JP5039330B2 - Mass spectrometry system - Google Patents

Description

  The present invention relates to an analysis system for a mass spectrometry spectrum using a mass spectrometer, and is optimal within the real time of measurement in order to accurately and efficiently identify the chemical structure of a biopolymer such as a polypeptide or a sugar. The present invention relates to a system for automatically optimizing various mass analysis conditions.

In general mass spectrometry, after a sample to be measured is ionized, various generated ions are sent to a mass spectrometer, and the mass-to-charge ratio m / z, which is the ratio of the mass number m and the valence z of the ions. Every time, the ionic strength is measured. The resulting mass spectrum consists of measured ion intensity peaks (ion peaks) for each mass to charge ratio m / z value. The mass analysis of the sample ionized as described above is called MS ¹ . In a tandem mass spectrometer capable of multistage dissociation, an ion peak having a specific mass-to-charge ratio m / z value is selected from the ion peaks detected by MS ¹ (the selected ion species is the parent). Further, the ions are dissociated and decomposed by collision with gas molecules or the like, and the generated dissociated ion species is subjected to mass spectrometry, and a mass spectrum is obtained in the same manner. Here, performing n-stage dissociation of the parent ion and mass analyzing the dissociated ion species is referred to as MS ^{n + 1} . In this way, in the tandem mass spectrometer, the parent ions are dissociated into multiple stages (1 stage, 2 stages,..., N stages), and the mass number of the ion species generated at each stage is analyzed (MS ² , MS ³ , ..., MS ^{n + 1} ).

  Here, with conventional tandem mass spectrometers, the analysis conditions are adjusted before analysis, and mass spectrometric measurement is performed with the conditions fixed (optimized values and conditional expressions) optimized. It had been.

  In the following Patent Document 1, the application time of the CID voltage is adjusted and set. In this invention, too, the time for applying the CID voltage is increased in proportion to the mass-to-charge ratio of ions to be dissociated. It is only adjusted by setting in advance.

Japanese Patent Laid-Open No. 2002-313276

In particular, when liquid chromatography (LC) is installed at the front stage of the mass spectrometer, the sample is eluted according to the ease of adsorption to the adsorbent packed in the LC column, After being dispersed, it flows into the mass spectrometer and is subjected to mass analysis. In this case, the analysis takes 2 to 3 hours, and during the analysis, the amount of the substance in the sample flowing into the mass spectrometer varies in various ways. When the mass spectrometry measurement is performed under the condition that is adjusted before the analysis and is optimized to the conditions (determined values and the predetermined conditional expressions) that are adjusted before the analysis as in the above prior art, the optimum The analysis conditions are likely to vary during an analysis time of 2-3 hours. In such a case, the mass spectrum acquired during a period in which the optimum condition has deviated contains only poor information, and sufficient structural information cannot be obtained, making the original structural analysis difficult. For example, when the sample is a protein in a living body and the information of sufficient dissociated ions cannot be obtained in the MS ² data, it is difficult to accurately estimate the original peptide structure or protein structure. Also in the said patent document 1, when the optimal conditions of CID voltage application time change, CID voltage application time cannot be corrected corresponding to this, and the quality of mass spectrum information cannot be improved.

To solve this problem, in each stage of MS ^n, effectively utilizing the information contained in the MS ⁿ spectrum analysis conditions to determine whether an optimum state, according to the result, the analytical conditions It is necessary to perform optimization with high efficiency and high accuracy within the real time of measurement.

In the present invention, in a mass spectrometer capable of tandem analysis, the following means (1) to (3) are mainly employed to dissociate target ions n-1 times to solve the above-mentioned problems, and mass spectrometry is performed. In this system, the mass spectrum (MS ⁿ ) obtained in this way is analyzed at high speed within the actual measurement time, and the next analysis content is determined.
(1) An analysis condition adjustment database for optimizing analysis conditions is provided in accordance with the acquired mass spectrum information.
(2) Information is analyzed at high speed for each ion peak in the mass spectrum (MS ⁿ ).
(3) When it is determined from the results of (2) that there is a change / adjustment of the analysis conditions, the analysis conditions are quickly changed / adjusted based on the database of (1).

