JP2007198882A - Mass-spectrometric system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass-spectrometric system capable of predicting a mass chromatogram of a precursor ion, and capable of executing MS<SP>n</SP>at timing of obtaining a favorable MS<SP>n</SP>data with high probability, using one mass spectrograph. <P>SOLUTION: In this mass-spectrometric system constituted of a separation means for a sample and a mass-spectrometric means, elution patterns for all the ion species are calculated based on the MS<SP>1</SP>data obtained in sample measurement up to the n-th time, and a priority order of the ion species analyzed mass-spectrometrically in the (n+1)-th of sample measurement is determined based on the ion species elution patterns and the MS<SP>n</SP>data. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a mass spectrometry system including a sample separation unit and a mass analysis unit.

In general mass spectrometry, after a sample to be measured is ionized, various ions generated are sent to a mass spectrometer, and the mass-to-charge ratio (m / z), which is the ratio of the mass number to the valence of ions, is measured. The measured intensity of ions is measured for each value. The resulting mass spectrum includes a measured intensity peak (ion peak) of the measured ions for each mass to charge ratio value. Thus, mass analysis of the ionized sample itself is called MS ^1, and the obtained data is called MS ¹ data. A mass spectrometer capable of multi-stage dissociation of the ion peaks detected by MS ^1, select the ion peaks having a specific mass-to-charge ratio (ion species selected is referred to as precursor ions), further, the The precursor ions are dissociated and decomposed by collision with gas molecules and the like, and the generated dissociated ion species (the dissociated ion species are referred to as dissociated ions) are subjected to mass spectrometry, and a mass spectrum is similarly obtained. Here, n-1 stage dissociation of the precursor ions and mass analysis of the dissociated ion species are referred to as MS ^n, and the obtained data is referred to as MS ⁿ data. Thus, in a mass spectrometer capable of multistage dissociation, precursor ions are dissociated in multiple stages (1 stage, 2 stages,..., N−1 stages), and the mass number of ion species generated in each stage is analyzed ( MS ² , MS ³ ,..., MS ⁿ ).

  Also, in the analysis of complex mixed samples such as biological samples, it is difficult to separate ionic species having the same or very close mass-to-charge ratio with a mass spectrometer alone. Mass spectrometry systems in which sample separation means such as liquid chromatograph (LC) and gas chromatograph (GC) are integrated with mass spectrometry are widely used.

  However, in the analysis of complex mixed samples, even if the ion species are separated by the sample separation means, the ionized sample in the same elution time often contains ion species that are a plurality of precursor ion candidates. was there. The following techniques have been proposed as means for solving this problem.

In Patent Document 1, in order to avoid duplicate measurement of precursor ions that have been executed MS ⁿ by the Nth time, information on precursor ions is registered as an internal database, and the number of measurements, the sum of ion intensities, integrated ion intensity, MS ⁿ data A mass spectrometric system having a function of excluding precursor ions subjected to MS ⁿ from selection in the real time during measurement at the time of the (N + 1) th and subsequent measurements is disclosed.

In Patent Document 2, (i) using a mass spectrometer as a main detector and a sub-detector (such as an ultraviolet detector), a sample eluted from the LC section first enters the sub-detector, and then for a predetermined time. In a mass spectrometry system configured so that a sample enters the mass spectrometer with a delay, the peak is detected from the chromatogram of the data obtained from the sub-detector, and the mass is determined according to the retention time of the peak in the chromatogram and the measured intensity. It has been proposed to control the analyzer. (Ii) The sample eluted from the LC section is branched in two directions by a splitter, the main detector is used on one side and the sub detector on the other side, and the main detector is controlled based on the data of the sub detector. Has been proposed. When this is applied, a peak is detected from the mass chromatogram of the MS ¹ data obtained by the sub-detector by using a mass spectrometer as the sub-detector, and according to the retention time and measurement intensity of the peak in the chromatogram, It is possible to control the mass spectrometer.

JP-A-2005-91344. Japanese Patent Application Laid-Open No. 2002-181784.

