WO2011010649A1 - Spectrométrie de masse et dispositif de dissociation des ions - Google Patents

Spectrométrie de masse et dispositif de dissociation des ions Download PDF

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WO2011010649A1
WO2011010649A1 PCT/JP2010/062207 JP2010062207W WO2011010649A1 WO 2011010649 A1 WO2011010649 A1 WO 2011010649A1 JP 2010062207 W JP2010062207 W JP 2010062207W WO 2011010649 A1 WO2011010649 A1 WO 2011010649A1
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ion
spectrum
ions
dissociation
precursor
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PCT/JP2010/062207
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Japanese (ja)
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直己 万里
宏之 佐竹
集 平林
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株式会社日立製作所
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/004—Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn

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  • 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.
  • mass spectrometry mass spectrometry
  • mass spectrometry 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.
  • 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.
  • mass spectrometry 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.
  • 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.
  • 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.
  • CID collision-induced-dissociation
  • IRMPD infrared multi-photon absorption
  • ECD Electron-capture-dissociation
  • ETD electron-transfer-dissociation
  • 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.
  • 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.
  • 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.
  • a, b, and c are molecules including the NH 2 terminal side
  • 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.
  • 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.
  • EDD electron desorption-dissociation
  • the method of reacting positively charged precursor ions with electrons is called the ECD method.
  • 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.
  • FIG. 3 shows the ECD reaction time dependence of a divalent peptide ion called substance P (Non-patent Document 4).
  • the vertical axis represents relative ionic strength
  • 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.
  • 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.
  • FIG. 4 shows schematic diagrams of ECD spectra acquired with various amounts of electrons.
  • the vertical axis represents ionic strength
  • 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.
  • a spectrum in which only the precursor ion 200 remains is obtained (FIG. 4B).
  • 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).
  • 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.
  • 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).
  • dissociation other than cz is likely to occur as in the HECD method.
  • the ETD method cannot increase the electron energy in principle.
  • 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.
  • the vertical axis represents ionic strength
  • 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.
  • 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).
  • shaft of FIG. 6 (A) is a sequence coverage
  • a horizontal axis is electron energy.
  • the vertical axis in FIG. 6B represents the relative ion intensity occupied by all ions
  • the horizontal axis represents the electron energy.
  • the CECD method is optimal because it provides a high sequence coverage without causing secondary dissociation.
  • 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.
  • 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.
  • 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).
  • ACE Automatic Collision Energy
  • 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.
  • the dissociation efficiency can be improved by applying an HECD method, an AI-ECD method, an ETcaD method, or the like to these ions.
  • 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.
  • the energy is changed in three stages, but it is not known whether the optimum dissociation energy has been set.
  • 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.
  • the mass spectrometry method 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 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
  • a control unit that variably controls the voltage applied to the.
  • the mass spectrometry method 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.
  • the ion dissociation apparatus 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
  • a control unit that variably controls the voltage applied to the.
  • 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 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 6 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
  • the schematic diagram which shows the reaction time in the predicted ion chromatogram and each ion intensity
  • 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 database mode control flow based on Embodiment 5 of invention.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • LIT4 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.
  • 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.
  • 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 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.
  • the amount of precursor ions can be adjusted by controlling the ion accumulation time in LIT4.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • a predetermined specific value may be given as a set value for the precursor ion analyzed for the first time.
  • the electron energy is set to 1 eV
  • the reaction time is set to 10 ms
  • the electron amount control voltage is set to 20V. The electron voltage control voltage will be described later.
  • 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).
  • 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.
  • 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.
  • 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.
  • 13 C 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.
  • the proton addition number corresponds to the valence z.
  • the m / z value of the monoisotopic peak 400 is (M + z) / z
  • 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.
  • 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].
  • the precursor ion 201 having an ionic strength Ix (FIG. 10 ( Assume that an ECD spectrum in which B)) remains is obtained.
