Mass analysis data processing method and mass analysis data processing device

Abstract

To provide a mass analysis data processing method and a mass analysis data processing device in which analysis work of mass spectral data that is acquired by performing mass analysis of a sample of an analysis object can be efficiently performed.SOLUTION: A mass analysis data processing method comprises: preparing MS / MS spectral data that is acquired by MS / MS scan measurement using a plurality of different precursor ions for each of one or more chemical compounds included in a sample (steps 1 to 3); and creating integrated MS / MS spectral data in which a plurality of MS / MS spectral data that are acquired by the MS / MS scan measurement using precursor ions derived from the chemical compound for each of the one to more chemical compounds of the plurality of MS / MS spectral data is integrated into one (step 6).SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for processing data obtained by mass spectrometry of compounds contained in a sample. [Background technology]

[0002] Development of nucleic acid drugs that use nucleic acids as medicines is underway for the purpose of treating genetic diseases, etc. Nucleic acid drugs are molecules with a mass of several thousand to tens of thousands that are produced by chemical synthesis, and have a chain shape in which several tens of nucleotides such as adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T), which constitute DNA and RNA, are linked via linkers (e.g., Non-Patent Document 1).

[0003] Chemically synthesized nucleic acids and peptides may contain, in addition to the desired nucleic acid or peptide (target compound), various contaminating compounds derived from the raw materials used in the synthesis, impurities generated during the synthesis, etc. Therefore, after chemically synthesizing nucleic acids or peptides, the presence or absence of not only the target compound but also contaminating compounds is confirmed by chromatographic mass spectrometry of the sample, and if contaminating compounds are present, they are identified.

[0004] Since a wide variety of compounds may be contained in nucleic acid or peptide samples, when performing chromatography mass spectrometry on such samples, mass spectrometry data is often obtained by DDA (Data Dependent Acquisition), which comprehensively measures compounds separated in a chromatographic column. In DDA, MS scan measurements are performed on ions generated from compounds in the sample, and when an ion with an intensity exceeding a predetermined threshold is detected, MS / MS scan measurements are performed with the ion as a precursor ion, and this process is repeated. The MS scan measurement is a measurement in which the mass-to-charge ratio of the ion to be measured among the ions generated from the compounds in the sample is scanned and detected within a predetermined mass-to-charge ratio range. The MS / MS scan measurement is a measurement in which the mass-to-charge ratio of the product ion to be measured, generated by dissociating the precursor ion, is scanned and detected within a predetermined mass-to-charge ratio range.

[0005] In DDA, MS / MS scan measurements are usually performed repeatedly during the time period when the target compound and impurity compounds are introduced into the mass spectrometer from the chromatograph column, and one MS / MS spectrum data is obtained from each measurement. After the measurement is completed, the data from multiple MS / MS spectra obtained for precursor ions with the same mass-to-charge ratio are integrated into one MS / MS spectrum data by averaging the intensities of mass peaks with the same mass-to-charge ratio.

[0006] For example, in the case of nucleic acids, dozens of nucleotides such as adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T) are linked together in a chain, and it is known that precursor ions dissociate at the binding sites (linkers) of these nucleotides, so the mass-to-charge ratio of the fragment ions generated from the precursor ions in MS / MS scan measurements can be theoretically calculated. In addition, the mass-to-charge ratio of the fragment ions of impurity compounds can also be theoretically calculated from information such as the reagents used in chemical synthesis. By comparing the theoretically calculated mass-to-charge ratio value with the mass-to-charge ratio of the mass peaks contained in the integrated MS / MS spectrum data, the fragment ions corresponding to each mass peak are identified, and the compounds are identified. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] "Confirmation of synthesis of nucleic acid drugs - Rapid and simple sequence confirmation using MALDI-TOF MS", [online], August 2017, Shimadzu Corporation, [Retrieved November 14, 2022], Internet<URL:https: / / www.an.shimadzu.co.jp / aplnotes / maldi / an_b067.pdf> Summary of the Invention [Problem to be solved by the invention]

[0008] When nucleic acids or peptides are ionized, ions of various charges are generated. When ions (precursor ions) with different charges are dissociated, even if the molecular structure is the same, fragments are generated at different positions where the precursor ions dissociate, and different patterns of MS / MS spectrum data are obtained. Therefore, the accuracy of compound identification can be improved by analyzing the MS / MS spectrum data obtained for each precursor ion of various charges.

[0009] On the other hand, nucleic acid and peptide samples contain hundreds to thousands of compounds (target compounds and impurity compounds), so creating multiple MS / MS spectrum data for each of these numerous compounds results in an enormous number of data, and analyzing them requires a lot of time and effort.

[0010] Here, we have used the example of obtaining mass spectrometry data for samples containing nucleic acids and peptides by DDA using a chromatography mass spectrometer. However, similar problems can also occur when obtaining mass spectrometry data using only a mass spectrometer, or when obtaining mass spectrometry data using a measurement method other than DDA, such as performing MS / MS scan measurements of precursor ions with a mass-to-charge ratio predetermined for each compound.

[0011] The problem to be solved by the present invention is to provide a mass spectrometry data processing method and a mass spectrometry data processing device that can efficiently analyze mass spectrum data acquired by mass spectrometry of a sample to be analyzed. [Means for solving the problem]

[0012] The mass spectrometry data processing method according to the present invention, which has been made to solve the above problems, comprises: preparing MS / MS spectrum data acquired by MS / MS scan measurement using a plurality of different precursor ions for each of one or a plurality of compounds contained in the sample; Among the plurality of MS / MS spectrum data, a plurality of MS / MS spectrum data acquired by MS / MS scan measurement using a precursor ion derived from each of the one to a plurality of compounds is integrated into one to generate integrated MS / MS spectrum data. It is something.