  According to the present invention, analysis conditions can be optimized within the analysis time of the structure of a substance to be measured, and analysis data with a small amount of information can be obtained with little failure.

  Embodiments of the present invention will be described below with reference to the drawings.

First, the first embodiment will be described. FIG. 1 is a flowchart for automatically optimizing analysis conditions in the mass spectrometry system according to the first embodiment of the present invention. The mass spectrometry data is data measured by the mass spectrometry system 19 shown in FIG. In the mass spectrometry system 19, a sample to be analyzed is preprocessed by a pretreatment system 11 such as liquid chromatography. For example, in the case of a protein that is the original sample, it is decomposed into a polypeptide size by a digestive enzyme in the pretreatment system 11, and separated and fractionated by gas chromatography (GC) or liquid chromatography (LC). Is done. Hereinafter, an example in which LC is employed as the separation / fractionation system in the pretreatment system 11 will be described. After sample separation / fractionation, the sample is ionized by the ionization unit 12 and separated by the mass analysis unit 13 according to the mass-to-charge ratio m / z of ions. Here, m is the ion mass, and z is the charge valence of the ion. The separated ions are detected by the ion detection unit 14, and the data processing unit 15 organizes and processes the data, and the mass analysis data 1 as the analysis result is displayed on the display unit 16. The control unit 17 controls the entire series of mass analysis processes--sample ionization, transport and incidence of the sample ion beam to the mass analysis unit 13, mass separation process, ion detection, and data processing.

The mass analysis method includes a method in which a sample is ionized and analyzed as it is (MS analysis method), and a tandem mass in which mass analysis is performed on dissociated ions generated by selecting a specific sample ion (parent ion) and dissociating it. There is an analysis method. In tandem mass spectrometry, an ion having a specific mass-to-charge ratio (precursor ion) is selected from dissociated ions, the precursor ion is further dissociated, and mass analysis of the generated dissociated ions is performed. As described above, there is a function (MS ⁿ ) for performing dissociation and mass spectrometry in multiple stages. In other words, after measuring the mass spectrometry distribution of the material in the sample as the source as mass spectrum data (MS ¹ ), select a parent ion having a certain m / z value, dissociate it, and After measuring the mass spectrometry data (MS ² ), the dissociation / mass is performed such as further dissociating the selected precursor ions in the MS ² data and measuring the mass analysis data (MS ³ ) of the obtained dissociated ions. The analysis is performed in multiple stages (MS ⁿ (n ≧ 3)). For each dissociation step, information on the molecular structure of the precursor ion that is in the state before dissociation is obtained, which is very effective for estimating the structure of the precursor ion. As the structure information of these precursors becomes more detailed, the estimation accuracy when estimating the parent ion structure, which is the original structure, is improved.

In the present embodiment, as a precursor ion dissociation method, first, a case where a collision dissociation method in which a precursor gas collides with a buffer gas such as helium is used will be described. Since neutral gas such as helium gas is required for collision dissociation, a collision cell 13A for collision dissociation is provided separately from the mass analyzer 13 as shown in FIG. In some cases, the mass analyzer 13 may be filled with a neutral gas, and the mass analyzer 13 may be subjected to collisional dissociation. In that case, the collision cell 13A becomes unnecessary. Further, as a dissociation means, electron capture dissociation in which target ions are dissociated by irradiating low energy electrons and allowing the parent ions to capture a large amount of low energy electrons may be employed.

  FIG. 3 shows a tuning method for a tandem mass spectrometer according to a conventional method. Before carrying out the sample analysis, by performing an analytical measurement on a specific substance, the result is optimized tuning of the analysis conditions of the apparatus. After that, during the mass analysis of the sample to be analyzed, the analysis conditions are set based on a constant or fixed conditional expression. In such a device tuning method, for example, when the concentration of the sample changes in time series, the optimal analysis conditions may fluctuate during sample analysis. In such a case, the mass obtained as a result is obtained. Analytical data often does not contain enough information. Therefore, according to the conventional method, since the situation of the changing sample is not dealt with flexibly, sufficient structural data (information) cannot be obtained, and the possibility of failure of analysis increases. The problem is particularly serious when the amount of sample to be reanalyzed cannot be secured, such as when the sample is a very small amount of a substance in the living body.

