JP7380515B2 - Sample analysis method and sample analysis system using mass spectrometry - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies: https://eventpilot. us/web/planner. php? id=ASMS20, May 6, 2020 [Publications, etc.] https://eventpilot. us/web/planner. php? id=ASMS20, June 1, 2020

The present invention relates to a method and system for analyzing a sample or analyzing compounds in a sample using mass spectrometry techniques. The present invention is particularly useful for, for example, metabolomics (metabolome analysis).

Metabolomics is a method for comprehensively analyzing the types and concentrations of various metabolites produced by life activities, and is expected to be applied to a variety of fields. For example, in the medical field, applications include the search for biomarkers for early detection of diseases and the identification of disease-causing substances, and in the food field, they are used to evaluate quality, such as comparing products between manufacturers and the origin of raw materials. It can be applied to prediction, search for functional components, etc.

As disclosed in Non-Patent Document 1, mass spectrometry and chromatography-mass spectrometry, which combines chromatography and mass spectrometry, are one of the representative analysis methods in metabolomics, and these have been widely used in recent years. It's being used. In addition, in metabolomics, when analyzing large amounts of data obtained by liquid chromatograph mass spectrometers, etc., methods such as principal component analysis (PCA) and partial least squares (PLS) are used. Statistical analysis methods are widely used, and there are various statistical analysis software for implementing them.

International Publication No. 2019/012589

Yoshida Yoshida and 2 others, “Metabolomics and Metabonomics”, Japanese Society for Analytical Chemistry, Bunseki, July 2009 issue, pp.371-378

In chromatography-mass spectrometry, multiple components contained in a sample can be separated by chromatography, but when there are many components, retention times and mass-to-charge ratios of different components (strictly speaking, italicized "m") /z", but in this specification, it is conventionally written as "mass-to-charge ratio" or "m/z") are often close, so MS/MS analysis with higher component selectivity is used. It is preferable. Furthermore, in order to improve the accuracy of statistical analysis based on mass spectral data obtained by mass spectrometry, it is preferable that the mass accuracy and mass resolution of the mass spectrum be as high as possible. For these reasons, in recent metabolomics, MS/MS analysis is possible, and quadrupole-time-of-flight mass spectrometers (hereinafter referred to as conventional mass spectrometers) have higher mass accuracy and mass resolution than triple quadrupole mass spectrometers. A mass spectrometer (referred to as a "Q-TOF type") is often used as a mass spectrometer.

As disclosed in Patent Document 1, a liquid chromatograph mass spectrometer using a Q-TOF mass spectrometer repeatedly generates MS/MS spectra in a predetermined mass-to-charge ratio range targeting a specific precursor ion. can be obtained. However, the data obtained with Q-TOF mass spectrometers contains many peaks with insufficient signal strength and poor reproducibility and quantification, and these noise peaks are combined with the original data for multivariate analysis. The accuracy of analysis will decrease if subjected to Therefore, it is necessary to perform data preprocessing to remove noise peaks as much as possible prior to multivariate analysis, and there is a problem that this data preprocessing is quite complicated and takes time and effort. In particular, in non-targeted metabolomics, which performs comprehensive analysis without limiting target components, the amount of data obtained by liquid chromatograph mass spectrometers is enormous, so the data preprocessing work described above is even more necessary. , which is a demanding task.

The present invention was made to solve the above-mentioned problems, and its main purpose is to simplify the complicated data preprocessing and to provide mass spectrometry that can achieve high comprehensiveness and reliability. An object of the present invention is to provide a sample analysis method and a sample analysis system.

One aspect of the sample analysis method using mass spectrometry according to the present invention, which has been made to solve the above problems, is as follows:
Using a mass spectrometer that is capable of MS/MS analysis and whose final stage mass separator is a time-of-flight mass separator, precursor ions are detected in accordance with predetermined standards for the target sample or a sample containing the target sample. a first measurement step of performing MS/MS analysis for;
A product ion peak is extracted from the MS/MS spectrum data obtained in the first measurement step, and includes a multiple reaction monitoring (MRM) transition that is a set of the mass-to-charge ratio of the product ion and the mass-to-charge ratio of the precursor ion. an analysis control information creation step for creating analysis control information;
a second measurement step of performing MS/MS analysis by MRM method on the target sample based on the analysis control information using a triple quadrupole mass spectrometer;
an analysis step of performing multivariate analysis on the MS/MS analysis data collected in the second measurement step to obtain information related to the target sample or information related to the compound contained in the target sample;
This includes:

Further, the sample analysis system according to the present invention, which has been made to solve the above problems, is a system for implementing the sample analysis method according to the above invention, and one aspect thereof is:
a first mass spectrometer that is capable of MS/MS analysis and whose final stage mass separator is a time-of-flight mass separator;
a second mass spectrometer that is a triple quadrupole type;
a first control unit that controls the operation of the first mass spectrometer to perform MS/MS analysis of precursor ions in accordance with predetermined standards on the target sample or a sample containing the target sample; ,
A product ion peak is extracted from the MS/MS spectrum data obtained by the first mass spectrometer under the control of the first control unit, and the mass-to-charge ratio of the product ion and the mass-to-charge ratio of the precursor ion are calculated. an analysis control information creation unit that creates analysis control information including multiple reaction monitoring (MRM) transitions that are a set of;
a second control unit that controls the operation of the second mass spectrometer to perform MS/MS analysis using the MRM method on the target sample based on the analysis control information;
Under the control of the second control unit, multivariate analysis is performed on the MS/MS analysis data collected by the second mass spectrometer, and information related to the target sample or compounds contained in the target sample is analyzed. an analysis unit that obtains information related to the
It is equipped with the following.

In the above aspect of the present invention, the first mass spectrometer is capable of MS/MS analysis and the final stage mass separator is a time-of-flight mass separator. The method or structure of the system does not matter. As the first mass spectrometer, for example, a quadrupole time-of-flight (Q-TOF) mass spectrometer, an ion trap time-of-flight mass spectrometer, or the like can be used.

Further, each of the first mass spectrometer and the second mass spectrometer may not be a standalone mass spectrometer, but may be a liquid chromatograph mass spectrometer connected to a liquid chromatograph upstream thereof. In that case, the first stage liquid chromatograph can be shared by the first mass spectrometer and the second mass spectrometer.
Moreover, the above-mentioned multivariate analysis includes statistical analysis and machine learning.

In the above aspects of the sample analysis method and sample analysis system according to the present invention, for example, instead of performing multivariate analysis on MS/MS spectrum data obtained with a Q-TOF mass spectrometer, For example, analysis control information including MRM transitions for MRM measurement is created based on data obtained by a method such as a data dependent acquisition (DDA) method. Then, using this analysis control information, data obtained by performing MRM measurement in a triple quadrupole mass spectrometer is subjected to multivariate analysis.

Although the first mass spectrometer, such as a Q-TOF mass spectrometer, has many noise elements in the data obtained, it has high detection coverage and can detect compounds contained in a sample with high mass accuracy and mass resolution. It can be detected without omission. Thereby, MRM transitions suitable for comprehensively detecting compounds can be obtained. On the other hand, in MS/MS analysis using MRM performed by the second mass spectrometer, ions in a specific MRM transition can be detected with high sensitivity and high selectivity.

According to the present invention, the highly reliable data obtained in this way can be subjected to multivariate analysis, so preprocessing of data such as noise peak removal prior to multivariate analysis is not necessary or necessary. However, it can be simplified considerably. On the other hand, since the comprehensiveness of detected compounds is high, even when a large number of compounds are included in a sample, accurate multivariate analysis results that reflect those many compounds can be obtained. That is, according to the present invention, it is possible to perform analysis with both high reliability and comprehensiveness, and the present invention is suitable for, for example, metabolomics.

FIG. 1 is a configuration diagram of main parts of an embodiment of a sample analysis system according to the present invention. FIG. 2 is an explanatory diagram of an example of a sample analysis method using the system shown in FIG. 1. The figure which shows an example of the chromatogram obtained by the actual measurement of the sample which mixed three types of red wine. The figure which shows an example of the score plot and loading plot obtained by PCA analysis based on the MRM measurement result about three types of red wine. The figure which shows the ANOVA test result of the metabolite in three types of red wine.

EMBODIMENT OF THE INVENTION Hereinafter, one embodiment of the sample analysis method and sample analysis system according to the present invention will be described with reference to the accompanying drawings.

[Configuration of sample analysis system of this embodiment]
The sample analysis system of this embodiment is a system that performs comprehensive analysis of metabolites in a sample, that is, metabolomics.
FIG. 1 is a schematic diagram of the sample analysis system of this embodiment.

This system includes a first measurement section 10 whose mass spectrometry section is a Q-TOF mass spectrometer, a second measurement section 100 whose mass spectrometry section is a triple quadrupole mass spectrometer, and a first measurement section 10 whose mass spectrometry section is a triple quadrupole mass spectrometer. a first control/processing section 20 that controls the second measuring section 100 and processes the data obtained by the first measuring section 10; and a second controlling/processing section 20 that controls the second measuring section 100 and processes the data obtained by the second measuring section 100. A control/processing section 200 is included.

