JP2013228211A - Mass spectroscope and mass spectroscopic method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve a throughput while increasing precision of structural analysis by automatically selecting a suitable precursor ion featuring the structure of a target compound.SOLUTION: The supply amount of a cleaning gas to an ion trap, i.e. a gas pressure condition is changed and mass spectrometric analysis of the same sample is executed to obtain mass spectra, respectively. Although a modification body such as a phosphate group is hardly desorbed because of a lower energy of ions in the presence of a high gas, energy of ions is still high in the presence of a low gas and the modification body is easily desorbed. Consequently, when a difference in mass between a peak at which a signal intensity increases among a plurality of mass spectra when gas pressure is varied and a peak at which the signal intensity decreases reaches the known mass of the modification body or its multiple, it can be estimated that those peaks have the same basic structure and differ in only the number of modification bodies. Here, those peaks are selected for precursor ions and MSis performed to take structure analysis.

Description

  The present invention temporarily holds ions to be analyzed in an ion trap, selects ions in the ion trap and performs a dissociation operation on the selected ions, and then generates product ions generated by the dissociation. The present invention relates to a mass spectrometer for mass spectrometry and a mass spectrometry method.

  An ion trap mass spectrometer equipped with a MALDI (matrix-assisted laser desorption / ionization) ion source and a three-dimensional quadrupole ion trap is widely used in the identification and structural analysis of polymer compounds such as sugar chains and peptides. ing. Mass spectrometry of various ions temporarily held in the ion trap includes the use of the mass separation function of the ion trap itself, and time-of-flight mass spectrometry that discharges ions from the ion trap all at once and is provided outside the ion trap. In some cases, mass separation of ions is performed by a detector and detection is performed, but here, these are included and referred to as an ion trap mass spectrometer.

A general analysis method for polymer compounds using an ion trap mass spectrometer is as follows.
After the target compound to be analyzed is ionized by the MALDI method and captured in the ion trap, ions having a specific mass-to-charge ratio m / z derived from the target compound are selectively left in the ion trap as precursor ions, An ion selection operation for discharging other unnecessary ions to the outside of the ion trap is performed. Thereafter, a collision-induced dissociation (CID) gas is introduced into the ion trap, and the precursor ions are excited by the action of a high-frequency electric field to collide with the CID gas, thereby promoting the dissociation of the precursor ions. In some cases, since the target structure is not sufficiently dissociated in one CID operation, the selection of the precursor ion and the CID operation may be repeated a plurality of times. For the product ions finely fragmented by performing one or more CID operations on the ions derived from the target compound, detection of ions with mass scanning is performed to obtain an MS ⁿ spectrum, and this MS ⁿ By using the spectrum for database search, for example, the compound is identified and its structure is estimated.

In order to improve the accuracy in the identification and structural analysis of the compounds as described above, or to improve the throughput by reducing the time required for the measurement and analysis, an appropriate mass-to-charge ratio as a precursor ion to be subjected to CID operation It is important to select the ions. As a conventional method for selecting a general precursor ion, a peak appearing in a mass spectrum (MS ¹ spectrum) obtained by performing a normal mass analysis without performing a CID operation is detected. For example, the signal intensity is greater than or equal to a threshold value. For example, a technique is adopted in which all peaks satisfying a predetermined criterion are extracted and ions corresponding to the peaks are selected as precursor ions. Usually, there are a large number of peaks on the mass spectrum that satisfy a predetermined standard. Therefore, when MS ² analysis is performed on all of them, it takes a lot of time for measurement alone. In addition, as the number of times of measurement increases, the amount of sample consumption increases accordingly, so a large number of samples must be prepared.

  In order to avoid the above problems, it is not necessary to extract all the peaks that satisfy a predetermined standard on the mass spectrum, but to restrict only the predetermined number of peaks by prioritizing in order of intensity, for example. The method of imposing can be considered. However, since the peak with relatively high signal intensity is not necessarily the ion peak that characterizes the structure of the target compound, precursor ions that are effective for identification and structural analysis can be obtained simply by mechanically extracting the peaks according to the order of intensity. May not be available.