Therefore, in the present invention, optimal analysis conditions are stored for the type and amount of ideal information of the MS spectrum and the MS ⁿ spectrum (n ≧ 2) for the substance expected to be contained in the sample. The analysis condition adjustment database 10 stores information on what kind of analysis condition adjustment is necessary when the ideal type and amount of information of the MS spectrum and MS ⁿ spectrum (n ≧ 2) are not satisfied. By analyzing the mass spectrum data just acquired in real time, it is determined whether the information satisfies the ideal type and amount of information. Even on the way, the analysis conditions of the apparatus can be optimized.

As shown in FIG. 4, in the MS ² mass spectrum data obtained for a certain precursor ion, the analysis is performed when the dissociated ion data is smaller than a predetermined value input in advance by the user input unit 18 of FIG. Based on the condition adjustment database 10, various voltages in the apparatus can be optimized and changed as analysis conditions, and the same precursor ions can be analyzed again by MS ² to obtain data that can provide a sufficient amount of information. 1 shows the real-time processing 9 of the analysis conditions for the mass spectrum of MS ⁿ (n ≧ 2) in FIG. 1. Similarly, the real-time processing 5 of the analysis conditions for the MS mass spectrum is as follows. Is possible. In this case, MS mass spectrum information includes mass range, resolution, sensitivity, and the like. If the information is less than the specified value, what kind of analysis condition adjustment is necessary is stored in the analysis condition adjustment database 10 and the analysis condition is adjusted and changed in real time based on this information. Then, MS analysis is performed again.

Here, the analysis of the MS, MS ⁿ mass spectrum is preferably performed during the preparation time until the next analysis or the dead time, and it is desirable that the analysis processing speed be fast so as not to affect the timing of the next analysis. For example, within one of 100msec, 10msec, 5msec, 1msec,
Analyze MS and MS ⁿ mass spectra and change analysis conditions. Further, here, the device condition corresponds to each voltage of each electrode in the device. For example, in the example of FIG. 4, to increase the number of dissociated ions, the CID voltage is increased. The amount of dissociated ions and how much the CID voltage should be increased is stored in the analysis condition adjustment database 10, and based on this, the CID voltage set value is changed in real time.

In addition, as other device voltages, other voltages in the device include a voltage for determining a mass range, a voltage to be applied to exclude ions other than dissociating ion species, and a plurality of voltages may be optimized and changed at the same time. . In addition to the voltage amplitude value, the voltage frequency may be optimized and changed. FIG. 5 shows a conceptual diagram of the contents stored in the analysis condition adjustment database 10. With respect to the MS ⁿ mass spectrum, information to be analyzed and evaluated (vertical axis) and analysis condition parameters (horizontal axis) correlated with the information are stored in a table. Here, as shown in FIG. 5, the arrival percentage of the information value obtained by the spectrum analysis is obtained with respect to the specified value previously designated by the user in the user input unit 18, and the percentage is less than 100%. Each adjustment parameter may be changed according to the size of the arrival%. In this case, excessive or excessive adjustment can be avoided.

  In the present embodiment, as shown in FIG. 4, the same mass spectrum is acquired before and after the analysis condition is changed. As post-processing, if the mass spectrum before changing the analysis conditions is replaced with the mass spectrum after changing the analysis conditions or merged with the mass spectrum after changing the analysis conditions, post-processing analysis as usual Can be implemented. For this purpose, it is desirable that the contents re-measured after changing the analysis conditions are discharged to a log or the like so that the relationship between the mass spectra before and after the analysis change can be identified later.