The first measurement section 10 includes a liquid chromatograph section (LC section) 11 and a Q-TOF mass spectrometry section (QTOFMS section) 12. The second measurement section 100 includes a liquid chromatograph section (LC section) 101 and a triple quadrupole mass spectrometry section (TQMS section) 102. In FIG. 1, the LC section 11 of the first measuring section 10 and the LC section 101 of the second measuring section 100 are separate bodies, but they are made common and the eluate eluted from the common LC section is divided into two parts. A flow path switching unit may be provided to selectively introduce the flow path into one of the two mass spectrometers. Alternatively, the eluate may be introduced from a common LC section to one of the mass spectrometer sections by having the user reconnect the piping.

Although not shown, both of the LC units 11 and 101 have a standard configuration for an LC, such as a mobile phase container, a liquid pump, an injector, a column, and a column oven. When performing gradient elution, additional configurations are required. As disclosed in Patent Document 1, the QTOFMS unit 12 has a configuration including a quadrupole mass filter at the front stage with a collision cell in between, and a time-of-flight mass separator at the rear stage, and the ion source is electrospray ionization. It performs atmospheric pressure ionization such as. The TQMS section 102 has a configuration in which quadrupole mass filters are provided at the front and rear stages with a collision cell in between, and the ion source performs atmospheric pressure ionization such as electrospray ionization.

The first control/processing section 20 includes an analysis control section 21, a data storage section 22, an MS/MS spectrum creation section 23, a product ion selection section 24, and an MS/MS information output section 25 as functional blocks. The second control/processing section 200 also includes, as functional blocks, an MRM measurement method creation section 201, an analysis control section 202, a data storage section 203, a multivariate analysis processing section 204, an analysis result output section 205, and a component identification/structural analysis section. 206 and an analysis database 207.

Generally, the entities of the first control/processing unit 20 and the second control/processing unit 200 are personal computers or higher-performance computers called workstations, and dedicated software (computer programs) are preinstalled on the computers. The functions of each of the above functional blocks can be realized by operating the above on the computer. In that case, the first control/processing section 20 and the second control/processing section 200 may be a combination of different software and another software that inputs/outputs data etc. between these two software, or , it may be one that integrates all software into one.

[Analysis operation using sample analysis system]
An example of sample analysis using the above sample analysis system will be described with reference to FIG. 2.
Figure 2 is an example of analyzing and analyzing three samples #1, #2, and #3 of the same type, and the purpose is to search for compounds that characterize the differences between the three samples and identify the compounds. do. The type of sample is not particularly limited, and may include, for example, agricultural and fishery products, biological samples (blood, saliva, urine, cell tissue, etc.) in addition to food and drink as in the experimental examples described later.

First, the user mixes three samples #1, #2, and #3 to prepare a mixed sample for analysis. Then, using the first measuring section 10, LC/MS/MS analysis is performed on this mixed sample. Here, the compounds contained in the mixed sample are unknown. Therefore, in order to comprehensively analyze the compound, the QTOFMS section 12 repeatedly performs MS/MS analysis using the DDA method.
That is, the analysis control section 21 controls the first measuring section 10 and injects the mixed sample into the mobile phase fed by the liquid pump in the LC section 11 at a predetermined timing. The mixed sample is introduced into the column along with the flow of the mobile phase, and various compounds in the mixed sample are temporally separated while passing through the column. The eluate from the column is continuously introduced into the QTOFMS section 12, and compounds in the eluate are detected in the QTOFMS section 12 by MS/MS analysis using the DDA method.

In the DDA method, first, normal MS analysis (analysis without performing ion selection with a quadrupole mass filter and ion dissociation operation with a collision cell) over a predetermined mass-to-charge ratio range is performed. In the example of FIG. 2, a mass spectrum is obtained by MS analysis at a retention time RT1. Data resulting from this MS analysis is stored in the data storage section 22. Furthermore, the analysis control unit 21 immediately analyzes the acquired mass spectrum and searches for a peak that meets preset conditions, such as peak intensity being equal to or higher than a predetermined threshold. When a plurality of peaks are detected in one mass spectrum, all of the peaks may be selected, or the number of peaks may be limited in order of intensity, for example.

Then, a mass-to-charge ratio corresponding to the selected peak is set as a precursor ion, and then MS/MS analysis targeting that precursor ion is performed to obtain an MS/MS spectrum ( product ion spectrum). The data obtained by this MS/MS analysis is stored in the data storage unit 22 in association with the data obtained by the MS analysis. In the example of FIG. 2, the peak marked with a circle is the selected peak in the mass spectrum obtained at the retention time RT1, and the MS/MS spectrum is obtained by performing MS/MS analysis using the ion corresponding to this peak as a precursor ion. is obtained.