[0013] In order to solve the above problems, the present invention provides a mass spectrometry data processing apparatus, comprising: A storage unit in which MS / MS spectrum data acquired by MS / MS scan measurements using a plurality of different precursor ions for each of one or a plurality of compounds contained in a sample is stored; an integrated MS / MS spectrum data creation unit that creates integrated MS / MS spectrum data by integrating, into one, a plurality of MS / MS spectrum data acquired by MS / MS scan measurements using precursor ions derived from the compounds for each of the one to multiple compounds among the plurality of MS / MS spectrum data; Equipped with. Effect of the Invention

[0014] In the mass spectrometry data processing method and mass spectrometry data processing device according to the present invention, for each of one or more compounds contained in a sample, MS / MS spectrum data acquired by MS / MS scan measurement using multiple different precursor ions derived from the compound are prepared. This may be done by actually performing a measurement to acquire data, or by storing data acquired in advance in a storage unit and reading it out from there. The multiple different precursor ions include, for example, ions with the same mass number but different valences, adduct ions, isotope ions, dehydrated ions, fragment ions, etc. Different patterns of MS / MS spectrum data are obtained by MS / MS scan measurement using each of the precursor ions.

[0015] In the present invention, the MS / MS spectrum data thus obtained is integrated with data obtained by MS / MS scan measurements using precursor ions derived from the same compound to generate one integrated MS / MS spectrum data. The MS / MS spectrum data may be integrated, for example, by averaging or summing the intensities of mass peaks having the same mass-to-charge ratio. With the mass spectrometry data processing method and mass spectrometry data processing device according to the present invention, it is only necessary to check the same number of integrated MS / MS spectrum data as the number of compounds contained in the sample, and therefore analysis work can be performed more efficiently than in the past. [Brief description of the drawings]

[0016] [Figure 1] 1 is a diagram showing the configuration of a main portion of a liquid chromatograph mass spectrometer including an embodiment of a mass spectrometry data processing device according to the present invention; [Diagram 2] 3 is a flowchart according to one embodiment of a mass spectrometry data processing method according to the present invention. [Diagram 3] FIG. 2 is a diagram illustrating a flow of a DDA in the present embodiment. [Figure 4] FIG. 4 is a diagram for explaining classification of precursor ions in this embodiment. [Diagram 5] FIG. 2 is a diagram for explaining an MS scan measurement and an MS / MS scan measurement performed in this embodiment. [Figure 6] FIG. 2 is a diagram for explaining MS spectrum and MS / MS spectrum data acquired in this embodiment. [Figure 7] An example of an integrated MS / MS spectrum created by averaging the intensities of mass peaks. [Figure 8] An example of creating an integrated MS / MS spectrum by extracting the maximum intensity of mass peaks. [Figure 9] 13 shows an example of an analysis result display screen in the present embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of a mass spectrometry data processing method and a mass spectrometry data processing device according to the present invention will be described with reference to the drawings.

[0018] 1 is a diagram showing the configuration of the main parts of a liquid chromatograph mass spectrometer 1 including a mass analysis data processing device of this embodiment. The liquid chromatograph mass spectrometer 1 of this embodiment includes a liquid chromatograph 10, a mass analyzer 20, and a control / processing unit 40 that controls the operations of these components. The control / processing unit 40 corresponds to one embodiment of the mass analysis data processing device according to the present invention.

[0019] The liquid chromatograph 10 comprises a mobile phase container 11 in which a mobile phase is stored, a pump 12 that draws in the mobile phase and delivers it at a constant flow rate, an injector 13 that injects a liquid sample into the mobile phase, and a column 14 that separates compounds contained in the liquid sample. When multiple liquid samples are to be analyzed continuously, an autosampler (not shown) is further provided, and multiple liquid samples set in the autosampler are introduced in sequence from the injector 13.

[0020] The mass spectrometer 20 includes an ionization chamber 21 and a vacuum chamber. The vacuum chamber is evacuated by a vacuum pump (not shown). Inside the vacuum chamber, from the ionization chamber 21 side, there are provided a first intermediate vacuum chamber 22, a second intermediate vacuum chamber 23, a third intermediate vacuum chamber 24, and an analysis chamber 25, in that order, and the system has a multi-stage differential pumping system in which the degree of vacuum increases in this order.

[0021] An electrospray ionization (ESI) probe 211 that applies an electric charge to a sample solution and sprays it is installed in the ionization chamber 21. The ionization chamber 21 and a first intermediate vacuum chamber 22 in the rear stage are communicated with each other through a capillary tube 212.

[0022] An ion guide 221 composed of multiple rod electrodes is disposed in the first intermediate vacuum chamber 22. The ion guide 221 converges the flight path of the ions along the ion optical axis C. The first intermediate vacuum chamber 22 and the second intermediate vacuum chamber 23 are separated by a skimmer 222 having a small hole at the top.

[0023] An ion guide 231 composed of a plurality of rod electrodes is disposed in the second intermediate vacuum chamber 23. Like the ion guide 221, the ion guide 231 also converges the flight path of the ions along the ion optical axis C. The second intermediate vacuum chamber 23 and the third intermediate vacuum chamber 24 are separated by a partition wall having small holes formed therein.