  A detailed example will be described. In many cases, biopolymer compounds such as peptides, which are often analyzed by ion trap mass spectrometers, contain modified products or functional groups that are easily detached. As typical modified products, sialic acid, sulfate group, phosphate group and the like are well known. For example, it is known that sialic acid is preferentially released by low energy CID in a sugar chain to which sialic acid, which is a kind of acidic sugar, is bonded or a glycopeptide to which a sialic acid-binding sugar chain is added. Such desorption of sialic acid is easily caused not only by the CID process but also by in-source decomposition and collision with a cooling gas in the ion trap (see, for example, Patent Documents 1 and 2). Therefore, when the sample includes a compound in which a plurality of modified products that are easily detached as described above are bound, the modified product is not removed from the basic structure on the mass spectrum obtained without performing CID. A peak derived from an ion that is not desorbed at all, a peak derived from an ion in which some of a plurality of modified products are desorbed from the basic structure, and a peak derived from an ion in which all the modified products are desorbed from the basic structure Appears mixed. This makes the mass spectrum quite complex. In addition, since the intensity of ions including the basic structure is dispersed in a plurality of peaks, the intensity of each peak may be relatively low. In such a case, as described above, in the method of extracting a predetermined number of peaks according to the order of intensity, there is a possibility that ions including a basic structure well representing the characteristics of the compound may not be selected as the precursor ions.

JP 2010-256101 A JP 2011-145089 A

The present invention has been made in view of the above problems, and its object is to appear in a mass spectrum when performing identification and structural analysis of a polymer compound having a modified compound or functional group that is easily detached. An object of the present invention is to provide a mass spectrometer and a mass spectrometry method capable of extracting an appropriate ion peak that characterizes a target compound from peaks and selecting it as a precursor ion to perform MS ² analysis.

The first invention made to solve the above problems is a mass spectrometer equipped with an ion trap that temporarily captures ions derived from a target compound to be analyzed and promotes dissociation of the captured ions. And
a) gas supply means for introducing a cooling gas for cooling the ions trapped in the internal space of the ion trap into the ion trap;
b) analysis execution means for executing a plurality of mass analyzes with different gas pressure conditions in the ion trap by the gas supplied into the ion trap by the gas supply means on the same sample, and acquiring a mass spectrum respectively; ,
c) By comparing the signal strengths of the peaks having the same mass-to-charge ratio for a plurality of mass spectra respectively acquired under the different gas pressure conditions, the same backbone structure is obtained and coupled to the backbone structure. A precursor ion selecting means for identifying a plurality of ions having different numbers of modified products and selecting at least one of the ions as a precursor ion;
It is characterized by having.

In addition, a second invention made to solve the above problems is a mass spectrometer having an ion trap that temporarily captures ions derived from a target compound to be analyzed and promotes dissociation of the captured ions. A mass spectrometry method used,
a) To cool the ions trapped in the internal space of the ion trap, multiple times of mass spectrometry with different gas pressure conditions in the ion trap are performed on the same sample by the cooling gas supplied into the ion trap. An analysis execution step for acquiring a mass spectrum,
b) By comparing the signal strengths of the peaks having the same mass-to-charge ratio for a plurality of mass spectra respectively acquired under the different gas pressure conditions, the same backbone structure is obtained and coupled to the backbone structure A precursor ion selection step of identifying a plurality of ions having different numbers of modifications, and selecting at least one of the ions as a precursor ion;
It is characterized by having.

  In an ion trap mass spectrometer, a cooling operation is generally performed to collect ions trapped in the ion trap near the center of the trapping space for the purpose of improving detection sensitivity and mass resolution. Cooling is an operation that attenuates the kinetic energy of ions by introducing a cooling gas such as He, which is an inert gas, into the ion trap and bringing the trapped ions into contact with these gases. Since ions having a small kinetic energy are easily affected by the trapping electric field, the spatial expansion of ions in the trapping space is suppressed, and the ions are likely to gather near the center of the trapping space. Generally, in a series of processes for mass spectrometry as described above, cooling is performed after ions are introduced into the ion trap from the outside or after precursor ions are dissociated by CID and the generated product ions are captured. Is executed.

  The compound to be analyzed in the first and second inventions typically includes an ionization process, a process in which ions are transported from an ion source provided outside to an ion trap, or a cooling process in the ion trap. In this case, one or a plurality of modified or functional groups that are easily eliminated are bonded to the backbone structure. Specifically, examples of such a modified product / functional group include sialic acid, sulfuric acid group, and phosphoric acid group.