The analysis condition adjustment database 10 stores the results of analyzing the correlation between the acquired data and the analysis conditions as needed. When a new optimum condition is obtained, the analysis condition adjustment database 10 stores the result in the analysis condition adjustment database 10. The contents may be expanded at any time. Or it is good also as a system automatically stored in the database 10 of analysis condition adjustment.

As described above, according to the present embodiment, the MS ⁿ spectrum is analyzed at high speed within the actual measurement time, it is determined whether or not the optimum value of the analysis condition has changed, and it is determined that the analysis condition needs to be optimized. In this case, the analysis conditions are automatically adjusted in the real time of analysis, so that analysis data can be acquired with little information failure and a large amount of information.

Next, a second embodiment of the present invention will be described with reference to FIG. Here, as the analysis conditions, various set times within the analysis measurement time are changed and adjusted. An example is shown in FIG. If the total ion count (TIC) value does not satisfy the specified value for the MS ⁿ (n ≧ 1) spectrum, the ion accumulation time or the allocation time for MS ⁿ analysis is increased and changed, and measurement is performed again. . As a result, data satisfying the TIC required by the user can always be acquired. Here, as various set times within the analytical measurement time, there are other times for the MS ² spectrum, such as time for selecting and isolating precursor ions, time for applying a CID voltage, and the like. By adjusting the above, it can be expected to improve the dissociation efficiency of the precursor ions.

  Therefore, according to the present embodiment, it is possible to acquire a mass spectrum having the amount of information required by the user by changing various setting times.

  Next, a third embodiment of the present invention will be described with reference to FIG. As shown in FIG. 7, here, as a method of changing the analysis condition, the analysis condition is scanned or varied within a specific range and measured. This embodiment is considered to be particularly effective when the optimal value of the analysis condition is not stored in the analysis condition adjustment database 10 or when it is difficult to adjust the pinpoint analysis condition.

  Next, a fourth embodiment of the present invention will be described with reference to FIGS. In the present embodiment, as shown in FIG. 8, the mass spectrometry system 19 is connected to a server 21 that centrally manages the analysis condition adjustment database 10 and a network 20 such as the Internet or an intranet, and communicates with the server 21. Obtain optimal information on analysis conditions. In this case, as shown in FIG. 9, a plurality of mass analysis systems 19 are connected to the server 21, and information is transmitted to the plurality of mass analysis systems 19 when adjustment of analysis conditions is necessary. According to the present embodiment, since a plurality of mass spectrometry systems 19 can be adjusted under the conditions stored in the same analysis condition adjustment database 10, data of almost the same quality can be acquired between apparatuses. Further, since it is not necessary to have a plurality of analysis condition adjustment databases 10, it is easy to manage the analysis condition adjustment database 10.

Next, a fifth embodiment of the present invention will be described with reference to FIGS. In this embodiment, as shown in FIG. 10, in order to adjust the analysis conditions, an existing substance is mixed into the sample as an adjustment marker and analyzed. If an adjustment marker is detected, an MS or MS ⁿ spectrum corresponding to the adjustment marker is acquired and analyzed in real time to determine whether the analysis conditions are appropriate when the adjustment marker is detected. Here, the analysis condition adjustment database 10 in FIG. 5 may store analysis condition adjustment data for MS data for the adjustment marker to be employed. For example, when liquid chromatography is connected as the pretreatment system 11 of the mass spectrometer, the substance in the sample is transferred to the mass analyzer 13 depending on the time (holding time) that passes through the liquid chromatography (LC) column. Inflow time zone is different. As shown in FIG. 11, in this case, if the adjustment marker is a substance that is detected with a long LC holding time, it is desirable that the analysis condition can be optimized and adjusted over a long period. Alternatively, as shown in FIG. 12, a plurality of adjustment markers having different m / z and holding times may be employed. In this case, an effect of avoiding adjustment bias due to the m / z value of ions can also be expected.