When a plurality of precursor ions are selected in the mass spectrum, MS/MS analysis is performed on each precursor ion to obtain an MS/MS spectrum. The analysis control unit 21 repeatedly executes a set of one MS analysis and one or more MS/MS analyzes as described above at predetermined time intervals until the end of a predetermined measurement time. The QTOFMS unit 12 is controlled as follows. If there is no peak satisfying the specified conditions in the mass spectrum, the MS/MS analysis corresponding to the MS analysis at that time is not performed.

After the measurement in the first measurement section 10 is completed (or from the middle of the measurement), the MS/MS spectrum creation section 23 in the first control/processing section 20 generates a spectrum at each retention time based on the data stored in the data storage section 22. Create an MS/MS spectrum. The product ion selection unit 24 selects one product ion peak in each MS/MS spectrum according to predetermined criteria. For example, among the peaks detected in the MS/MS spectrum, a peak with the highest signal intensity at the top of the peak may be selected. However, it may be possible to select a plurality of product ion peaks from one MS/MS spectrum. As a result, a set of at least one precursor ion and one product ion (for example, (RT1, M1, M2) in FIG. 2) is determined at the retention time at which the MS/MS analysis was performed. The MS/MS information output unit 25 generates MS/MS information including a set of retention time, precursor ion mass-to-charge ratio, and product ion mass-to-charge ratio, and sends this to the second control/processing unit 200. do.

In the second control/processing unit 200, the MRM measurement method creation unit 201 receives the above MS/MS information and creates a measurement method including an MRM transition for performing MRM measurement in the TQMS unit 102. Specifically, a detection time range is determined by adding a predetermined time margin before and after the retention time in MS/MS information, and during that detection time range, the mass-to-charge ratio of the precursor ion corresponding to the retention time is determined. A measurement method is created to perform an MRM transition including the mass-to-charge ratio of product ions. For example, the detection time range is defined as RT1-Δt=t1 to RT1+Δt=t2, in which Δt is secured as a time margin before and after holding time RT1, and for that detection time range t1 to t2, M1>M2 (however, M1 : m/z of precursor ion, M2: m/z of product ion) can be used as an MRM transition. Here, Δt can be determined as appropriate, for example, 1 minute, 0.5 minutes, etc.

Generally, the number of sets of retention time, precursor ion mass-to-charge ratio, and product ion mass-to-charge ratio listed in MS/MS information is quite large, so if the detection time range is determined as described above, multiple The detection time ranges of the two will overlap. Since the TQMS unit 102 can perform multiple MRM measurements at the same time (strictly speaking, by time division), it is not a practical problem even if a certain number of detection time ranges overlap. The more overlapping detection time ranges there are, the shorter the dwell time (data acquisition time) for one MRM transition becomes, which is disadvantageous in terms of sensitivity. Therefore, the detection time ranges may be adjusted so as to appropriately limit the number of overlapping detection time ranges. Alternatively, the number of MRM measurements to be performed may be limited so that the number of overlapping detection time ranges is equal to or less than a specified number.

The measurement method at this time was created based on the results of measurements on a mixture of three types of samples #1, #2, and #3, so the three types of samples #1, #2, and #3 were mixed. This is a measurement method that can comprehensively detect various compounds included in #2 and #3.

The analysis control unit 202 controls the operation of the TQMS unit 102 according to the measurement method created by the MRM measurement method creation unit 201, and performs LC/MS/MS analysis on each of the three types of samples #1, #2, and #3. The second measurement unit 100 is controlled to perform the following. That is, the second measuring section 100 sequentially measures three types of samples one by one without mixing them. The separation conditions of the LC section 101 at this time (type of mobile phase, flow rate (flow rate), gradient elution conditions, column temperature, etc.) are the same as those for the measurement of the mixed sample in the first measurement section 10. This is to ensure that the elution times of the same compounds are approximately the same.

LC/MS/MS analysis of one sample #1 in the second measuring section 100 is performed as follows. The analysis control unit 202 controls the second measurement unit 100 and injects sample #1 into the mobile phase fed by the liquid pump in the LC unit 101 at a predetermined timing. Sample #1 is introduced into the column along with the flow of the mobile phase, and various compounds in sample #1 are temporally separated while passing through the column. The eluate from the column is continuously introduced into the TQMS section 102, and compounds in the eluate are detected in the TQMS section 102 by MRM measurement (MS/MS analysis).