If the mass spectrometry system disclosed in Patent Document 1, it is possible to exclude the ionic species MS ⁿ already executed precursor ions and the user has specified in advance by the N-th from MS ⁿ target in N + 1 th. However, it is unclear at which point in the mass chromatogram of the precursor ion to be the analysis target the MS ⁿ is performed. Therefore, in the sense that there is a high possibility of contributing to the identification of the analytical ion species at the time of database search / collation, there is a possibility that the MS ⁿ data of the precursor ion is performed at a good timing, and there is a possibility that MS ⁿ is performed at a bad timing. ⁿ may also be performed. Then, it is unclear whether or not good MS ⁿ data can be obtained when the sum of ion intensity, integrated ion intensity, quality of MS ⁿ data, etc. are limited, and precursor ions are excluded from the MS ⁿ target. Further, even if good MS ⁿ data is obtained as a result, if MS ⁿ is executed at a timing when the quality of the MS ⁿ data is bad before that, the number of useless MS ⁿ times increases. There is a possibility that the number of precursor ion species for performing MS ⁿ is decreased during the measurement of each sample.

In Patent Document 2, (i) an ultraviolet detector is used as a sub-detector, but in the analysis of a complicated mixed sample such as a biological sample, a plurality of precursor ion candidates are included in a sample ionized at the same elution time. Therefore, there is a problem that a mass chromatogram of precursor ions cannot be determined with an ultraviolet detector. This problem is addressed above (ii
This can be solved by a technique in which the sample eluted from the LC section is branched by a splitter, supplied to the main detector and the sub detector, and the main detector is controlled based on the data of the sub detector. However, when the sample is branched by the splitter, it is difficult to keep the pressure constant due to clogging or the like, and there is a possibility that the mass spectrometer cannot be operated normally while keeping the predetermined time accurate. Further, since two mass spectrometers are used, there is a problem that twice as many samples are required for each measurement as compared with the case where one mass spectrometer is used. Furthermore, there is a problem that the cost increases because two mass spectrometers are prepared.

The object of the present invention is to solve the above-described problems by predicting the mass chromatogram of precursor ions using a single mass spectrometer, and at a timing when there is a high possibility that good MS ⁿ data can be obtained. It is intended to provide a mass spectrometry system that can perform MS ⁿ .

As a result of diligent research in view of the above-mentioned problems, the present inventor has found that, in a mass spectrometry system composed of a sample separation means and a mass analysis means, all ion species are obtained from MS ¹ data obtained by n-th sample measurement. calculating the elution pattern was conceived to determine the ionic species priority to mass spectrometry during sample measurements of (n + 1) th based on said ionic species elution pattern and MS ⁿ data.

  That is, the present invention provides a mass spectrometry system including a sample separation means and a mass analysis means for mass spectrometry of a sample separated by the separation means, wherein the mass analysis means is n (n is an integer of 1 or more). Means for generating mass spectral data including data obtained by mass spectrometry of an ionized sample as it is in the first mass analysis and data obtained by selectively dissociating a specific ion species and performing mass spectrometry; Based on the ion species elution pattern calculation means for calculating the elution pattern of all ion species from the data obtained by mass spectrometry of the ionized sample as it is, and the mass of the n + 1th time based on the ion species elution pattern and the mass spectrum data. An analysis priority determining means for determining the priority of the ion species to be analyzed. It is intended to provide a Temu.

  In the mass spectrometric system of the present invention, the ion species elution pattern calculating means calculates, for each ion species, the peak time of measured intensity or the time of elution when the measured ion intensity is within a predetermined range, and the analysis priority determining means Is characterized in that the calculation result gives a high priority to ion species that match the analysis timing in the (n + 1) th mass analysis.

  The mass spectrometric system of the present invention further includes reanalysis determination means for determining whether or not it is necessary to reanalyze each ion species for the result data obtained by mass analysis by the mass analysis means. And

  The mass spectrometric system of the present invention also includes a mass spectrometric condition determining means for determining the mass spectrometric condition in the n + 1th mass analysis based on the data obtained by mass spectrometry of the ionized sample as it is and the mass spectrum data. Is further provided.