Next, a sixth embodiment of the present invention will be described with reference to FIG. In the present embodiment, when the sample is a protein-derived sample such as a biological sample, the number of decoded amino acids is used as information derived by analyzing the MS ² spectrum in real time. Therefore, in the case of the present embodiment, in the analysis condition adjustment database 10, as analysis information of the MS ² spectrum, what analysis conditions are changed and adjusted when the prescribed value is not satisfied with respect to the number of amino acids is stored. Is done. In particular, when analyzing proteins by mass spectrometry, the number of amino acids decoded in post-processing analysis is linked to peptide identification and protein identification. Therefore, we can identify more peptides and proteins.

  Next, a seventh embodiment of the present invention will be described with reference to FIG. In the present embodiment, an input interface in the user input unit 18 as shown in FIG. 14 is shown with respect to user input necessary in the present invention such as a prescribed value for determining whether or not to perform real-time adjustment of analysis conditions. Here, in order not to complicate the user input, there may be a default value and a recommendation mode selection. Such user input by the user input unit 18 enables optimization of analysis conditions desired by the user.

Next, an eighth embodiment of the present invention will be described with reference to FIGS. 15, 16, and 17. FIG. In this embodiment, as shown in FIG. 15, the system is provided with an internal database 22 in which information about ion species analyzed once in MS ² is stored, and MS ⁿ (n ≧ 1) spectrum data just acquired is real-time. In the system for automatically determining the next mass analysis content according to the result of the comparison and comparison with the data stored in the internal database 22, as shown in FIG. By giving priority to the desired analysis content, it is possible to determine the optimal analysis content based on the priority in real time. Further, in this case, by having the user prioritize, only one internal database 22 is provided, and it can be handled in an exclusion or inclusion manner. In particular, the example shown in FIG. 16C relates to the case where the sample is a protein-derived sample and, as shown in FIG. 17, an isotope-labeled sample and an unlabeled sample are mixed. In the case as shown in FIG. 17, peaks are detected in pairs, but when the intensity ratio is different between pairs or when a single peak is detected, it is peculiar to either sample (labeled sample or unlabeled sample). Since these peptides are usually peptides, these are usually subjected to MS ² analysis for identification analysis. In such a case, there is a need to perform quantitative analysis (MS analysis) for quantitative evaluation of quality. Therefore, in this embodiment, the identification / quantitative analysis mode can be selected. In that case, the MS ² analysis is minimized,
The analysis content is automatically determined to perform the MS analysis. Therefore, FIG. 16C shows an example in which the user prioritizes MS ² analysis. According to this embodiment, the analysis content desired by the user is executed. In addition, when MS ^{2 is used for} an unanalyzed peak, the internal database 22 is an exclusion database, and stored components that have already been analyzed are excluded from the MS ² target. Or if the strength is significantly different from the intensity stored in the internal database, the internal database 22
It is treated as an inclusion database. Therefore, according to the present embodiment, the internal database 22 can be handled in an exclusion or inclusion manner, and the analysis content desired by the user is automatically determined and implemented, so that a highly efficient analysis can be expected.

It is the schematic of the flow of the automatic determination process of the mass spectrometry flow by 1st Example of this invention. It is the schematic of the whole mass spectrometry system which measures mass spectrometry data by the 1st example of the present invention. It is the schematic of the analysis condition tuning method by the conventional method. 1 is a schematic diagram of an automatic tuning method during analysis according to a first embodiment of the present invention. FIG. It is the schematic of the analysis condition adjustment database storage content of this invention. FIG. 5 is a schematic diagram of an automatic tuning method during analysis according to a second embodiment of the present invention. FIG. 5 is a schematic diagram of an automatic tuning method during analysis according to a third embodiment of the present invention. It is the schematic of the whole mass spectrometry system which measures mass spectrometry data by the 4th example of the present invention. It is the schematic of the whole mass spectrometry system which measures mass spectrometry data by the 4th example of the present invention. It is the schematic of the handling with respect to the sample of the adjustment marker of this invention. It is an example of the point of the adjustment marker in LC holding time-detection m / z value mapping of this invention. It is an example of the point of the adjustment marker in LC holding time-detection m / z value mapping of this invention. It is the schematic of the analysis content adjustment database storage content by 6th Example of this invention. It is an example of the user input screen by 7th Example of this invention. It is the schematic of the whole mass spectrometry system which measures mass spectrometry data by the 8th example of the present invention. It is an example of the user input screen by 8th Example of this invention. It is the schematic regarding the flow of the analysis of the sample which mixed the isotope labeling and the non-labeling sample.