[0024] A quadrupole mass filter 241 having a pre-rod electrode and a main rod electrode, which separate ions according to their mass-to-charge ratio, a collision cell 243 having a multipole ion guide 244 therein, and an ion guide 245 consisting of a plurality of ring electrodes are arranged in the third intermediate vacuum chamber 24. A collision-induced dissociation (CID) gas such as argon or nitrogen is supplied into the collision cell 243 from a gas source (not shown) as required.

[0025] In the analysis chamber 25, an ion guide 251 composed of multiple ring electrodes, an orthogonal acceleration electrode 252 composed of a pusher electrode 2521 and a puller electrode 2522, a second acceleration electrode 253, a reflectron 254, a flight tube 256, a back plate 257, and an ion detector 255 are arranged. The pusher electrode 2521 is a plate-shaped electrode, and the puller electrode 2522 is a plate-shaped electrode as a whole with an ion passage portion formed in the center. The second acceleration electrode 253 has multiple ring-shaped electrodes and a slit located on the rear side. The reflectron 254 is composed of a first reflectron 2541 and a second reflectron 2542, both of which are multiple ring-shaped electrodes. The flight tube 256 is a cylindrical electrode, and the back plate 257 is a plate-shaped electrode.

[0026] The mass spectrometer 20 can perform MS scan measurement and MS / MS scan measurement. In the MS scan measurement, the quadrupole mass filter 241 does not select ions (does not function as a mass filter), and ions generated from the sample are allowed to enter the analysis chamber 25 without being selected.

[0027] The ions that have entered the analysis chamber 25 are focused along the ion optical axis C by the ion guide 251, and then enter the space between the push electrode 2521 and the pull electrode 2522 (orthogonal acceleration space).

[0028] A pulse voltage is applied to the orthogonal acceleration electrode 252 at a constant period. The application of this pulse voltage forms an electric field in the orthogonal acceleration space that deflects the flight direction of the ions in a direction perpendicular to the ion optical axis C (direction from the pusher electrode 2521 to the puller electrode 2522). The ions whose flight direction is deflected by the orthogonal acceleration electrode 252 are given a certain amount of kinetic energy by an acceleration electric field formed by application of a voltage to the second acceleration electrode 253, and then fly along a return flight path defined by the reflectron 254, flight tube 256, and back plate 257 and enter the ion detector 255. At this time, since ions with smaller mass-to-charge ratios fly faster, the ions are separated according to their respective mass-to-charge ratios while flying along the flight path, and are incident on the ion detector 255 in order of the ions with the smallest mass-to-charge ratios and detected.

[0029] In the MS / MS scan measurement, the quadrupole mass filter 241 also selects ions (functions as a mass filter). The quadrupole mass filter 241 passes only ions set as precursor ions. In addition, CID gas is supplied to the inside of the collision cell 243, and the precursor ions are accelerated and introduced into the collision cell 243, where they collide with the CID gas to promote dissociation of the precursor ions. Product ions generated by dissociation of the precursor ions are converged by the ion guide 245 to fly along the ion optical axis C and enter the analysis chamber 25. In the analysis chamber 25, the product ions are made to fly back and forth in the flight space, as in the case of the MS scan measurement, and the ions separated according to their mass-to-charge ratio during this time are sequentially detected by the ion detector 255.

[0030] The control / processing unit 40 has a storage unit 41. The storage unit 41 stores a compound database describing the measurement conditions (retention time, MS / MS scan measurement conditions, etc.) of various compounds, and information on various conditions (intensity threshold, mass scanning range, etc.) when performing DDA.

[0031] The control / processing unit 40 includes, as functional blocks, a measurement condition setting unit 42, a measurement execution unit 43, a precursor ion classification unit 44, an integrated MS / MS spectrum data creation unit 45, a compound identification unit 46, and an analysis result display unit 47. The actual entity of the control / processing unit 40 is a personal computer, and the above-mentioned units function by executing a dedicated program preinstalled in the computer. Furthermore, an input unit 5 consisting of a mouse, a keyboard, etc., and a display unit 6 consisting of a liquid crystal display, etc. are connected to the control / processing unit 40.

[0032] Next, a flow of analysis using the mass spectrometry data processing device of this embodiment will be described. Fig. 2 is a flowchart of one embodiment of the mass spectrometry data processing method according to the present invention. Here, an example will be described in which MS / MS spectrum data is obtained by actually measuring a sample, but MS / MS spectrum data obtained by a previous measurement may be stored in a storage unit and read out to prepare MS / MS spectrum data.

[0033] When the user issues an instruction to start analysis of a sample, the measurement condition setting unit 42 displays target MS / MS and DDA as measurement methods on the screen of the display unit 6, and allows the user to select one of them (step 1).

[0034] When the user selects a measurement method, the measurement condition setting unit 42 has the user set the measurement conditions required to execute the selected measurement method (step 2). Specifically, when the user selects target MS / MS, a list of compounds is read from the compound database stored in the storage unit 41, and the user is prompted to select a compound to be measured. When the user selects a compound, the measurement conditions of the compound (retention time, mass-to-charge ratio of precursor ion, mass scan range during MS / MS scan measurement, etc.) are read and the measurement conditions are set. When the user selects DDA, the measurement conditions such as the mass scan range during MS scan measurement, the measurement intensity value (threshold) serving as a criterion for selecting precursor ions to be measured in MS / MS scan, and the mass scan range during MS / MS scan measurement are read from the storage unit 41 and the measurement conditions are set. These measurement conditions may be changed by the user as necessary.

[0035] After the measurement method and measurement conditions have been determined, when the user sets a sample and issues an instruction to start measurement, the measurement execution unit 43 executes mass analysis of the sample based on the measurement conditions set by the measurement condition setting unit 42 (step 3). In the case of target MS / MS, MS / MS scan measurements of the compound selected as the target are repeatedly performed at the retention time of the compound. If there are compounds with overlapping retention times, MS / MS scan measurements of those compounds are repeatedly performed in order during the overlapping time period.