As described above, according to the mass spectrometry system of the present invention, it is possible to predict the optimal timing for performing MS ⁿ by determining the analysis priority of ion species to be subjected to mass analysis at the time of the (n + 1) th sample measurement. It becomes possible. If MS ⁿ can be performed at an optimal timing based on the prediction result, high-quality MS ⁿ data can be acquired. Furthermore, in the case where a predetermined limit is imposed on the number of times of measurement of one ion species, by obtaining high-quality MS ⁿ data, the number of times of analysis of one ion species is reduced, and the number of times of one sample measurement is increased. It becomes possible to acquire MS ⁿ data of ion species.

  The best mode for carrying out the mass spectrometry system of the present invention will be described below in detail with reference to the accompanying drawings. FIGS. 1-11 is a figure which illustrates embodiment of this invention, In these figures, the part which attached | subjected the same code | symbol represents the same thing, and a basic structure and operation | movement are the same. To do.

  FIG. 1 is a diagram schematically illustrating a configuration example of a mass spectrometry system according to an embodiment of the mass spectrometry system of the present invention. In FIG. 1, the mass spectrometry system includes a sample separation device 101, an ionization unit 102, a mass analysis unit 103, a detector 104, a data processing unit 105, a display unit 106, and a control unit 107.

  The sample separation device 101 preferably includes a sample separation device such as a liquid chromatograph (LC), a gas chromatograph (GC), or an electrophoresis device. The ionization unit 102 includes, for example, an electrospray (ESI) ion source, an atmospheric pressure chemical ionization (APCI) ion source, an atmospheric pressure photoionization (APPI) ion source, a laser desorption ionization (LDI) ion source, and an inductively coupled plasma (ICP). Ion source, electron impact (EI) ion source, chemical ionization (CI) ion source, field ionization (FI) ion source, fast atom bombardment (FAB) ion source, liquid secondary ion mass spectrometry (LSIMS) ion source, ordinary Piezoelectric ionization (API) ion source, field desorption (FD) ion source, Matrix Assisted laser desorption ionization (MALDI) ion source, desorption / ionization on silicon (DIOS) ion source, etc. It is preferable to include an ion source. May be a continuous ion source or a pulsed ion source, for example, a time of flight mass spectrometer, a quadrupole mass spectrometer, Penning or Fourier transform ion cyclotron resonance (FTICR). It preferably includes a mass spectrometer, such as a mass spectrometer, a two-dimensional or linear quadrupole ion trap, a pole or a three-dimensional quadrupole ion trap.

  In the mass spectrometry system shown in FIG. 1, first, when a measurement sample is introduced into the sample separation device 101, it is separated according to its characteristics and sent to the ionization unit 102. The sample ionized by the ionization unit 102 is separated into each mass number component by the mass analysis unit 103 and detected by the detector 104. The detection signal obtained in the detector 104 is sent to the data processing unit 105 and processed, and then displayed on the display unit 106 as mass spectrum data. As illustrated, the ionization unit 102, the mass analysis unit 103, the detector 104, and the data processing unit 105 are controlled by the control unit 107.

The mass spectrometry method performed in this embodiment will be described with reference to FIG. As shown in FIG. 2, in the mass spectrometry system of the present embodiment, MS ¹ that performs mass analysis of the ionized sample itself and a specific ion species (precursor ion) are selected, and the precursor ions are further dissociated and generated. MS ⁿ which performs mass analysis by dissociating precursor ions in n-1 stages, such as mass analysis of dissociated ion species (dissociated ions), can be performed. Thus, in a mass spectrometer capable of multistage dissociation, precursor ions are dissociated in multiple stages (1 stage, 2 stages,..., N−1 stages), and the mass number of ion species generated in each stage is analyzed ( MS ² , MS ³ ,..., MS ⁿ ). Here, as the precursor ion dissociation method, for example, a dissociation method such as a collision dissociation method or an electron capture dissociation method is preferably used.