  • the precursor ion residual ratio I can be expressed by the following equation using the time constant ⁇ determined by the properties of the sample and the reaction time t [ms].
  • 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 ⁇ and the precursor ion residual rate. Can do.
  • 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.
  • 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.
  • 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.
  • the precursor ion 102 is held along the central axis of the LIT4.
  • 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.
  • the control unit 9 adjusts the amount of electrons introduced by controlling the voltage applied to the electron amount control electrode 103.
  • FIG. 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).
  • FIG. 12A shows an example in which the ion trap voltage 106 is 20V
  • FIG. 12B shows an example in which the ion trap voltage 106 is 28V.
  • electrons are not incident on the ion trap while the applied voltage to the electron amount control electrode 103 is up to 14V.
  • the ion trap voltage is 20V
  • the maximum current flows when the electron amount control voltage is around 20V.
  • the ion trap voltage is 28V
  • the maximum current flows when the electron amount control voltage is 23V.
  • the amount of electrons incident on the LIT 4 can be adjusted by controlling the electron amount control electrode 103.
  • 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.
  • the DC voltage applied to the electron quantity control electrode 103 is varied, but the control method differs depending on the ion dissociation part.
  • 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.
  • the electron amount control voltage 103 is fixed at 23 V, and the amount of electrons is adjusted only by the reaction time.
  • 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.
  • sequence example when ECD is executed multiple times show sequence examples according to Embodiment 1 of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • the time constant ⁇ 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 ⁇ 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 ⁇ obtained.
  • the optimal reaction time can be calculated if the ion amount can be predicted.
  • 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 ⁇ .
  • a function primary equation
  • ⁇ the relationship between the ion intensity and the optimum reaction time for each ion is compiled into a database.
  • 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).
  • 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.
  • 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.
  • the storage time may be adjusted by the ECD cell 6 instead of the LIT4.
  • 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.
  • 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.
  • FIG. 13 shows an ECD reaction control flow based on the second embodiment of the present invention.
  • the same reference numerals are given to the portions corresponding to those in FIG. 1.
  • the charge-reduced species ratio was about 0.2.
  • 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%.
  • the charge-reduced species ratio was reduced to about 0.01 and the sequence coverage was 100%.
  • 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.
  • 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)
  • 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.
  • the overall processing unit 10 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.
  • FIG. 14 shows an ECD reaction control flow based on Embodiment 3 of the present invention.
  • the same reference numerals are given to the portions corresponding to FIG. 1.
  • the electron energy is increased using the charge-reduced species ratio as an index.
  • the electron energy may be increased by using one or both of the fragment ion ratio and the fragment type as an index.
  • the overall processing unit 10 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.
  • FIG. 15 shows an ECD reaction control flow based on the fourth embodiment of the present invention.
  • the electron energy is increased using one or both of the fragment ion ratio and the fragment type as an index.
  • amino acid sequence analysis de novo analysis
  • 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.
  • the overall processing unit 10 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.
  • Embodiment 5 ECD method is used as an ion dissociation method (part 5)
  • 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.
  • these analysis conditions are determined in advance, and mass spectrometry is executed based on these analysis conditions.
  • 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)).
  • 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.
  • the introduced ions are accumulated in a linear ion trap (LIT) 4 and all ions (or certain ions) are isolated and ejected.
  • LIT linear ion trap
  • 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.
  • 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.
  • 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.
  • 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.
  • LIT4 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.
  • 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 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.
  • 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.
  • 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.
  • FIG. 28 is a display example of an operation screen.
  • the operator selects the data dependence analysis mode through an input device (not shown).
  • 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. .
  • the set parameters are stored in the database 2102, analysis by the mass spectrometer is started.
  • the ECD reaction is performed according to the set ECD analysis conditions.
  • FIG. 29 is a display example of an operation screen.
  • the operator selects a database (DB) mode through an input device (not shown).
  • DB database
  • the operator selects a DB to be used (selects mouse_plasma_Trp_f3 in FIG. 29).
  • the contents of the parameter of the selected DB are displayed on the data display unit 18.
  • 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.
  • 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.
  • FIG. 30 shows a processing flow when the database shown in FIG. 29 is used.
  • 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).
  • 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.
  • 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.
  • 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.