[0036] In the case of DDA, first, an MS scan measurement is performed in a mass-to-charge ratio range defined as a measurement condition to obtain MS spectrum data. Then, it is determined whether or not a mass peak having an intensity equal to or greater than a threshold value defined as a measurement condition exists in the MS spectrum data. If a mass peak having an intensity equal to or greater than the threshold value does not exist, an MS scan measurement is performed again. On the other hand, if a mass peak having an intensity equal to or greater than the threshold value exists, an ion having a mass-to-charge ratio of the mass peak is set as a precursor ion, and MS / MS scan measurements are performed in sequence in a mass-to-charge ratio range defined as a measurement condition to obtain MS / MS spectrum data. If a single MS spectrum data contains a plurality of mass peaks having an intensity exceeding the threshold value, an MS / MS scan measurement is performed in which ions having mass-to-charge ratios of the plurality of mass peaks are set as precursor ions, and MS / MS spectrum data is obtained for each precursor ion. When performing an MS / MS scan measurement, an ion having a mass-to-charge ratio within a range (mass window) having a certain width (for example, ±1 to several Da) centered on the mass-to-charge ratio of the precursor ion is selected as a precursor ion in the quadrupole mass filter 241. This allows isotope ions to be simultaneously selected as precursor ions and subjected to MS / MS scan measurement.

[0037] FIG. 3 shows a schematic flow of DDA. The upper part of FIG. 3 is a total ion current chromatogram (TICC) showing the time change of the total ion intensity in the MS scan measurement. The TICC represents the time change of the amount of the compound in the sample introduced into the mass spectrometer 20. In time period t0, substantially no compound in the sample is introduced into the mass spectrometer 20, and in time periods t1, t2, and t3, the compound in the sample is introduced into the mass spectrometer 20. FIG. 3 shows an example of an MS spectrum (middle) and an MS / MS spectrum (lower). In each of time periods t1, t2, and t3, the type and amount of the compound subjected to mass analysis change with the passage of time, and the position and intensity of the mass peak appearing in the MS / MS spectrum change accordingly.

[0038] After the measurement is started, no mass peaks exceeding the threshold value appear until the compounds in the sample are introduced into the mass spectrometer 20 (time period t0), so MS scan measurements are repeatedly performed. After that, when the compounds in the sample start to be introduced into the mass spectrometer 20 (time period t1), mass peaks with intensities exceeding the threshold value appear in the MS spectrum data. In the example shown in FIG. 3 (the MS spectrum shown on the right side of the middle row), the intensities of seven mass peaks (1 to 7) exceed the threshold value, so MS / MS scan measurements are performed once for each of the ions with mass-to-charge ratios corresponding to these seven mass peaks (1 to 7) as precursor ions. The MS / MS scan measurements of the seven precursor ions are performed, for example, in descending order of intensity, or in descending order of mass-to-charge ratio. In the example of FIG. 3, the MS / MS scan measurements are performed in descending order of intensity, that is, in the order of precursor ions 1 to 7, and MS / MS spectrum data (lower row of FIG. 3) is obtained for each of them. Pre1 to Pre7 mean that they are MS / MS spectra for precursor ions 1 to 7, respectively. The dashed peaks in the MS / MS spectrum indicate the positions (mass-to-charge ratios) of the precursor ions. After the MS / MS scan measurements are completed for each of the seven precursor ions, an MS scan measurement is performed again, and the same process as above is repeated.

[0039] Mass spectrum (MS spectrum, MS / MS spectrum) data acquired by the above measurements (target MS / MS or DDA) are sequentially stored in the storage unit 41 together with the time at which the data was acquired. The MS / MS spectrum data is also associated with information on the mass-to-charge ratio of the precursor ion and stored in the storage unit 41.

[0040] After the measurement is completed, if the MS / MS spectrum data was acquired by DDA (YES in step 4), the precursor ion classification unit 44 reads out data in which multiple mass peaks with intensities exceeding the threshold appear from the MS spectra (i.e., MS spectra in which mass peaks with intensities exceeding the threshold appear) acquired during each time period (t1, t2, t3) and which were the basis for performing the MS / MS scan measurement.

[0041] The precursor ion classification unit 44 determines whether the ions corresponding to the mass peaks included in the read MS spectrum data are derived from the same compound based on the mass-to-charge ratio of the mass peaks. Specifically, the precursor ions are classified into [M+nH] n+ (n is an integer of 1 or more), pairs of mass peaks with a common mass-to-charge ratio that has a different n are identified and classified as originating from the same compound. In other words, ions with the same mass number but different valences are classified as originating from the same compound.

[0042] An example is shown in FIG. 4. The MS spectrum shown in FIG. 4 is the same as the MS spectrum shown in the middle of FIG. 3. Seven mass peaks having intensities exceeding the threshold appear in this MS spectrum, of which five are divalent to hexavalent ions that satisfy the above-mentioned requirements. The precursor ion classification unit 44 then classifies these five ions as originating from the same compound (step 5). In the example of FIG. 4, multiple ions are classified for only one compound, but during a time period when multiple compounds are simultaneously subjected to mass spectrometry, multiple mass peaks appearing in one MS spectrum may be classified into multiple compounds, respectively.