In the mass spectrometry system of this embodiment, as shown in FIG. 3A, the analysis content of the second measurement is determined using the data of the first measurement between the first measurement and the second measurement. On the other hand, in the prior art, as shown in FIG. 3B, the second measurement is performed as it is after the first measurement. Here, the first measurement, the second measurement,..., The measurement nth mean from the introduction of the sample to the end of the analysis. Hereinafter, only the first measurement and the second measurement will be described, but the same can be applied to the measurement n-1 and measurement n. In addition, the analysis content of the nth measurement is not determined based only on the data of the (n-1) th measurement. For example, to calculate the ionic species elution pattern from the measured n-1 th data, as such excludes precursor ions MS ⁿ analysis until the (n-1) th from the measured first from the selection, n-1 from the measured first The analysis content of the nth measurement may be determined using a part or all of the data of the second time.

FIG. 4 is a diagram showing in detail the mass spectrometry procedure in the mass spectrometry system of the present embodiment. First, information of each ion species is registered in an internal database (hereinafter, the internal database is referred to as an ion species list) from MS ¹ data obtained in the first measurement. Here, the ion species registered in the ion species list is preferably only a monoisotopic peak, but is not limited thereto. Moreover, it is preferable to limit the ion species to be registered depending on the mass-to-charge ratio, valence, measurement intensity, and the like of the ion species. Although the ion species list is created from the MS ¹ data in FIG. 4, the ion species of the MS ⁿ data may be added to the ion species list.

  The information of each ion species registered in the ion species list preferably includes the mass-to-charge ratio, the number of charges, the elution start time, the time when the measurement intensity reaches the peak, the measurement intensity at the peak, and the like. Or what corresponds to said information, such as time to detect ion, and the mass number of ion itself, may be included. Furthermore, regarding the ion species to be registered, it is preferable that the user can designate a range to be registered in the ion species list based on each information.

Next, MS ² executed precursor ions are registered in the ion species list as precursor ion exclusion targets at the second measurement, and other ion species are registered as precursor ion selection targets at the second measurement. Instead of registering each ion species in the ion species list as a precursor ion selection target or exclusion target, either one of them is registered, or the ion species subject to exclusion is deleted from the ion species list. May be. Further, instead of the precursor ion exclusion of the MS ² already precursor ions in the measurement second, conversely, to register the MS ² already precursor ion as a selection target, and excluded the other ionic species You can also. Alternatively, all ion species in the ion species list can be selected for the second measurement regardless of whether MS ² is executed. Moreover, it is also possible to register MS ² -completed precursor ions as ions to be preferentially analyzed.

Next, an analysis priority order is determined for the ion species selected as a precursor ion selection target, and registered in the ion species list. As described above, in the conventional mass spectrometry system, some may give MS ² (or MS ⁿ⁾ in the quality is good timing for MS ² data precursor ions, bad timing quality MS ² (or MS ⁿ ). In the mass spectrometry system of this embodiment, the analysis priority is determined so that the mass analysis is performed in the vicinity of the peak of the precursor ion expected to have good quality of the MS ² data. Specifically, as shown in FIG. 5, with reference to the peak time of each ion species registered in the ion species list, the analysis priorities are set so that MS ² is preferentially given to precursor ions that become peaks at the measurement timing. Can be determined. For example, in FIG. 6A, the analysis priority order can be determined so that each of the eluted ion species 601 to 603 can be preferentially analyzed at the timing when the measured intensity is near the peak. On the other hand, as shown in FIG. 6B, when the same ion species 601 to 603 are measured by a conventional mass spectrometry system, the measurement is performed in the order of the measurement intensity at the measurement timing, so that the precursor ions 602 and 603 are changed. Mass spectrometry could not be performed at each peak position.

The second measurement is performed based on the analysis priority order thus determined. FIG. 7 is a diagram showing details of the analysis process in the second measurement. In FIG. 7, first, MS ¹ data is acquired, the ion species in the MS ¹ data are compared with the ion species list, the ion species with the highest priority is selected in real time, and MS ² is performed. In the comparison, an error in the first measurement and the second measurement may be taken into consideration, and it may be determined that the values match within a predetermined likelihood or range. It is preferable to configure the system so that the same operation is repeated an arbitrary number of times. In FIG. 7, MS ¹ or MS ² is determined as the determination of the next analysis content after MS ^2. However, MS ³ or the like may be included in the determination target. Furthermore, when performing MS ² continuously, it is preferable that the precursor ion which performed MS ² immediately before can be excluded from a precursor ion selection object only in this case.