  When the target sample contains a modified product that easily desorbs or a compound in which one or more functional groups are bonded to the backbone structure, the cooling action will occur if the gas pressure in the ion trap is low (the degree of vacuum is high). However, the kinetic energy of the trapped ions is relatively large. Due to this energy, the modified products and functional groups bonded to the backbone structure are easily detached. Conversely, if the gas pressure in the ion trap is high (the degree of vacuum is low), the cooling action is strong and the kinetic energy of the ions is relatively low. For this reason, elimination of the modified product or functional group bonded to the backbone structure is unlikely to occur. For this reason, between a plurality of mass spectra acquired for the same sample under different gas pressures, ions from which one or more modified products are added to the same basic structure or ions from which all modified products are desorbed A difference occurs in the intensity of the peak. For example, the peak at which the signal intensity increases when the gas pressure is changed from a high state to a low state with the same mass-to-charge ratio can be estimated to be a peak corresponding to the ion from which the modification product is desorbed.

Therefore, in the mass spectrometer according to the first aspect of the invention, the precursor ion selection means calculates, for example, a peak intensity difference for each mass-to-charge ratio for two mass spectra having different gas pressure conditions, and the intensity difference has a positive value. A set of peaks having a negative intensity difference is searched for such that the mass-to-charge ratio difference of the peaks matches the mass of a known modification or functional group or an integral multiple thereof. Since the peak set obtained by such a search is considered to be an ion having the same basic structure and different only in the number of modified products and functional groups, for example, one of the obtained peak sets Select as a precursor ion. The precursor ion thus selected should be a good representation of the structural characteristics of the target compound. Therefore, the accuracy of identification and structural analysis can be improved by using the MS ² spectrum obtained by dissociating the precursor ion by CID or the like for identification of the target compound and structural analysis.

According to the mass spectrometer according to the first invention and the mass spectrometry method according to the second invention, one or more modified or functional groups that are easily detached, such as a peptide subjected to post-translational modification, are bonded to the backbone structure. When performing identification or structural analysis of a compound, a precursor ion that well represents the characteristics of the structure can be selected accurately. Thereby, the accuracy of identification and structural analysis of the polymer compound is improved. Further, since execution of MS ² analysis for inappropriate precursor ions that are not useful for identification and structural analysis can be avoided, it is effective in reducing the number of times of MS ² analysis and shortening the measurement time.

The block diagram of the principal part of MALDI-IT-TOFMS which is one Example of this invention. The schematic explanatory drawing of precursor ion selection in MALDI-IT-TOFMS of a present Example. The figure which shows the comparison of the measurement mass spectrum obtained under different gas pressure conditions. The figure which shows the difference spectrum based on the measured mass spectrum obtained under different gas pressure conditions.

  Hereinafter, a matrix-assisted laser desorption ionization ion trap time-of-flight mass spectrometer (MALDI-IT-TOFMS) and a characteristic mass spectrometry method implemented by the apparatus according to an embodiment of the present invention will be described with reference to the accompanying drawings. To explain.

  FIG. 1 is a configuration diagram of a main part of the MALDI-IT-TOFMS of this embodiment. This MALDI-IT-TOFMS includes an ion source 1 that ionizes a target sample, a three-dimensional quadrupole ion trap 2 that retains ions and dissociates ions therein, and an ion in a vacuum chamber (not shown). And a time-of-flight mass analyzer 3 that detects and separates according to the mass-to-charge ratio.

  The ion source 1 is a MALDI ion source, a laser irradiation unit 11 that emits a pulsed laser beam, a sample plate 12 to which a sample S containing a target compound is attached, and the sample S that has been emitted from the sample S by laser beam irradiation. It includes an ion optical system 13 that draws out ions and guides the extracted ions.

  The ion trap 2 includes a ring-shaped ring electrode 21 and an inlet end cap electrode 22 and an outlet end cap electrode 24 which are arranged to face each other so as to sandwich the ring electrode 21. A space surrounded by the electrodes 21, 22, and 24 becomes a capturing region. An ion incident port 23 is bored substantially at the center of the inlet end cap electrode 22, and ions emitted from the ion source 1 are introduced into the ion trap 2 through the ion incident port 23. On the other hand, an ion emission port 25 is formed substantially at the center of the exit-side end cap electrode 24, and ions are emitted from the ion trap 2 to the time-of-flight mass analysis unit 3 through the ion emission port 25. In the ion trap 2, cooling gas or CID gas is supplied from the gas supply source 28 through the gas introduction pipe 26, and the gas amount is controlled by the opening time of the pulse valve 27.