[0043] If the MS / MS spectrum data is acquired by target MS / MS (NO in step 4), the MS / MS spectrum is acquired based on measurement conditions that are predetermined for each compound, and it is known which compound the MS / MS spectrum belongs to. Therefore, the above-mentioned process by the precursor ion classification unit 44 is not performed.

[0044] After completing the above-mentioned processing for the MS spectra acquired in each time period, the integrated MS / MS spectrum data creation unit 45 integrates the data of the multiple MS / MS spectra derived from the same compound into one (step 6). In this processing, for the mass peaks in each MS / MS spectrum, the one with the maximum intensity is extracted from among the mass peaks with a common mass-to-charge ratio, and integrated into one MS / MS spectrum data.

[0045] Here, as an example, a case will be described in which the measurement method is DDA, and MS scan measurement and MS / MS scan measurement are performed in time period t1 at the timing shown in Fig. 5. In Fig. 5, MS means MS scan measurement, and Pre1 to Pre7 mean MS / MS scan measurements for precursor ions 1 to 7, respectively. For ease of understanding, Fig. 5 describes a case in which one target compound and two impurity compounds are measured in time period t1, which is a unimodal peak, but in reality, multiple target compounds and many impurity compounds may be subjected to mass analysis at the same time (for example, time period t2).

[0046] As shown in FIG. 5, when a mass peak exceeding the threshold is detected in the MS scan measurement, an MS / MS scan measurement is performed with the ion corresponding to the mass peak as a precursor ion. At this time, even if there is no change in the compounds (target compound and impurity compounds) subjected to mass analysis, the intensity of the ion detected exceeding the threshold may change. In the example shown in FIG. 5, the intensities of ions 1 to 7 exceed the threshold in the first (1st cycle) and fourth (4th cycle) MS scan measurements, and MS / MS scan measurements are performed for each of them, while in the second (2nd cycle) MS scan measurement, only the intensities of ions 2 to 4, 5, and 7 exceed the threshold, and in the third (3rd cycle) MS scan measurement, only ions 2 to 6 exceed the threshold, and only MS / MS scan measurements with these as precursor ions are performed.

[0047] In addition, the amount of a compound subjected to mass spectrometry changes over time, so even if an MS / MS scan measurement is performed on the same precursor ion, the intensity of the mass peak in the MS / MS spectrum will differ depending on the timing.

[0048] FIG. 6 shows an example of an MS / MS spectrum obtained by the MS / MS scan measurement shown in FIG. 5. However, FIG. 6 shows only the MS / MS spectra of precursor ions 1, 3, 4, 6, and 7, which are classified as originating from the same compound and are the targets for creating integrated MS / MS spectrum data. In this example, four cycles of measurements including MS scan measurement and MS / MS scan measurement are performed, and the second and third cycles are performed near the peak of TICC, that is, during the time period when the amount of the compound subjected to mass analysis is large. On the other hand, the first and fourth cycles are near the start and end of the peak, so the amount of the compound subjected to mass analysis is small. Therefore, the mass peak intensity of the MS / MS spectrum obtained by the MS / MS scan measurement performed in the first and fourth cycles is smaller than the mass peak intensity of the MS / MS spectrum obtained by the MS / MS scan measurement performed in the second and third cycles.

[0049] One method for creating an integrated MS / MS spectrum for one compound is to average the mass peak intensities in multiple MS / MS spectra. When this method is used in the example shown in Figures 5 and 6, the mass peak intensities for each mass-to-charge ratio are divided by 16 (the total number of MS / MS spectra). In this case, for precursor ion 1, MS / MS scan measurements are performed only in the first and fourth cycles. Therefore, the mass peak intensities that appear only in the MS / MS spectrum of precursor ion 1 are smaller than the mass peak intensities of the MS / MS spectra acquired in all cycles, such as precursor ions 3 and 4. The same is true for precursor ions 6 and 7, for which MS / MS scan measurements were performed three times each.

[0050] In addition, the MS / MS scan measurement of precursor ion 1 is performed only near the start and end of the TICC peak, the amount of compound subjected to mass analysis is small, and the intensity of the mass peak in the MS / MS spectrum acquired in each MS / MS scan measurement is also small. Therefore, when the mass peak intensities are averaged, the intensity of the mass peak that appears only in the MS / MS spectrum acquired in the MS / MS scan measurement of precursor ion 1 becomes extremely small. On the other hand, the intensity of the mass peaks that appear regardless of the type of precursor ion is relatively large.

[0051] Although the present invention also includes an embodiment in which integrated MS / MS spectrum data is created by averaging the intensities of mass peaks, in a more preferred embodiment of the present invention, integrated MS / MS spectrum data is created by extracting the maximum value of mass peak intensities at each mass-to-charge ratio. Note that, in order to absorb some mass errors that may occur in mass analysis, some degree of likelihood is provided when creating integrated MS / MS spectrum data, and for example, mass peaks with a mass-to-charge ratio difference of 1 Da or less are regarded as mass peaks with substantially the same mass-to-charge ratio.

[0052] FIG. 7 shows an example of an integrated MS / MS spectrum created by averaging the intensities of mass peaks of each mass-to-charge ratio. FIG. 8 shows an example of an integrated MS / MS spectrum created by extracting the maximum value of the intensities of mass peaks of each mass-to-charge ratio. In FIG. 7, the two mass peaks marked with arrows appear only in the MS / MS spectrum of precursor ion 1, so that the intensity in the integrated MS / MS spectrum becomes very small when the intensity values ​​are averaged. The mass spectrum shown in the schematic diagram here has only a few mass peaks, but an integrated MS / MS spectrum obtained from an actual measurement has many mass peaks, so that only mass peaks exceeding a certain threshold are often analyzed to distinguish them from noise peaks. In this case, the mass peaks marked with arrows in FIG. 7 are judged to be noise peaks and are not subjected to analysis, and it becomes impossible to obtain fragment information that should be obtained from the mass peaks. On the other hand, in the integrated MS / MS spectrum of FIG. 8, the two mass peaks marked with arrows that appear only in the MS / MS spectrum of precursor ion 1 are mass peaks with sufficient intensity.