  Hereinafter, more specific examples of the above-described mass spectrometry system will be described.

In the present embodiment, as ion species information to be registered in the ion species list, an elution time within a predetermined measurement intensity range is registered in the ion species list in addition to the peak time of a certain ion species. As shown in FIG. 8A, when the measurement intensity at the peak time of a certain precursor ion species is equal to or greater than the predetermined measurement intensity, a range of time that is equal to or greater than the predetermined measurement intensity is registered in the ion species list in addition to the peak time. If the measured intensity is below, only the peak time is registered. At this time, in the determination of the analysis priority, it is preferable to configure the system so that the MS ² analysis is preferentially performed if a certain precursor ion species has a predetermined intensity or higher even if it is not the peak time. Further, as shown in FIG. 8B, in the case of the ion species 801 and the ion species 802 having the same peak time, MS ² is executed with the ion species 802 as the peak time, and the ion species 801 executes MS ² before and after that. The analysis priority order may be determined.

  As another example, as shown in FIG. 9, an ion species whose peak intensity of a certain ion species has a measured intensity of 901 or higher is registered with a peak time and a time within the range, and the peak intensity is less than the measured intensity of 901 For ion species having a measurement intensity of 902 or higher, the peak time may be registered, and for ion species having a measurement intensity of less than 902, the peak time may not be registered and registered as a precursor ion exclusion target in the ion species list.

In this embodiment, the system is configured such that the ion species designated in advance by the user are preferentially MS ² or excluded from precursor ions. At this time, it is preferable to determine the timing of MS ² by the determination method as shown in the first embodiment even for the ion species designated by the user.

In the present embodiment, a method for determining the necessity of reanalysis will be described. In this mass spectrometry system, there is no ion species list at the first measurement. For this reason, the first measurement MS ² does not always perform the MS ² at the timing when the quality of the MS ² data is expected to be good in the mass chromatogram of the precursor ions as shown in FIG. Therefore, in this embodiment, the necessity of reanalysis of the MS ² completed precursor ions is determined by a procedure as shown in FIG.

In FIG. 10, first, the quality of the obtained MS ² data is determined, and the necessity of reanalysis is determined. For the determination of the quality of the MS ² data, for example, the result of collation with the De Novo sequencing method or database search, or the number of ion peaks included in the MS ² data can be used. Next, it is calculated at which timing of the elution pattern of the precursor ions the MS ² analysis is performed, and the necessity of reanalysis is further determined based on the result. For the determination of the timing of the MS ² analysis, for example, it can be used whether the analysis is performed at the peak of the elution pattern. In both judgments, if it is judged that either one of them needs to be reanalyzed, it will be registered in the ion species list as a precursor ion selection target ion. Register to the ion species list. Note that the determination of reanalysis may be performed by determining either the quality of MS ² data or the timing of MS ² analysis.

In the present embodiment, a method for automatically determining the predetermined measurement intensity described in the first embodiment will be described. First, the quality of data is determined in the first measurement MS ² data. From the result and the measured intensity of the precursor ion at the time of analysis, it is determined which measured intensity is higher than the possibility of obtaining good quality data, and the measured intensity is adopted as the predetermined measured intensity. Further, as a more preferable form, not only the measurement intensity of the precursor ion at the time of analysis but also more detailed MS ² information, such as the mass and measurement intensity of the precursor ion, the total ion amount at the time of selecting the precursor ion, and further at the time of analysis Predetermined measurement intensity is estimated from apparatus conditions and the like.