  • FIG. 31 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.
  • 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.
  • the electron control voltage is set to a fixed value, and different values of ECD reaction time are set according to the ion intensity.
  • 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.
  • 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).
  • 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.
  • 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).
  • LIT2001 having an ion guide function is used as the electron current measuring unit 2100.
  • 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).
  • other methods may be used for measuring the electron current.
  • 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.
  • 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).
  • 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 ⁇ A) described in the database by a certain value (for example, ⁇ 0.02 ⁇ A) 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 ⁇ A 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 ⁇ A 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).
  • a predetermined value for example, 1 ms
  • 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.
  • 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 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 ⁇ A) described in the database by a certain value (for example, ⁇ 0.02 ⁇ A) 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).
  • the overall processing unit 10 creates a peak list including the retention time, m / z, valence, and ion intensity (3003).
  • 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.
  • 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.
  • 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.
  • FIG. 36 shows a processing flow executed when correcting the time.
  • 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 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 ⁇ A) described in the database by a certain value (for example, ⁇ 0.02 ⁇ A) 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).
  • the overall processing unit 10 creates a peak list including the retention time, m / z, valence, and ion intensity (3003).
  • 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).
  • 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.
  • FIG. 16 shows a mass spectrometry system using the ETD method for ion dissociation.
  • the same reference numerals are given to portions corresponding to FIG. 7.
  • the sample 1 as an analysis target is separated by the pretreatment of the sample separation device 2 (in the figure, liquid chromatography (LC)).
  • LC liquid chromatography
  • GC gas chromatogram
  • the separated sample is ionized in the ion source 3 and then introduced into the mass spectrometer.
  • the introduced positive ions 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.
  • 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.
  • 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.
  • LIT low-density organic compound
  • 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.
  • a plurality of LIT detectors 606 may be installed.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • a precursor ion is isolated in the front stage 600 of LIT (704).
  • 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). .
  • 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.
  • 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. .
  • CID is performed by exciting only the charge-reducing species.
  • IRMPD may be used instead of CID.
  • 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.
  • FIG. 22 shows a mass spectrometry system using the CID method for ion dissociation.
  • 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.
  • a sample separation device 2 in the figure, liquid chromatography (LC)
  • LC liquid chromatography
  • GC gas chromatogram
  • the separated sample is ionized in the ion source 3 and introduced into the mass spectrometer.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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).
  • a precursor ion is selected from the unfragmented spectrum (305).
  • 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).
  • 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).
  • 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).
  • FIG. 24 shows the dependence of precursor ions and fragment ions on the ion dissociation strength.
  • 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.
  • the ion dissociation strength is further increased, the amount of fragment ions decreases.
  • 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).
  • the vertical axis represents the precursor ion residual rate
  • the horizontal axis represents the ion dissociation strength.
  • 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.
  • FIGS. 26A to 26C show sequence examples according to Embodiment 7 of the present invention.
  • a precursor ion is selected and pre-CID is performed a plurality of times with various ion dissociation intensities.
  • 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.
  • 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.
  • the 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
  • 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.
  • 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.
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Electron Tubes For Measurement (AREA)

Abstract

Cette invention concerne une méthode de spectrométrie de masse permettant d’obtenir des données relatives à la structure d’une substance avec une meilleure efficacité, et de raccourcir le délai nécessaire pour mesurer et identifier ladite substance. L’invention concerne spécifiquement une méthode de spectrométrie de masse caractérisée en ce qu’elle consiste, en cours de dissociation ionique, à comparer la quantité d’ions mesurés avant la réaction et la quantité d’ions mesurés après la dissociation et à estimer ainsi la force de dissociation ionique optimale.
PCT/JP2010/062207 2009-07-24 2010-07-21 Spectrométrie de masse et dispositif de dissociation des ions WO2011010649A1 (fr)

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WO2018037484A1 (fr) * 2016-08-23 2018-03-01 株式会社島津製作所 Dispositif de traitement de données de spectrométrie de masse, procédé de traitement de données de spectrométrie de masse et programme de traitement de données de spectrométrie de masse
JPWO2018037484A1 (ja) * 2016-08-23 2019-01-10 株式会社島津製作所 質量分析データ処理装置、質量分析データ処理方法、及び質量分析データ処理プログラム
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