ionization. LIT4 can accumulate many ions. For this reason, highly sensitive detection is possible. However, instead of LIT4, a three-dimensional quadrupole ion trap, a quadrupole mass filter, a collision cell, or a Fourier transform ion cyclotron resonance mass separation unit may be used.

In this apparatus configuration, the CID method is used as the ion dissociation method, but the IRMPD method may be used. TOF7 is preferable because it has high resolution and high mass accuracy, but it is preferable to use three-dimensional quadrupole ion trap, LIT, quadrupole mass filter, magnetic field mass analyzer, Fourier transform ion cyclotron resonance mass analyzer, orbitrap mass analysis. A vessel may be used.

Each ion detected by the TOF detector 8 is arranged and / or processed by the overall processing unit 10 together with the m / z value. The overall processing unit 10 can be realized not only when it has a hardware circuit structure but also through a processing function of a program that operates on a computer system. The ion dissociation parameter determination unit 12 of the overall processing unit 10 includes an ion dissociation intensity determination unit 13, a precursor ion intensity measurement unit 15, a precursor ion residual ratio calculation unit 16, and an ion dissociation strength calculation determination unit 1001.

The precursor ion intensity measuring unit 15 measures the intensity of each precursor ion in the non-fragmented spectrum and the fragmented spectrum. The precursor ion residual ratio calculation unit 16 calculates a precursor ion residual ratio based on the measured precursor ion intensity. In the case of the CID method, the amount of fragment ions takes a peak value at the lowest ion dissociation intensity at which the precursor ion residual ratio is 0 (zero). Details will be described later. The ion dissociation strength calculation constant unit 1001 calculates the ion dissociation strength when the precursor ion residual ratio becomes 0 (zero). The ion dissociation strength determining unit 13 is supplied with a control signal corresponding to the calculated ion dissociation strength to the LIT 4 via the control unit 9. That is, LIT4 is controlled to the determined intensity. The mass spectrum, the non-fragmented spectrum and the precursor ion intensity, precursor ion residual ratio, ion dissociation intensity, etc. in the fragmented spectrum are displayed on the data display unit 18.

(Outline of processing operation)
FIG. 23 shows a CID reaction control flow based on the seventh embodiment of the present invention. A sample 1 which is a mixture of a plurality of substances is introduced into a sample separation device 2 (liquid chromatography in the figure). Chromatography is equipped with a separation column for separation according to the nature of the substance, and the sample that has passed through the separation column elutes at different times for each component. Each component of the eluted sample is ionized by the ion source 3 (302). The ions are introduced into the LIT 4 in the mass spectrometer, accumulated, and discharged (303). The ejected ions are detected by the TOF detector 8 to obtain a non-fragmented spectrum in which the horizontal axis is m / z and the vertical axis is the ion intensity (304).

Next, a precursor ion is selected from the unfragmented spectrum (305). In general, the precursor ions are selected in the order of ionic strength. However, as described above, it may be selected in consideration of the valence and molecular weight. The ion intensity of the precursor ion selected at this time is measured (308).

Next, precursor ions are isolated with LIT4 (309), CID is performed on the isolated precursor ions at various ion dissociation intensities (800), and a CID spectrum for each dissociation intensity is acquired (311). Next, the precursor ion intensity present in the CID spectrum is measured (308).

Next, the residual ratio of the precursor ions is obtained based on the precursor ion intensity in the non-fragmented spectrum and the precursor ion intensity in the CID spectrum (801). Furthermore, the calculated residual rate is plotted on a chart with the horizontal axis representing the ion dissociation strength and the vertical axis representing the precursor ion residual rate, and the lowest ion dissociation strength at which the precursor ion residual rate is 0 is estimated (802) Then, CID is performed with the ion dissociation strength (803).

Here, CID was carried out at various ion dissociation strengths (CID gain) using substance P with ion strengths of 1000, 4000 and 12000. FIG. 24 shows the dependence of precursor ions and fragment ions on the ion dissociation strength. As shown in FIG. 24, the precursor ion decreases with increasing ion dissociation intensity almost without depending on the ion intensity. The amount of fragment ions becomes maximum when the precursor ions first become zero. When the ion dissociation strength is further increased, the amount of fragment ions decreases. Thus, if the precursor ion residual ratio is measured under various ion dissociation strength conditions, the ion dissociation strength at which the precursor ions become 0 can be estimated (FIG. 25). In FIG. 25, the vertical axis represents the precursor ion residual rate, and the horizontal axis represents the ion dissociation strength.