[0053] When the integrated MS / MS spectrum data is created for each compound, the compound identification unit 46 extracts mass peaks present in the integrated MS / MS spectrum of each compound (step 7). In addition, the compound identification unit 46 theoretically calculates the mass-to-charge ratios of fragment ions that may be generated from compounds assumed to be contained in the sample, and compares them with the mass-to-charge ratios of the extracted mass peaks to identify fragments corresponding to each mass peak in the MS / MS spectrum (step 8).

[0054] For example, in the case of nucleic acids, dozens of nucleotides such as adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T) are linked together in a chain, and it is known that precursor ions dissociate at the binding sites (linkers) of these nucleotides. Specifically, it is known that product ions (fragment ions) are generated as a, b, c, and d ions (5'-end side) and W, X, Y, and Z ions (3'-end side) depending on the dissociation position in the linker. Therefore, it is possible to theoretically calculate the mass-to-charge ratio of the fragment ions generated from the precursor ions in the MS / MS scan measurement. In addition, it is possible to theoretically calculate the mass-to-charge ratio of the fragment ions of impurity compounds from information such as the reagents used in chemical synthesis. By comparing the theoretically calculated mass-to-charge ratio value with the mass-to-charge ratio of the mass peaks contained in the integrated MS / MS spectrum data, the fragment ions corresponding to each mass peak are identified, and the compounds contained in the sample are identified (step 9).

[0055] When the identification of the compounds contained in the sample is completed, the analysis result display unit 47 displays an analysis result display screen on the display unit 6. The analysis result display screen 70 includes a chromatogram display unit 71 and a mass spectrum display unit 72.

[0056] When the user issues an instruction to start analysis, the analysis result display unit 47 displays a chromatogram display section 71 on the screen of the display unit 6. In the chromatogram display section 71, a total ion current chromatogram (TICC) of all compounds, a total ion current chromatogram (TICC) for each compound, or an extracted ion current chromatogram (XICC) can be displayed in response to a user's operation via the input unit 5.

[0057] When the user specifies a specific time on any of the chromatograms displayed on the chromatogram display section 71 using the input section 5, the mass spectrum display section 72 is also displayed on the screen of the display section 6. The mass spectrum display section 72 displays the mass spectrum acquired at the time specified by the user on that chromatogram. For example, when a specific time is specified on the TICC of all compounds, the MS spectrum acquired at that time (or the cycle including that time) is displayed. In addition, when the TICC or XIC of each compound is selected, the integrated MS / MS spectrum of that compound is displayed. FIG. 9 shows an example of a state in which the TICC of all compounds (two-dot chain line) and the TICC of each compound (solid line or dashed line) are displayed on the chromatogram display section 71, the user selects the TICC of one compound (solid line), and the integrated MS / MS spectrum of that compound is displayed on the mass spectrum display section 72. The user can check the time change of the amount of all compounds contained in the sample, the time change of the amount of each compound, and the information of the integrated MS / MS spectrum of each compound on the analysis result display screen 70. Here, an example has been described in which the chromatogram display section 71 and the mass spectrum display section 72 are displayed on the screen of the display section 6, but in addition to these, information on the compound identified by the compound identification section 46 may also be displayed.

[0058] When analyzing product ions (fragment ions) generated from precursor ions of various valences for samples that contain hundreds to thousands of compounds (target compounds and impurity compounds), such as chemically synthesized nucleic acids or peptides, if one were to individually analyze the MS / MS spectral data obtained from MS / MS scan measurements of each precursor ion, the number of data would be enormous, making the task of analyzing them time-consuming and labor-intensive.

[0059] In contrast, in the mass spectrometry data processing method and mass spectrometry data processing device of this embodiment, it is necessary to analyze only one integrated MS / MS spectrum data for each compound, which significantly reduces the time and effort required for analysis.

[0060] In this embodiment, when creating integrated MS / MS spectrum data, the maximum value of the mass peak intensity for each mass-to-charge ratio contained in the data of multiple MS / MS spectra acquired by MS / MS scan measurements is extracted, so that integrated MS / MS spectrum data can be created without losing information on mass peaks of product ions that are generated only from specific precursor ions or mass peaks of product ions that appear infrequently.

[0061] 5 to 8, the case where MS / MS spectrum data is acquired by DDA has been described, but even when MS / MS spectrum data is acquired by targeted MS / MS, if a large number of compounds must be measured in the same time period, precursor ions may occur for which MS / MS scan measurement can only be performed in a time period when the amount of the compound is small. Even in such cases, by extracting the maximum value of the mass peak in the MS / MS spectrum data acquired in that time period, integrated MS / MS spectrum data containing useful mass peak information can be created regardless of the timing at which the MS / MS scan measurement is performed.

[0062] The above embodiment is merely an example and can be modified as appropriate in accordance with the spirit of the present invention.

[0063] In the above embodiment, a liquid chromatograph mass spectrometer is used, but the same method as above can be used when a gas chromatograph mass spectrometer is used, or when only a mass spectrometer is used without using a chromatograph to obtain mass analysis data. In the above embodiment, ions are generated using an ESI probe 211, but an appropriate ion source may be used depending on the characteristics of the sample. Furthermore, in the above embodiment, a triple quadrupole mass spectrometer 20 is used, but a mass spectrometer equipped with a mass separator of any configuration may be used as long as it is capable of performing MS / MS scan measurement.