The TQMS unit 102 repeatedly acquires the ion intensity of a specific MRM transition during a predetermined detection time range according to the measurement method. Therefore, as shown in FIG. 2, a chromatogram (extracted ion chromatogram) showing a temporal change in ion intensity with an MRM transition of M1>M2 is obtained, for example, during the time period t1 to t2. Data resulting from this MS/MS analysis is stored in the data storage unit 203. For each MRM transition, chromatogram data for a predetermined detection time range as described above can be obtained. As mentioned above, even if the separation conditions in the LC section 11 of the first measurement section 10 and the separation conditions in the LC section 101 of the second measurement section 100 are made exactly the same, there will be some difference in the elution time of the same compound. It is inevitable that it will occur. On the other hand, here, the detection time range is set with sufficient time margin for the actual retention time (elution time) during measurement in the first measuring section 10, so that the sample can correspond to the retention time. If a compound is included, product ions derived from that compound can be reliably detected.

As mentioned above, the measurement method is a measurement method that can comprehensively detect various compounds contained in the three types of samples #1, #2, and #3. Chromatogram data corresponding to the compounds contained in #1, #2, and #3, respectively, can be collected without omission and with high sensitivity. On the other hand, since MRM measurement allows highly sensitive analysis, it is possible to collect highly reliable data with good reproducibility and quantitative performance.

Once data corresponding to compounds that are likely to be included in each sample have been obtained, the multivariate analysis processing unit 204 reads the data as appropriate from the data storage unit 203 and performs, for example, principal component analysis or partial analysis. Analysis is performed using an appropriate method such as the least squares method or hierarchical cluster analysis. Furthermore, analysis may be performed using machine learning including deep learning. The analysis method to be used may be appropriately selected depending on the purpose of the analysis. For example, when principal component analysis is used, it is possible to create score plots and loading plots as analysis results, and based on the analysis results, multiple samples can be classified into multiple groups, and at the same time they can be used to contribute to the classification or to separate each group. Characterizing elements, ie, compounds, can be estimated.

The analysis result output unit 205 outputs the analysis results based on the multivariate analysis or machine learning to a display unit (not shown). The user who sees the analysis results specifies the compound of interest and instructs the start of execution of the identification process by performing a predetermined operation on the operation unit (not shown). Then, the component identification/structure analysis unit 206 traces the original MS/MS information from the MRM transition information corresponding to the designated compound, and calculates the MS/MS spectrum when the MS/MS information was obtained. 1 obtained from the control/processing unit 20. Then, using the analysis database 207 containing MS/MS spectra of various compounds, a search is made for compounds whose spectral patterns are similar to the actually measured MS/MS spectra, and the designated compound is identified. , or obtain structural information of the compound. The analysis result output unit 205 also outputs these identification results and structural analysis results to the display unit.

As described above, the sample analysis system of this embodiment can search for compounds that characterize the differences between multiple types of samples, identify the compound, and obtain information on the chemical structure of the compound. .

In the above explanation, a mixed sample prepared by mixing a plurality of samples was measured by the first measuring section 10, but the samples were individually measured by the first measuring section 10 without being mixed, and each MS/MS MRM transitions may be determined based on the analysis results.

[Experiment example]
Experimental examples conducted according to the above procedure will be described next.
In this experiment, "LCMS-9030" manufactured by Shimadzu Corporation (LC section 11 is "Nexera X3 system") was used as the first measuring section 10, and "LCMS-8060NX" manufactured by Shimadzu Corporation was used as the second measuring section 100. Three types of commercially available red wines (bottled wine, paper-packed wine, and non-alcoholic wine) were also prepared as samples. In order to comprehensively analyze the hydrophilic metabolites contained in each wine, a mixture of these three types of red wine was used as a mixed sample to be analyzed in the first measuring section 10. As sample pretreatment, a mixed sample was prepared by centrifuging the mixed red wine at a rotation speed of 12,000 rpm for 5 minutes, collecting the supernatant, and diluting it 10 times with ultrapure water.

Comprehensive analysis of metabolites was performed on this mixed sample using the first measuring section 10 under the LC analysis conditions shown in Table 1 and the MS analysis conditions shown in Table 2.

FIG. 3 is a diagram showing an example of a chromatogram obtained by actual measurement of the mixed sample. FIG. 3(A) is the positive ion mode, and FIG. 3(B) is the negative ion mode. FIG. 3(A) is the result of adding up the time changes of the observed ion signal intensities in the negative ion mode. MS/MS spectrum data could be obtained with sufficiently high intensities for 1443 peaks in the positive ion mode and 759 peaks in the negative ion mode as precursor ions.

A measurement method for LC/MS analysis in the second measurement section 100 was created by performing the processing explained in FIG. 2 on the MS/MS spectrum data obtained by the first measurement section 10 as described above. . However, here, a measurement method including 1443 MRM transitions was created using MS/MS spectrum data obtained in positive ion mode. Then, each of the three types of red wine was used as a sample, and LC/MS analysis of each sample was performed by the second measuring section 100 using the above measurement method. As sample pretreatment, samples were prepared by centrifuging each red wine at a rotation speed of 12,000 rpm for 5 minutes, collecting the supernatant, and diluting it 100 times with ultrapure water.