  The time-of-flight mass spectrometer 3 includes a flight space 31 in which ions are turned back by the reflectron 32 and an ion detector 4 that detects ions flying in the flight space 31. Ions released simultaneously from the inside of the ion trap 2 through the ion emission port 25 fly in the flight space 31, but each ion has a flight speed corresponding to the mass-to-charge ratio. As a result, a time difference is generated and the ion detector 4 is reached. The ion detector 4 generates a detection signal corresponding to the amount of incident ions and sends the signal to the data processing unit 5.

The data processing unit 5 converts the input signal as described above into a digital value and creates a mass spectrum (MS ⁿ spectrum) based on the data, or qualitative (identification) and quantification using the mass spectrum. Or, structural analysis or the like is executed. In particular, in order to execute data processing characteristic of the present invention, the data processing unit 5 includes a data collection unit 51, a data storage unit 52, a spectrum comparison unit 53, a precursor ion determination unit 54, an identification / structure analysis processing unit 55, and the like. Includes functional blocks.

  The analysis control unit 6 has a function of controlling the above-described units such as the ion source 1, the ion trap 2, the time-of-flight mass analysis unit 3, and the data processing unit 5. Further, an input unit 8 and a display unit 9 are connected to a central control unit 7 that performs overall control higher than the input / output control and the analysis control unit 6. The data processing unit 5, the analysis control unit 6 and the central control unit 7 use a personal computer as a hardware resource and execute dedicated processing / control software installed in the personal computer in advance on the computer. It can be embodied.

  Next, processing and control operations for structural analysis of a compound characteristic of MALDI-IT-TOFMS of this example will be described with reference to FIG. Here, the compounds subject to structural analysis are high-molecular compounds containing modified products and functional groups that can be easily detached, and typical modified products and functional groups include sialic acid, sulfate groups, and phosphate groups. is there. Typical compounds to be analyzed include sialic acid-bonded sugar chains, glycopeptides to which sialic acid-bonded sugar chains are added, sulfated sugar chains, sulfated peptides, phosphorylated sugar chains, phosphorylated peptides, etc. It is. Such a compound is mixed with a predetermined matrix to prepare a sample S.

  When the analysis is started by an instruction from the input unit 8 by the analyst, first, under the condition that the cooling gas introduction time into the ion trap 2 is set to T1 under the control of the analysis control unit 6. Mass analysis is performed on the sample S containing the compound, and a mass spectrum is created based on data obtained by the analysis.

  More specifically, the laser irradiation unit 11 emits laser light for a short time under the control of the analysis control unit 6. The laser light is irradiated to the sample S, and the matrix in the sample S is rapidly heated to vaporize with the target compound. At this time, the target compound is ionized. At substantially the same time or in advance, the pulse valve 27 is opened for a predetermined time T1 (which is sufficiently longer than a time T2 described later), and the cooling gas is supplied into the ion trap 2 through the gas introduction pipe 26. Ions generated by the laser irradiation are converged by an electrostatic field formed by the ion optical system 13 and introduced into the ion trap 2 through the ion incident port 23. The introduced ions are trapped in the internal space of the ion trap 2 by an electric field formed by a high frequency voltage applied to the ring electrode 21.

  Although ions have a relatively large kinetic energy when introduced into the ion trap 2, the trapped ions come into contact with the cooling gas and give kinetic energy because the ion trap 2 is filled with a cooling gas. lose. Since the flow rate per unit time of the gas supplied through the gas introduction pipe 26 is constant, the longer the opening time of the pulse valve 27, the larger the total amount of the cooling gas, and the higher the density of the cooling gas in the ion trap 2. As shown in FIG. 2 (a-1), when the gas density in the ion trap 2 is high, the probability that the ions come into contact with the gas increases, and the kinetic energy of the ions decreases accordingly. A compound ion to which a modified product that is easily desorbed is bonded is more likely to spontaneously desorb the modified product as its internal energy increases. Therefore, when the gas density in the ion trap 2 is high and the energy of ions is low, the desorption of the modification product is not promoted so much. As a result, as shown in FIG. 2A-2, the state in which the modified product 101 is bonded to the ionic backbone structure 100 is easily maintained.