[0064] In the above embodiment, assuming a sample containing nucleic acid or peptide, the MS / MS spectrum data of precursor ions with the same mass number but different valences are integrated to generate integrated MS / MS spectrum data. However, the ions (precursor ions) generated from the same compound may include, for example, adduct ions ([M+X] + ), isotope ions, dehydrated ions ([M+H-H2O] + ), fragment ions ([Ma+H] + , [Mb+H] + For samples in which these are expected to be produced, these ions can be classified as ions derived from the same compound based on their mass-to-charge ratio values, and the MS / MS spectrum data obtained by MS / MS scan measurements using these ions as precursor ions can be integrated.

[0065] [Aspects] It will be apparent to those skilled in the art that the above-described exemplary embodiments are illustrative of the following aspects.

[0066] (Section 1) A mass spectrometry data processing method according to one aspect of the present invention includes the steps of: preparing MS / MS spectrum data acquired by MS / MS scan measurement using a plurality of different precursor ions for each of one or a plurality of compounds contained in the sample; Among the plurality of MS / MS spectrum data, a plurality of MS / MS spectrum data acquired by MS / MS scan measurement using a precursor ion derived from each of the one to a plurality of compounds is integrated into one to generate integrated MS / MS spectrum data. It is something.

[0067] (Section 8) A mass spectrometry data processing apparatus according to claim 8 according to another aspect of the present invention comprises: A storage unit in which MS / MS spectrum data acquired by MS / MS scan measurements using a plurality of different precursor ions for each of one or a plurality of compounds contained in a sample is stored; an integrated MS / MS spectrum data creation unit that creates integrated MS / MS spectrum data by integrating, into one, a plurality of MS / MS spectrum data acquired by MS / MS scan measurements using precursor ions derived from the compounds for each of the one to multiple compounds among the plurality of MS / MS spectrum data; Equipped with.

[0068] In the mass spectrometry data processing method according to paragraph 1 and the mass spectrometry data processing device according to paragraph 8, for each of one or more compounds contained in a sample, MS / MS spectrum data obtained by MS / MS scan measurement using multiple different precursor ions derived from the compound are prepared. This data may be obtained by actually performing a measurement, or data obtained in advance may be read out. The multiple different precursor ions include, for example, ions with the same mass number but different valences, adduct ions, isotope ions, dehydrated ions, fragment ions, etc., and different patterns of MS / MS spectrum data are obtained by MS / MS scan measurement using each of the precursor ions.

[0069] In the mass spectrometry data processing method according to paragraph 1 and the mass spectrometry data processing device according to paragraph 8, the MS / MS spectrum data thus obtained is integrated with data obtained by MS / MS scan measurements using precursor ions derived from the same compound to generate one integrated MS / MS spectrum data. The MS / MS spectrum data may be integrated, for example, by averaging or summing the intensities of mass peaks having the same mass-to-charge ratio. In the mass spectrometry data processing method according to paragraph 1 and the mass spectrometry data processing device according to paragraph 8, it is only necessary to check the same number of integrated MS / MS spectrum data as the number of compounds contained in the sample, so that the analysis work can be performed more efficiently than in the past.

[0070] (Section 2) The mass spectrometry data processing method according to paragraph 2 is the mass spectrometry data processing method according to paragraph 1, The compound is identified by predicting a partial structure of the compound corresponding to the mass peak based on the mass-to-charge ratio of the mass peak contained in the integrated MS / MS spectrum data.

[0071] In the mass spectrometry data processing method according to the second aspect, compounds contained in a sample can be identified from integrated MS / MS spectrum data.

[0072] (Section 3) The mass spectrometry data processing method according to paragraph 3 is the mass spectrometry data processing method according to paragraph 1 or 2, The MS / MS spectrum data was obtained by MS / MS scan measurement of compounds separated in a chromatographic column.

[0073] In the mass spectrometry data processing method according to paragraph 3, compounds are separated in a chromatographic column, so that even when ions having similar mass-to-charge ratios are generated from different compounds, they can be individually subjected to MS / MS scan measurement to obtain MS / MS spectrum data.

[0074] (Section 4) The mass spectrometry data processing method according to paragraph 4 is the mass spectrometry data processing method according to paragraph 3, The integrated MS / MS spectrum data is generated by collecting the mass peak with the maximum intensity from among the mass peaks having a common mass-to-charge ratio contained in the plurality of MS / MS spectrum data.

[0075] In the mass spectrometry data processing method according to the third aspect, the amount of compounds separated in the column and subjected to mass analysis changes over time, so that the measured intensity of ions varies depending on the timing of the MS / MS scan. Therefore, when processing such as averaging the intensities of mass peaks contained in the data of multiple MS / MS mass spectra is performed, the intensity value of the mass peaks contained in the data of MS / MS spectra acquired at a timing when the amount of compounds subjected to mass analysis was small becomes small. In the mass spectrometry data processing method according to the fourth aspect, the data of the integrated MS / MS spectrum is created by collecting the mass peak with the maximum intensity from among the mass peaks having a common mass-to-charge ratio contained in the data of the multiple MS / MS spectra, so that the data of the integrated MS / MS spectrum can be created including mass peaks with sufficient intensity regardless of the timing when the data of the MS / MS spectrum was acquired.