Also in this embodiment, the calculation of the precursor ion residual ratio, the estimation of the optimum ion dissociation intensity, and the setting of various parameters are performed in real time and do not hinder the analysis.

(Sequence example when CID is executed multiple times)
FIGS. 26A to 26C show sequence examples according to Embodiment 7 of the present invention. In the case of the sequence example shown in FIG. 26A, after obtaining a non-fragmented spectrum (after obtaining MS1), a precursor ion is selected and pre-CID is performed a plurality of times with various ion dissociation intensities. Next, the precursor ion residual rate is calculated independently, the ion dissociation intensity estimated to be 0 when the precursor ion residual rate is zero, and CID is performed under the conditions.

For example, the ion dissociation intensity for executing CID is such that when the vertical axis is the precursor ion residual rate and the horizontal axis is the ion dissociation intensity, two adjacent points are connected by a straight line, the slope is negative, and the line is closest to zero. And the ion dissociation intensity at which the precursor ion residual ratio becomes 0 on the straight line is selected.

Since the ion dissociation efficiency by CID does not depend on the amount of ions, this sequence can also be applied to liquid chromatography analysis.

Further, after selecting a precursor ion in the non-fragmentation spectrum, there may be a step of isolating the precursor ion alone and measuring the precursor ion intensity. In this case, since loss occurring during the isolation process or ion transport is also taken into account, more accurate ionic strength can be measured.

The sequence example shown in FIG. 26B is common to FIG. 26A in that a plurality of pre-CIDs are acquired at various ion dissociation intensities. However, in the case of this sequence example, a CID spectrum is acquired for the ion dissociation intensity calculated with the largest amount of fragment ions. Or you may select the lowest ion dissociation intensity | strength among the ion dissociation intensity | strengths in which the precursor ion residual rate became zero.

The sequence example shown in FIG. 26C is an example in which the sequence is divided into two times. That is, in the first analysis, only the pre-CID spectrum of each ion is acquired, and the ion dissociation intensity estimated to be optimal for each ion is calculated and databased. In the second analysis, pre-CID is not performed and only the CID spectrum is acquired.

(Effect of embodiment)
By applying the mass spectrometry method according to the above-described embodiment, optimum ion dissociation intensity data can be accumulated for each precursor ion. These data can be stored for each mass, valence, and m / z value. This is an index of the ion dissociation intensity of ions to be analyzed for the first time. Using these data, pre-CID may be performed with the ion dissociation intensity from the lower limit to the upper limit of the optimum ion dissociation intensity at a certain m / z value. Thereby, the time required to derive the optimum ion dissociation strength can be shortened.

Also, the calculation of the precursor ion residual ratio and the estimation and setting of the optimum ion dissociation intensity are carried out in real time and need not interfere with the analysis.

DESCRIPTION OF SYMBOLS 1 ... Sample, 2 ... Sample separation apparatus (LC or GC), 3 ... Ion source, 4 ... Linear ion trap, 5 ... Q deflector, 6 ... ECD cell, 7 ... TOF, 8 ... TOF detector, 9 ... Control part DESCRIPTION OF SYMBOLS 10 ... Whole process part, 12 ... Ion dissociation parameter determination part, 13 ... Ion dissociation intensity determination part, 15 ... Precursor ion intensity measurement part, 16 ... Precursor ion residual ratio calculation part, 18 ... Data display part, 19 ... Parameter input 100,

end electrode

1, 101 ...