In this example, the limitation on the number of MS ² times of the same precursor ion species in the second measurement will be described. As shown in FIG. 8A, when an analysis priority order is determined so that MS ² is preferentially performed if a certain precursor ion species has a predetermined intensity or higher, there is a possibility that MS ² may be performed at a plurality of MS ² analysis timings. . For this reason, as shown in FIG. 11, after the MS ^{2 and} before the selection of the next precursor ion species, for example, the number of MS2 executions of the precursor ion species, the sum of the measured intensities, the integrated value of the measured intensities, or the MS ^{2 It} is preferable to register in the ion species list as a precursor ion exclusion target according to the quality of the data.

In this embodiment, control of the apparatus conditions of the mass spectrometer will be described. Measured from information of mass spectrum data at the first measurement, for example, mass information and intensity of precursor ions, total ion amount at the time of selecting precursor ions, quality of MS ² data, and information on mass spectrometry such as apparatus conditions at the time of analysis 2 It is preferable that the mass spectrometry system is configured so that the optimal apparatus conditions at each ² o'clock MS can be predicted.

  As mentioned above, although the specific embodiment was shown and demonstrated about the mass spectrometry system of this invention, this invention is not limited to these. A person skilled in the art can make various changes and improvements to the configurations and functions of the invention according to the above-described embodiments or other embodiments without departing from the gist of the present invention.

It is a figure showing roughly the example of composition of the mass spectrometry system concerning one embodiment of the mass spectrometry system of the present invention. It is a figure explaining the mass spectrometry method by this invention. It is a figure which shows the comparison of the procedure of mass spectrometry in the mass spectrometry system of this invention, and the procedure of mass spectrometry in the conventional mass spectrometry system. It is a figure which shows the procedure of mass spectrometry in detail in the mass spectrometry system of this invention. Is a diagram showing the relationship between the timing of the quality and MS ² of MS ² data precursor ions. Is a diagram showing the relationship between the timing of the quality and MS ² of MS ² data precursor ions. It is a figure which shows the detail of the analysis process at the time of performing the 2nd measurement in the mass spectrometry system of this invention. It is a figure explaining 1st Example of the mass spectrometry system of this invention. It is a figure explaining 1st Example of the mass spectrometry system of this invention. It is a figure explaining 3rd Example of the mass spectrometry system of this invention. It is a figure explaining 5th Example of the mass spectrometry system of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 ... Sample separation device, 102 ... Ionization part, 103 ... Mass spectrometry part, 104 ... Detector, 105 ... Data processing part, 106 ... Control part, 601 ... Mass chromatogram of precursor ions, 602... 601 mass chromatogram of precursor ions different from 601... 601, mass chromatogram of precursor ions different from 602,. 802 ... a mass chromatogram of a precursor ion different from 801, 901 ... a certain measurement intensity, 902 ... a measurement intensity different from 901

Claims (4)

In a mass spectrometry system comprising a sample separation means and a mass analysis means for mass spectrometry of a sample separated by the separation means,
The mass spectrometry means includes
In a mass analysis of n (n is an integer of 1 or more), a mass including data obtained by mass analysis of an ionized sample as it is and data obtained by mass dissociation by selectively dissociating a specific ion species Means for generating spectral data;
Ion species elution pattern calculation means for calculating elution patterns of all ion species from data obtained by mass spectrometry of the ionized sample as it is,
A mass spectrometry system comprising: analysis priority determining means for determining a priority of ion species to be subjected to n + 1-th mass analysis based on the ion species elution pattern and the mass spectrum data. The ion species elution pattern calculation means calculates, for each ion species, the peak of measured intensity or the time of elution when the measured ion intensity is within a predetermined range,
2. The mass spectrometry system according to claim 1, wherein the analysis priority determination unit gives a high priority to an ion species whose calculation result matches an analysis timing in the (n + 1) th mass analysis.   The re-analysis determination means for determining whether or not it is necessary to re-analyze each ion species with respect to the result data obtained by mass analysis by the mass analysis means. Mass spectrometry system. The apparatus further comprises mass analysis condition determining means for determining mass analysis conditions in the (n + 1) th mass analysis based on data obtained by mass spectrometry of the ionized sample as it is and the mass spectrum data. The mass spectrometry system according to any one of claims 1 to 3.

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