[0076] (Section 5) The mass spectrometry data processing method according to paragraph 5 is a mass spectrometry data processing method according to paragraph 3 or 4, The MS / MS spectrum data was obtained by DDA, which repeats the process of performing an MS scan measurement of the compounds separated in the column, and when an ion having an intensity exceeding a predetermined threshold is detected, performing an MS / MS scan measurement using that ion as a precursor ion.

[0077] In the mass spectrometry data processing method according to paragraph 5, since compounds contained in a sample are comprehensively measured by DDA, even if a sample is expected to contain a large number of compounds, MS / MS spectrum data of the large number of compounds can be obtained without setting MS / MS scan measurement conditions for each of the large number of compounds. The mass spectrometry data processing method according to paragraph 5 can be suitably used for measuring samples that contain a large number of impurity compounds in addition to the target compound, such as chemically synthesized nucleic acid or peptide samples.

[0078] (Section 6) A mass spectrometry data processing method according to claim 6, in which the mass spectrometry data processing method according to claim 5 is Data of a plurality of MS / MS spectra acquired by MS / MS scan measurements using precursor ions having the same mass number but different valences is integrated into one to generate integrated MS / MS spectrum data.

[0079] When nucleic acids or peptides are ionized, ions with different charge numbers are generated. When ions (precursor ions) with different charge numbers, even if they have the same molecular structure, are dissociated, fragments are generated where the precursor ion dissociates at different positions in some cases, and different patterns of MS / MS spectrum data are obtained. In the mass spectrometry data processing method according to paragraph 6, by integrating MS / MS spectrum data acquired for precursor ions with different charge numbers, integrated MS / MS spectrum data containing mass peaks corresponding to various fragments is created, thereby enabling compounds to be identified with high accuracy.

[0080] (Section 7) A mass spectrometry data processing method according to claim 7 is a mass spectrometry data processing method according to any one of claims 3 to 6, further comprising: For each compound contained in the sample, the chromatogram and the integrated MS / MS spectrum are displayed on the screen.

[0081] In the mass spectrometry data processing method according to paragraph 7, the chromatogram and MS / MS spectrum of the compounds contained in the sample can be confirmed on the screen. When combined with the mass spectrometry data processing method according to paragraph 2, the identification results of the compounds can also be displayed on the screen, making it possible to further confirm the identification results on the screen. [Explanation of symbols]

[0082] 1...Liquid chromatograph mass spectrometer 10...Liquid chromatograph 11...Mobile phase container 12…Pump 13...Injector 14…Column 20...Mass spectrometer 21…Ionization chamber 211…ESI probe 22…First intermediate vacuum chamber 221…Ion Guide 23…Second intermediate vacuum chamber 231…Ion Guide 24…Third intermediate vacuum chamber 241...Quadrupole mass filter 243…Collision cell 244...Multipole ion guide 245…Ion Guide 25…Analysis room 251…Ion Guide 252...Orthogonal acceleration electrode 2521…Extrusion electrode 2522…Retraction electrode 253…Second acceleration electrode 254...Reflectron 2541…Reflectron No. 1 2542…Reflectron No. 2 255…Ion detector 256…Flight tube 257…Back plate 40...Control and processing section 41...Storage section 42…Measurement condition setting section 43…Measurement execution unit 44…Precursor ion sorting section 45…Integrated MS / MS spectrum data creation section 46…Compound Identification Section 47…Analysis result display section 5. Input section 6...Display section 70…Analysis result display screen 71... Chromatogram display section 72...Mass spectrum display section C…Ion optical axis

Claims

1. preparing MS / MS spectrum data acquired by MS / MS scan measurements using a plurality of different precursor ions for each of one or more compounds contained in the sample; For each of the one or more compounds, data of a plurality of MS / MS spectra acquired by MS / MS scan measurements using precursor ions derived from the compound are integrated into one data of an integrated MS / MS spectrum. A mass spectrometry data processing method.

2. 2. The mass spectrometry data processing method according to claim 1, wherein the compound is identified by estimating a partial structure of the compound corresponding to the mass peak based on the mass-to-charge ratio of the mass peak included in the integrated MS / MS spectrum data.

3. 2. The mass spectrometry data processing method according to claim 1, wherein the MS / MS spectrum data is obtained by MS / MS scan measurement of compounds separated in a chromatographic column.

4. 4. The mass spectrometry data processing method according to claim 3, wherein the integrated MS / MS spectrum data is created by collecting the mass peak with the greatest intensity from among the mass peaks having a common mass-to-charge ratio contained in the plurality of MS / MS spectrum data.

5. 4. The mass spectrometry data processing method according to claim 3, wherein the MS / MS spectrum data is acquired by a DDA that repeats a process of performing an MS scan measurement of a compound separated in the column, and when an ion having an intensity exceeding a predetermined threshold is detected, performing an MS / MS scan measurement using that ion as a precursor ion.

6. Creates integrated MS / MS spectrum data by integrating multiple MS / MS spectrum data acquired by MS / MS scan measurements using precursor ions with the same mass number but different valences. The mass spectrometry data processing method according to claim 5 .

7. moreover, 4. The mass spectrometry data processing method according to claim 3, wherein a chromatogram and the integrated MS / MS spectrum are displayed on a screen for each compound contained in the sample.

8. a storage unit in which MS / MS spectrum data acquired by MS / MS scan measurements using a plurality of different precursor ions for each of one or more compounds contained in a sample is stored; an integrated MS / MS spectrum data creation unit that creates integrated MS / MS spectrum data by integrating multiple MS / MS spectrum data acquired by MS / MS scan measurements using precursor ions derived from each of the one to multiple compounds into one; A mass spectrometry data processing device comprising:

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