end electrode

2, 102 ... precursor ion trapped in a linear ion trap, 103 ... electron quantity control electrode, 104 ... electron source, 105 ... electron source voltage, 106 ... linear trap voltage, 200 ... Precursor ion, 201 ... Charge reduction species, 202 ... Fragment ion, 400 ... Monoisotopic 401 ... isotope ion series, 600 ... linear ion trap first stage, 601 ... linear ion trap middle stage, 602 ... linear ion trump latter stage, 603 ... ion source, 604 ... negative ion reagent, 606 ... linear ion trap detector, 607 ... negative ion amount determination unit, 1000 ... reaction time calculation unit, 1001 ... ion dissociation intensity calculation unit. 2001 ... Linear trap

Claims

An ion source that ionizes a sample, an isolation unit that isolates ions having a specific mass-to-charge ratio, an electron source that supplies electrons, an ion dissociation unit that dissociates ions, and a mass analysis unit that performs mass analysis of ions A mass spectrometric method using a mass spectrometer having a control unit for controlling ion dissociation and a calculation processing unit,
Obtaining a non-fragmented spectrum obtained without dissociating ions;
Obtaining a fragmentation spectrum in which ions are dissociated at a certain ion dissociation intensity;
In the calculation processing unit, measuring each precursor ion intensity existing in the unfragmented spectrum and the fragmented spectrum spectrum,
Calculating a ratio of a precursor ion intensity of a fragmented spectrum to a precursor ion intensity of a non-fragmented spectrum in the calculation processing unit;
In the calculation processing unit, based on the calculated ratio and the set value of the ion dissociation strength, calculating an optimum value of the ion dissociation strength for obtaining a desired ratio;
A step of acquiring an actual fragmentation spectrum based on the calculated optimum value of the ion dissociation intensity in the calculation processing unit.
Calculating the optimum value of the ion dissociation intensity,
Giving the reaction time ta set to the ratio Ia to I = e ^ (t / τ) and calculating a time constant τ specific to the precursor ion;
Providing the calculated time constant τ and the desired ratio Ib to I = e ^ (t / τ), and calculating the reaction time tb necessary to obtain the desired ratio Ib. The mass spectrometric method according to claim 1.
The amount of electrons used for the reaction is controlled by controlling a voltage applied to an electrode having pores arranged between the ion dissociation region of the ion dissociation part and the electron source. The mass spectrometric method described in 1.
The mass spectrometry method according to claim 1 or 2, wherein the reaction time of the precursor ion to be analyzed for the first time is given by a specific reaction time set in advance.
The mass spectrometric method according to claim 1 or 2, wherein the ion dissociation strength of the precursor ion analyzed for the first time is given by a specific electrode voltage set in advance.
The mass spectrometry method according to claim 1, wherein the ion dissociation is controlled during the measurement.
The mass spectrometry method according to claim 1, wherein a plurality of fragmentation spectra are acquired for the same precursor ion.
When the ratio of the precursor ionic strength is out of the setting range, calculate the reaction time to obtain the desired ratio, perform ion dissociation with the calculated reaction time,
The mass spectrometric method according to any one of claims 1 to 3, wherein when the ratio of precursor ion intensities is within a set range, an actual fragmentation spectrum is acquired with a reaction time in which the fragmentation spectrum is acquired. .
The correlation characteristic recognized between the precursor ion intensity and the time constant is calculated as a linear equation based on the precursor ion intensity in the unfragmented spectrum and the calculated time constants. Item 8. The mass spectrometry method according to Item 1, 2 or 7.
Calculating the total amount of ions present in the fragmentation spectrum;
Calculating the amount of charge reduce species;
A step of calculating a ratio of the charge-reducing species amount to the total ion amount,
If the ratio of charge-reduced species amount is smaller than the set threshold, switch to analysis of another precursor ion,
The mass spectrometry method according to claim 1, wherein the electron energy is changed when the ratio of the charge-reducing species amount is larger than a set threshold value.
Calculating the total amount of ions present in the fragmentation spectrum;
Calculating the amount of charge reduce species;
Calculating the difference of the sum of the charge reduce species amount and the precursor ion amount from the total ion amount, and calculating the fragment ion amount;
Calculating the ratio of the fragment ion amount to the total ion amount,
If the fragment ion ratio is greater than the set threshold, switch to another precursor ion analysis,
The mass spectrometry method according to claim 1, wherein the electron energy is changed when the ratio of fragment ion amounts is smaller than a set threshold value.