After the measurement is completed, principal component analysis is performed on the MS/MS analysis data obtained by the second measuring section 100 using "Traverse-MS ^TM ", which is multivariate analysis software compatible with MRM data manufactured by Rafis Co., Ltd. and one-way analysis (One-way ANOVA). FIG. 4 is a score plot (A) and a loading plot (B) obtained by principal component analysis. Each plot on the score plot corresponds to a sample, and it can be seen from FIG. 4(A) that the three types of red wine are clearly classified. Further, each plot on the loading plot corresponds to an MRM transition, that is, a compound, and from FIG. 4(B), the MRM transition that contributes to the classification of the three types of red wine is revealed.

Figure 5 shows the peaks of ornithine, glutathione, and 4-aminobutyric acid, which are metabolites that were determined to have a significant difference in abundance (p value < 0.05) and were identified in each of the three types of red wines mentioned above. This is a comparative example of height. These results reveal the metabolites, or markers, that characterize each of the three types of red wine.
The above experimental examples show that by using the sample analysis system described above, it is possible to accurately extract compounds such as metabolites that are useful for classifying multiple samples, and it is possible to identify the compounds. I understand.

The sample analysis system of the above embodiment can be modified as appropriate. For example, in the embodiment described above, both the first measuring section 10 and the second measuring section 100 are liquid chromatograph mass spectrometers, but it is not essential to provide a compound separation means such as a liquid chromatograph upstream of the mass spectrometer. do not have.

In addition, the mass spectrometer in the first measuring section 10 may be a Q-TOF mass spectrometer, if it is a mass spectrometer that is capable of MS/MS analysis and capable of mass separation with high mass accuracy and mass resolution. Not limited to devices. For example, it is also possible to use an ion trap time-of-flight mass spectrometer that performs ion selection and ion dissociation operations using an ion trap instead of the front-stage quadrupole mass filter and collision cell. On the other hand, in the second measuring section 100, since it is necessary to perform MRM measurement with high sensitivity, a triple quadrupole mass spectrometer is used as the mass spectrometer.

Furthermore, in the above embodiment, the first measurement unit 10 executes MS/MS analysis using the DDA method in order to comprehensively detect ions, but the data independent acquisition (DIA) method MS/MS analysis may also be performed.

In MS/MS analysis using the DIA method, for example, MS/MS analysis is performed by shifting windows with a certain mass-to-charge ratio width and combining multiple types of ions contained in each window as precursor ions. Repeat the process of acquiring a spectrum. In this case, although the comprehensiveness of detection can be improved compared to the DDA method, on the other hand, one MS/MS spectrum is a mixture of MS/MS spectra derived from multiple types of ions. However, in general, by using a well-known deconvolution technique, it is possible to separate overlapping MS/MS spectra and estimate the mass-to-charge ratio of precursor ions corresponding to the MS/MS spectra. . Therefore, by adding such data processing, it is also possible to utilize MS/MS analysis using the DIA method in the first measuring section 10.

Furthermore, it is clear that the above-described embodiments and the various modified examples described above are examples of the present invention, and that any modifications, changes, or additions made as appropriate within the scope of the spirit of the present invention will still fall within the scope of the claims of the present application. It is.

[Various aspects]
It will be apparent to those skilled in the art that the exemplary embodiments described above are specific examples of the following aspects.

(Section 1) One aspect of the sample analysis method using mass spectrometry according to the present invention is
Using a mass spectrometer that is capable of MS/MS analysis and whose final stage mass separator is a time-of-flight mass separator, precursor ions are detected in accordance with predetermined standards for the target sample or a sample containing the target sample. a first measurement step of performing MS/MS analysis for;
A product ion peak is extracted from the MS/MS spectrum data obtained in the first measurement step, and includes a multiple reaction monitoring (MRM) transition that is a set of the mass-to-charge ratio of the product ion and the mass-to-charge ratio of the precursor ion. an analysis control information creation step for creating analysis control information;
a second measurement step of performing MS/MS analysis by MRM method on the target sample based on the analysis control information using a triple quadrupole mass spectrometer;
an analysis step of performing multivariate analysis on the MS/MS analysis data collected in the second measurement step to obtain information related to the target sample or information related to the compound contained in the target sample;
This includes:

(Section 10) Furthermore, one aspect of the sample analysis system according to the present invention is
a first mass spectrometer that is capable of MS/MS analysis and whose final stage mass separator is a time-of-flight mass separator;
a second mass spectrometer that is a triple quadrupole type;
a first control unit that controls the operation of the first mass spectrometer to perform MS/MS analysis of precursor ions in accordance with predetermined standards on the target sample or a sample containing the target sample; ,
A product ion peak is extracted from the MS/MS spectrum data obtained by the first mass spectrometer under the control of the first control unit, and the mass-to-charge ratio of the product ion and the mass-to-charge ratio of the precursor ion are calculated. an analysis control information creation unit that creates analysis control information including multiple reaction monitoring (MRM) transitions that are a set of;
a second control unit that controls the operation of the second mass spectrometer to perform MS/MS analysis using the MRM method on the target sample based on the analysis control information;
Under the control of the second control unit, multivariate analysis is performed on the MS/MS analysis data collected by the second mass spectrometer, and information related to the target sample or compounds contained in the target sample is analyzed. an analysis unit that obtains information related to the
It is equipped with the following.

According to the sample analysis method using mass spectrometry described in Section 1 or the sample analysis system described in Section 10, highly reliable data obtained by MRM measurement with a triple quadrupole mass spectrometer Since the data can be subjected to multivariate analysis, data preprocessing such as noise data removal before multivariate analysis becomes unnecessary, or even if it is necessary, it can be simplified. This simplifies data processing as a whole and improves the efficiency of analysis work. In addition, the creation of control information for MS/MS analysis using the MRM method uses the MS/MS analysis results from the first measurement step, which has high detection coverage, so the number of compounds contained in the sample can be reduced. Even in the case of a large number of compounds, accurate multivariate analysis results that reflect the large number of compounds can be obtained. That is, it is possible to perform analysis with both high reliability and comprehensiveness, and is suitable for, for example, metabolomics.

(Section 2) In the sample analysis method according to Item 1, a quadrupole-time-of-flight mass spectrometer may be used in the first measurement step.

According to the sample analysis method described in item 2, a highly sensitive MS/MS spectrum can be obtained in the first measurement step. Thereby, for example, an MRM transition can be created even for a compound contained in a small amount in a sample, and the compound can be subjected to MS/MS analysis in the second measurement step.

(Section 3) In the sample analysis method according to Item 1 or 2, in both the first measurement step and the second measurement step, a liquid chromatograph mass spectrometer is used to separate components using a liquid chromatograph. MS/MS analysis can be performed on the sample.

According to the sample analysis method described in Section 3, even if there are a large number of compounds contained in the sample, many of these compounds can be separated by the liquid chromatograph in the first stage. It becomes difficult for a plurality of compounds to be introduced into the mass spectrometer at the same time in the step, and even if only one of the plurality of compounds introduced at the same time can be subjected to MS/MS analysis, leakage in MS/MS analysis can be reduced.

(Section 4) In the sample analysis method according to any one of Items 1 to 3, in the first measurement step, MS/MS analysis using a data dependent acquisition (DDA) method is performed in a mass spectrometer. may be carried out.

In the first measurement step, MS/MS analysis can be performed using a data independent acquisition (DIA) method in a mass spectrometer; Data processing is required to separate the MS/MS spectra derived from each ion. On the other hand, if MS/MS analysis is performed using the DDA method, product ions can be extracted immediately from the MS/MS spectrum obtained by MS/MS analysis, although it is disadvantageous in terms of comprehensiveness compared to the DIA method. This simplifies data processing because there is no need to calculate the mass-to-charge ratio of precursor ions. Furthermore, since the mass-to-charge ratio of precursor ions and product ions can be determined accurately, highly reliable MRM transitions can be obtained.

(Section 5) In the sample analysis method according to any one of Items 1 to 4, in the analysis control information creation step, a product ion peak showing the maximum signal intensity in the MS/MS spectrum is extracted. , the MRM transition including the product ion can be determined.

According to the sample analysis method described in Section 5, highly reliable MRM transitions can be obtained through simple data processing.

(Section 6) In the sample analysis method according to any one of Items 1 to 5, in the analysis step, analysis processing is performed on MS/MS analysis data obtained for each of a plurality of target samples. By doing so, it is possible to extract compounds that are characteristic for classifying the plurality of target samples.

According to the sample analysis method described in Section 6, compounds that are important markers that contribute to sample classification, such as disease-related biomarkers and markers for identifying the production area of agricultural and marine products, can be efficiently detected. can be found.

(Section 7) In the sample analysis method described in Section 6, the compound is identified or the structure of the compound is identified based on an MS/MS spectrum using an ion derived from the characteristic compound as a precursor ion. The method may further include a compound analysis step of analyzing.