  In the ion trap 2, after cooling is performed for the introduced ions for a predetermined time, a predetermined DC voltage is applied to the end cap electrodes 22 and 24, so that the trapped ions are released all at once from the ion trap 2. And introduced into the time-of-flight mass spectrometer 3. Various ions are separated according to the mass-to-charge ratio while flying in the flight space 31 and reach the ion detector 4 to be detected. The data collection unit 51 receives a signal from the ion detector 4, creates a time-of-flight spectrum indicating ion intensity with respect to the time of flight, and then creates a mass spectrum by converting the time of flight to a mass-to-charge ratio. As described above, since the modified product is relatively difficult to desorb at this time, as shown in FIG. 2 (a-3), on the mass spectrum, an ion in a state where the modified product 101 is bonded to the backbone structure 100. A peak corresponding to is observed with a large signal intensity. On the other hand, the signal intensity of the peak corresponding to the ion from which the modified product 101 is detached from the backbone structure 100 is small. The mass spectrum data thus obtained is stored in the data storage unit 52.

  Next, under the control of the analysis controller 6, mass analysis is performed on the sample S containing the target compound under the condition that the introduction time of the cooling gas into the ion trap 2 is set to T2 which is sufficiently shorter than T1. Then, a mass spectrum is created based on the data obtained by the analysis. At this time, since the opening time of the pulse valve 27 is short, the density of the cooling gas in the ion trap 2 is low as shown in FIG. Therefore, the probability that the ions come into contact with the gas is low, so that the kinetic energy of the ions is not attenuated so much and is maintained in a high state. As a result, spontaneous desorption of the modified product 101 from the ionic backbone structure 100 proceeds, and as shown in FIG. 2 (b-2), all the modified products 101 are detached from the ionic backbone structure 100. The number of ions that are in a state in which only the selected ions and some of the modified products 101 are bonded increases.

  Therefore, on the mass spectrum generated by the data collection unit 51 at this time, as shown in FIG. 2 (b-3), almost no ion peak from which the modified product 101 is not desorbed is observed, and instead, In addition, peaks corresponding to ions from which all the modified products 101 have been released and peaks corresponding to ions from which some of the modified products 101 have been released appear with a large signal intensity. As shown in FIG. 2, when the mass-to-charge ratio of an ion in which two same modification products 101 are bonded to a certain basic structure 100 is M 3, the ion from which one modification product 101 is desorbed is its modification. The mass-to-charge ratio M2 is small by an amount corresponding to the mass-to-charge ratio Δ of the object 101. Further, all, that is, ions from which two modified products 101 have been desorbed have a smaller mass-to-charge ratio M1 corresponding to the mass-to-charge ratio Δ of the modified product 101.

  As described above, if mass spectra are obtained under different gas pressures in the ion trap 2, the spectrum comparison unit 53 compares the two mass spectra. Specifically, for example, a differential mass spectrum obtained by subtracting the peak signal intensity on one mass spectrum from the peak signal intensity on the other mass spectrum for each mass-to-charge ratio. FIG. 2 (c) is a differential mass spectrum obtained by subtracting the mass spectrum of FIG. 2 (b-3) from the mass spectrum of FIG. 2 (a-3).

Next, the precursor ion determination unit 54 selects a precursor ion suitable for MS ² analysis by analyzing the differential mass spectrum. For example, in the differential mass spectrum shown in FIG. 2 (c), a positive peak having a mass-to-charge ratio of M3 is a peak whose signal intensity is greatly reduced by changing the condition from high gas pressure to low gas pressure. On the contrary, the negative peak having a mass-to-charge ratio M1 is a peak in which the signal intensity is greatly increased by changing the condition from high gas pressure to low gas pressure. Since the difference between the mass-to-charge ratios of the two peak sets coincides with twice the mass of the known modification 101, it can be estimated that these are peaks derived from ions having the same basic structure. Moreover, since there is no peak separated by a mass corresponding to the mass of the modified product 101 below the peak having a mass to charge ratio of M1, the peak having a mass to charge ratio of M1 is an ion from which all of the modified product 101 has been desorbed. It can be estimated that there is. Therefore, one of these two peaks is selected as the precursor ion. Which peak is selected may be determined depending on whether the opening time of the pulse valve 27 is set to T1 or T2 for cooling during MS ² analysis. This is because the strength of the precursor ion at the time of MS ² analysis is as high as possible, that is, it is preferable that the amount of ions is large.