After obtaining a fragmented spectrum, obtain a non-fragmented spectrum, calculate an average value of the precursor ion intensity existing in each non-fragmented spectrum, and calculate a reaction time for obtaining the desired ratio for the average value. The mass spectrometric method according to claim 1 or 2, wherein
In the case where a liquid chromatograph or a gas chromatograph is disposed in front of the ion source, the method further comprises a step of reducing the flow rate of the liquid chromatograph or the gas chromatograph when a desired precursor ion is detected. Item 3. The mass spectrometry method according to Item 1 or 2.
When the first mass analysis process is executed, a linear equation given by the ion intensity and time constant τ of each precursor ion is calculated, the calculated primary equation, the mass-to-charge ratio of each precursor ion, the retention time, Record ionic strength and
The mass spectrometry method according to claim 1, 2, or 13, wherein an actual fragmentation spectrum is acquired with a reaction time suitable for the ionic strength of each precursor ion at the time of execution of mass spectrometry processing for the second time and thereafter.
The mass spectrometry method according to claim 1, 2, or 7, wherein a fragmentation spectrum is obtained under a plurality of ion dissociation conditions, and an actual fragmentation spectrum is obtained under an ion dissociation condition closest to the desired ratio. .
When performing mass analysis processing, record the mass-to-charge ratio, valence, mass number, calculated reaction time, and electron energy of the precursor ion. When performing subsequent mass analysis processing, record the ion dissociation strength of the precursor ion. The mass spectrometry method according to claim 1, wherein the mass spectrometry method is extracted from
The mass spectrometry method according to claim 1, wherein the ion dissociation intensity is adjusted by a change in frequency, amplitude, or direct current voltage of the high-frequency voltage.
8. The mass spectrometry method according to claim 1, wherein an actual fragmentation spectrum is acquired using the lowest applied voltage among the applied voltage values at which the ratio of precursor ions is zero.
When the vertical axis is the ratio of the precursor ion intensity and the horizontal axis is the applied voltage value, when two adjacent sampling points are connected by a straight line, the slope of the straight line is negative and the straight line Selecting the straight line closest to 0 among
The method of mass spectrometry according to claim 1, 2 or 7, further comprising: calculating a voltage value at which the ratio of the precursor ion intensities is 0 using a selected straight line.
An electron source, a ring-shaped first end electrode disposed on the far end side of the ion trap region with respect to the electron source, and a ring shape disposed on the near end side of the ion trap region with respect to the electron source An ion dissociation part having a second end electrode of the first electrode and a ring-shaped electron quantity control electrode disposed on the electron source side with respect to the second end electrode;
An ion dissociation apparatus comprising: a control unit that variably controls a voltage applied to the electronic control electrode.
An ion source that ionizes a sample, an isolation unit that isolates ions having a specific mass-to-charge ratio, an electron source that supplies electrons, an electron current measurement unit that measures the amount of electron current, and an ion dissociation A mass spectrometry method using a mass spectrometer having an ion dissociation part, a mass analysis part for mass analysis of ions, a control part for controlling ion dissociation, and a calculation processing part,
Registering the database;
Obtaining a non-fragmented spectrum obtained without dissociating ions;
In the calculation processing unit, searching for whether or not ions described in a database exist;
If the ion described in the database does not exist, the process returns to the step of acquiring the non-fragmented spectrum. If the ion described in the database exists, the actual fragmented spectrum is acquired with the ion dissociation intensity described in the database. A mass spectrometry method comprising the steps of:
The mass spectrometry method according to claim 21, wherein the database includes a mass-to-charge ratio, a retention time, a valence, an ionic strength, a reaction time, electron energy, an electron amount control voltage, and an electron current reference value. .
The mass spectrometry method according to claim 14 or 21, wherein an actual fragmentation spectrum is obtained by changing a reaction time according to ionic strength.
A step of comparing the electronic current value measured by the electronic current measuring unit with the electronic current reference value described in the database, and a difference of a certain value or more between the electronic current value and the electronic current reference value is If there is, the mass spectrometry method according to claim 21 or 22, wherein the reaction time or the electron quantity control voltage described in the database is changed.