In the sample analysis method described in Section 7, for example, a technique such as database search using a database containing MS/MS spectra of a large number of known compounds can be used. According to the sample analysis method described in Section 7, it is possible, for example, to identify the compound itself that serves as a marker that contributes to the classification of a sample, or to clarify the chemical structure of the compound.

(Section 8) In the sample analysis method according to Item 6 or 7, the characteristic compound may be a metabolite.

According to the sample analysis method described in Section 8, this method can be used for metabolomics.

(Section 9) In the sample analysis method according to any one of Items 1 to 8, the multivariate analysis may be statistical analysis or machine learning.

According to the sample analysis method described in Section 9, it is possible to classify a plurality of samples with high accuracy and to identify compounds contributing to the classification with high accuracy.

10...First measurement section 11...LC section 12...QTOFMS section 100...Second measurement section 101...LC section 102...TQMS section 20...First control/processing section 21...Analysis control section 22...Data storage section 23...MS/ MS spectrum creation section 24...Product ion selection section 25...MS/MS information output section 200...Second control/processing section 201...MRM measurement method creation section 202...Analysis control section 203...Data storage section 204...Multivariate analysis processing section 205...Analysis result output section 206...Component identification/structure analysis section 207...Analysis database

Claims (9)

Using a mass spectrometer that is capable of MS/MS analysis and whose final stage mass separator is a time-of-flight mass separator, precursor ions are detected in accordance with predetermined standards for the target sample or a sample containing the target sample. a first measurement step of performing MS/MS analysis for;
A product ion peak is extracted from the MS/MS spectrum data obtained in the first measurement step, and includes a multiple reaction monitoring (MRM) transition that is a set of the mass-to-charge ratio of the product ion and the mass-to-charge ratio of the precursor ion. an analysis control information creation step for creating analysis control information;
a second measurement step of performing MS/MS analysis by MRM method on the target sample based on the analysis control information using a triple quadrupole mass spectrometer;
an analysis step of performing multivariate analysis on the MS/MS analysis data collected in the second measurement step to obtain information related to the target sample or information related to the compound contained in the target sample;
In the analysis step, compounds that are characteristic for classifying the plurality of target samples are extracted by performing analysis processing on the MS/MS analysis data obtained for each of the plurality of target samples. , a sample analysis method using mass spectrometry. The sample analysis method using mass spectrometry according to claim 1, wherein a quadrupole-time-of-flight mass spectrometer is used in the first measurement step. 3. The method according to claim 1, wherein in both the first measurement step and the second measurement step, a liquid chromatograph mass spectrometer is used to perform MS/MS analysis on a sample whose components have been separated by a liquid chromatograph. Sample analysis method using mass spectrometry. The sample analysis method using mass spectrometry according to any one of claims 1 to 3, wherein in the first measurement step, MS/MS analysis is performed using a data dependent acquisition (DDA) method in a mass spectrometer. The mass spectrometer according to any one of claims 1 to 4, wherein in the analysis control information creation step, a product ion peak exhibiting the maximum signal intensity in the MS/MS spectrum is extracted, and an MRM transition including the product ion is determined. Sample analysis method using analysis. The method according to claims 1 to 5, further comprising a compound analysis step of identifying the compound or analyzing the structure of the compound based on an MS/MS spectrum using an ion derived from the characteristic compound as a precursor ion. A sample analysis method using mass spectrometry according to any one of the items . The sample analysis method using mass spectrometry according to any one of claims 1 to 6 , wherein the characteristic compound is a metabolite. The sample analysis method using mass spectrometry according to any one of claims 1 to 7 , wherein the multivariate analysis is statistical analysis or machine learning. a first mass spectrometer that is capable of MS/MS analysis and whose final stage mass separator is a time-of-flight mass separator;
a second mass spectrometer that is a triple quadrupole type;
a first control unit that controls the operation of the first mass spectrometer to perform MS/MS analysis of precursor ions in accordance with predetermined standards on the target sample or a sample containing the target sample; ,
A product ion peak is extracted from the MS/MS spectrum data obtained by the first mass spectrometer under the control of the first control unit, and the mass-to-charge ratio of the product ion and the mass-to-charge ratio of the precursor ion are calculated. an analysis control information creation unit that creates analysis control information including multiple reaction monitoring (MRM) transitions that are a set of;
a second control unit that controls the operation of the second mass spectrometer to perform MS/MS analysis using the MRM method on the target sample based on the analysis control information;
Under the control of the second control unit, multivariate analysis is performed on the MS/MS analysis data collected by the second mass spectrometer, and information related to the target sample or compounds contained in the target sample is analyzed. an analysis unit that obtains information related to the
The analysis unit extracts compounds that are characteristic for classifying the plurality of target samples by performing analysis processing on MS/MS analysis data obtained for each of the plurality of target samples. Sample analysis system.

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