The mass-to-charge ratio of the precursor ions selected by the precursor ion determination unit 54 is transmitted to the analysis control unit 6. Subsequently, the analysis control unit 6 controls each unit so that the MS ² analysis in which the mass-to-charge ratio of the selected precursor ions is set is performed on the same sample S. Thus, after ions of the target compound in the sample S are once trapped in the ion trap 2, only the selected precursor ions remain in the ion trap 2 and other ions are discharged or removed. A voltage is applied to the electrodes 21, 22 and 24. Thereafter, CID gas is supplied into the ion trap 2 and the ions are resonantly excited, whereby dissociation of the ions is promoted and the generated product ions are trapped in the ion trap 2. Then, product ions are released from the ion trap 2 and subjected to mass analysis by the time-of-flight mass analysis unit 3, so that the data collection unit 51 creates an MS ² spectrum. The identification / structure analysis processing unit 55 uses the mass-to-charge ratio and the intensity of each peak observed on the MS ² spectrum to estimate the structure of the target compound by performing a database search, for example.

In the MALDI-IT-TOFMS of this example, when a precursor ion is selected, an ion that well represents the characteristics of the structure of the target compound can be selected as the precursor ion. Thereby, since a lot of useful information can be used for the database search, the accuracy of the search is improved, and the structure estimation can be performed more accurately. In addition, since it is possible to avoid selecting a plurality of ions having different numbers of additions of modified products as precursor ions, unnecessary MS ² analysis is not performed and analysis time is shortened. It is effective for.

  In the above embodiment, two mass spectra were acquired by changing the opening time of the pulse valve 27 for supplying the cooling gas into the ion trap 2 in two stages, but the same was obtained under a larger number of different gas pressure conditions. It is also possible to acquire a mass spectrum for the sample S, obtain a plurality of difference mass spectra between two mass spectra among the many mass spectra, and select a precursor ion based on the plurality of difference mass spectra.

  In the above embodiment, the gas pressure in the ion trap 2, that is, the gas density is changed by changing the opening time of the pulse valve 27 for supplying the cooling gas to the ion trap 2. If a valve that can be changed is used, the gas flow rate may be changed.

[Measurement example]
An actual measurement example to which the processing according to the above embodiment is applied will be described. The equipment used was Shimadzu / Kratos AXIMA-QIT, the sample was 0.5 [μL] Casein digests 1 pmol, the matrix 0.5 μL of DHBA 12.5 mg / mL solution in 50/50 0.1% TFA / MeCN solvent. Further, there are five types of opening time of the pulse valve when introducing the cooling gas: 0 [μsec], 25 [μsec], 50 [μsec], 75 [μsec], and 95 [μsec]. The normal pulse valve opening time in the mass spectrometer used here is 95 [μsec].

  FIG. 3 shows measured mass spectra when the opening time of the pulse valve is 0 [μsec], 50 [μsec], and 95 [μsec]. At the opening time of 95 [μsec], the gas pressure is high and the desorption of the modified product does not easily occur. Therefore, the peak signal intensity of m / z 3122.29 is large, and at a position away from the peak by 98 Da × 4 No peak is observed. On the other hand, since the gas pressure is low at the opening time of 0 [μsec] and the modification product is easily detached, the peak of the signal intensity is high at a position 98 Da × 4 away from the peak at m / z 3122.29. On the contrary, the signal strength of the peak at m / z 3122.29 is very low. The difference in mass-to-charge ratio of these two peaks is equal to four times the mass of the known phosphate group, and a discrete peak is observed at a position 98 Da apart between the two peaks. From the above, it can be estimated that the peak at m / z 3122.29 is a phosphorylated peptide, and the peak at a position separated downward by 98 Da × 4 is a peptide from which all phosphate groups are eliminated.