Explanation of symbols

1 ... Mass spectrometry data (MS ^n), 2 ... MS ⁿ data analysis, 3 ... evaluation of MS ⁿ data include information, 4 ... determination of MS ⁿ data include information amount, for 5 ... MS mass spectrum, real-time processing of analytical conditions , 6 ... Analysis condition adjustment table or database collation, 7 ... Analysis condition change / adjustment, 8 ... Precursor ion selection, 9 ... Real time processing, 10 ... Database, 11 ... Pretreatment system, 12 ... Ionization unit, 13 ... Mass spectrometry , 14 ... ion detector, 15 ... data processing unit, 16 ... display unit, 17 ... control unit, 18 ... user input unit, 19 ... mass spectrometry system, 20 ... network such as the Internet and Intranet, 21 ... server, 22 ... internal database.

Claims (9)

Means for ionizing the substance to be measured;
Means for selectively dissociating ionic species having a specific mass-to-charge ratio from ions generated by the ionizing means;
Means for measuring a mass spectrum of the dissociated ion species represented by an intensity peak with respect to a mass-to-charge ratio;
A mass spectrometry system using a tandem mass spectrometer that repeats the selection, dissociation, and measurement of the ion species in a plurality of stages,
Based on the n-th stage mass spectrum result measured by selecting and dissociating n-1 times (n is an integer of 2 or more) ionic species,
Within the analysis time of the structure of the substance to be measured,
For the same ionic species, it has rows measurement of mass spectrum of n-stage analysis conditions and again selected and dissociated by changing in real time of the tandem mass spectrometer,
The analysis condition of the tandem mass spectrometer based on the mass spectrum result is changed within 100 msec, 10 msec, 5 msec, or 1 msec,
The analysis condition of the tandem mass spectrometer is a mass spectrometry system that is a value of various voltages in the mass spectrometer or a set value of various voltage application times in the mass spectrometer. Changes in the analysis conditions of the tandem mass spectrometer based on the mass spectrum results are as follows:
The mass spectrometry system according to claim 1, wherein the mass spectrometry system is performed at the same time as measurement of continuous mass spectra.   The change in the analysis conditions of the tandem mass spectrometer based on the mass spectrum result is performed in a preparation time from the measurement of one mass spectrum to the start of the next mass spectrum measurement. Mass spectrometry system. Changes in the analysis conditions of the tandem mass spectrometer based on the mass spectrum results are as follows:
The mass spectrometry system according to claim 1, wherein the mass spectrometry system is performed at a dead time in continuous mass spectrum measurement. What are the values of various voltages in the mass spectrometer?
The mass spectrometric system according to claim 1 , wherein the mass spectrometric system is at least one of a voltage value for determining a range (mass range) of ion species for measuring a mass spectrum and a voltage value for excluding ions other than dissociating ion species. What is the set value of various voltage application times in the mass spectrometer?
The mass spectrometry system according to claim 1 , wherein the mass spectrometric system is at least one of a set value of a voltage application time for excluding ions other than dissociating ion species and a set value of a voltage application time for storing dissociated ion species. The substance to be measured is a protein-derived substance,
The mass spectrum result has amino acid sequence information;
The mass spectrometry system according to claim 1, wherein the sequence information of the amino acid is analyzed with respect to a second-stage mass spectrum measured by selecting and dissociating ion species once.   2. The analysis condition of the tandem mass spectrometer is changed only when the mass spectrum result is compared with a predetermined value set in advance, and the mass spectrum result is less than the specified value. Mass spectrometry system. The mass spectrometry system according to claim 8 , wherein information obtained by analyzing the mass spectrum result and analysis conditions changed based on the information are recorded in a log.

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