For further confirmation, mass spectra with different gas pressure conditions were compared by the following procedure.
(1) In the mass spectrum acquired by setting the pulse valve opening time to 95 [μs], a peak having a peak intensity (% Area) of 0.2% or more is extracted. This peak is U.
(2) The peak and mass charge extracted in (1) in each of the mass spectra obtained by changing the pulse valve opening time to 0 [μsec], 25 [μsec], 50 [μsec], and 75 [μsec]. Extract the intensity of the peaks with the matching ratio. The peak at this time is N.
(3) For each peak, the intensity ratio P is calculated by the following equation (1).
P = {% Total of (N) −% Total of (U)} / {% Total of (U)} (1)

  FIG. 4 shows the calculation result in the form of a mass spectrum. In the figure, the signal intensity of the peptide ion from which the phosphate group has been removed indicated by ▲ increases as the pulse valve opening time is shortened (when the gas pressure is lowered). On the other hand, the signal intensity of the phosphorylated peptide ion indicated by the mark ● decreases as the pulse valve opening time is shortened (when the gas pressure is lowered). In addition to the peaks marked with ●, there are some peptides with reduced signal intensity. However, it can be seen that such a peak does not have a peak in which the mass-to-charge ratio matches 98 Da. Therefore, when the pulse valve opening time is shortened, if a pair of peaks having a mass-to-charge ratio difference of 98 Da is found between the peak in which the signal intensity increases and the peak in which the signal intensity decreases, the peak in which the signal intensity decreases It can be estimated that the peak is derived from a phosphorylated peptide. In this way, by searching for a pair of peaks where the signal intensity is increasing and peaks where the signal intensity is decreasing as a pair, it is possible to accurately detect peaks where a known modification or functional group is bound or eliminated. Precursor ions that can be extracted well and are useful for identification and structural analysis can be selected.

  It should be noted that the above embodiment is merely an example of the present invention, and it is natural that any modification, correction, addition, or the like as appropriate within the scope of the present invention is included in the scope of the claims of the present application.

DESCRIPTION OF SYMBOLS 1 ... Ion source 11 ... Laser irradiation part 12 ... Sample plate 13 ... Ion optical system 2 ... Ion trap 21 ... Ring electrode 22 ... Inlet end cap electrode 23 ... Ion entrance port 24 ... Outlet end cap electrode 25 ... Ion exit port 26 ... Gas introduction pipe 27 ... Pulse valve 28 ... Gas supply source 3 ... Time-of-flight mass analysis unit 31 ... Flight space 32 ... Reflectron 4 ... Ion detector 5 ... Data processing unit 51 ... Data collection unit 52 ... Data storage unit 53 ... Spectrum comparison unit 54 ... Precursor ion determination unit 55 ... Identification / structure analysis processing unit 6 ... Analysis control unit 7 ... Central control unit 8 ... Input unit 9 ... Display unit

Claims (2)

A mass spectrometer having an ion trap that temporarily captures ions derived from a target compound to be analyzed and promotes dissociation of the captured ions,
a) gas supply means for introducing a cooling gas for cooling the ions trapped in the internal space of the ion trap into the ion trap;
b) analysis execution means for executing a plurality of mass analyzes with different gas pressure conditions in the ion trap by the gas supplied into the ion trap by the gas supply means on the same sample, and acquiring a mass spectrum respectively; ,
c) By comparing the signal strengths of the peaks having the same mass-to-charge ratio for a plurality of mass spectra respectively acquired under the different gas pressure conditions, the same backbone structure is obtained and coupled to the backbone structure. A precursor ion selecting means for identifying a plurality of ions having different numbers of modified products and selecting at least one of the ions as a precursor ion;
A mass spectrometer comprising: A mass spectrometry method using a mass spectrometer equipped with an ion trap that temporarily captures ions derived from a target compound to be analyzed and promotes dissociation of the captured ions,
a) To cool the ions trapped in the internal space of the ion trap, multiple times of mass spectrometry with different gas pressure conditions in the ion trap are performed on the same sample by the cooling gas supplied into the ion trap. An analysis execution step for acquiring a mass spectrum,
b) By comparing the signal strengths of the peaks having the same mass-to-charge ratio for a plurality of mass spectra respectively acquired under the different gas pressure conditions, the same backbone structure is obtained and coupled to the backbone structure A precursor ion selection step of identifying a plurality of ions having different numbers of modifications, and selecting at least one of the ions as a precursor ion;
A mass spectrometer comprising:

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