WO2021130840A1

WO2021130840A1 - Imaging data processing method, imaging data processing device, and imaging data processing program

Info

Publication number: WO2021130840A1
Application number: PCT/JP2019/050515
Authority: WO
Inventors: 龍太松本; 潔小河
Original assignee: 株式会社島津製作所
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2021-07-01

Abstract

In this method for processing imaging data that is three-dimensional data having three axes for the retention times, mass-to-charge ratios, and ion intensities obtained for each of a plurality of measurement points on a sample through chromatograph mass spectrometry: two-dimensional data is created for each of the plurality of measurement points through the accumulation or averaging of ion intensity values in the three-dimensional data corresponding to the measurement point along the axial direction of one from among the retention times and mass-to-charge ratios (step 4); the input of a designated value for the other from among the retention times and mass-to-charge ratios is received (step 7); and image data is created by, for each of the plurality of measurement points, reading the ion intensity value corresponding to the designated value from the two-dimensional data corresponding to the measurement point (step 13).

Description

Imaging data processing method, imaging data processing device, and imaging data processing program

The present invention relates to a technique for processing imaging data obtained by using a chromatographic mass spectrometer.

Mass spectrometric imaging is used to investigate the distribution of target compounds contained in samples such as biological tissue sections. In the mass spectrometry imaging method, a plurality of measurement points are set in the analysis target area of the sample, mass spectrometry is performed at each measurement point, and mass spectrometry data is acquired.

Mass spectrometric imaging is generally performed using a matrix-assisted laser desorption / ionization (MALDI) -time-of-flight mass spectrometer (TOFMS: Time of Flight Mass Spectrometer) (TOFMS). For example, Patent Document 1). In MALDI-TOFMS, a matrix substance is applied to a sample in advance, laser light is irradiated at each measurement point, and the ions generated by the laser beam are subjected to mass spectrometry. The distribution of the target compound is shown by collecting the intensity data of the mass peak having the mass-to-charge ratio corresponding to the ion derived from the target compound from the mass spectrum data obtained at each measurement point and associating it with the position information of the measurement point. An MS imaging image is obtained.

In MALDI-TOFMS, since the ions generated from the sample are subjected to mass spectrometry as they are, many compounds other than the target compound are ionized at the same time. For example, in the case of a sample containing a large number of compounds such as a biological tissue section, a compound that is easier to ionize than the target compound absorbs energy from the matrix substance and is ionized before the target compound, so that the target compound is ionized. It becomes difficult. This phenomenon is called ion suppression. As a result, the detection sensitivity of the target compound may be deteriorated or the quantitativeness may be poor.

Recently, in order to solve the above points, a chromatographic mass spectrometry imaging method that combines a sampling method called a laser microdissection (LMD) method and a liquid chromatograph mass spectrometry method has been proposed. For example, Patent Document 2 describes a mode in which the hot melt LMD method is used as the LMD method.

In the hot melt LMD method, first, a film that melts by heat is attached to a slide glass, the slide glass is placed on the sample so that the film is on the sample side, and the film is brought into close contact with the sample to approach the measurement point. Irradiate infrared laser light. Then, the sample adheres to the film melted by the heat generated by the irradiation of the laser beam, and when the slide glass is separated from the sample, the sample is collected on the slide glass side. This is dissolved in an appropriate liquid to prepare a liquid sample. A sample is collected on a slide glass in this way at each of the plurality of measurement points, a liquid sample is prepared, and the sample is analyzed by a liquid chromatograph mass spectrometer. As a result, three-dimensional data centered on the holding time, mass-to-charge ratio, and ionic strength can be obtained for each measurement point. By extracting the ionic strength at the retention time and mass-to-charge ratio corresponding to the target compound from this three-dimensional data and associating it with the position information of the measurement point, a liquid chromatograph mass spectrometric imaging image showing the distribution of the target compound can be obtained. .. In this mass spectrometric imaging method, since the target compound contained in the sample is separated from other compounds and ionized in the liquid chromatograph, the above-mentioned problem caused by ion suppression can be solved.

International Publication No. 2018/037941 International Publication No. 2016/1633385 Japanese Unexamined Patent Publication No. 2009-8582

Generally, when examining the distribution of the target compound, at least 1,000 or more measurement points are set in consideration of the visibility of the image and the like. Therefore, in the mass spectrometric imaging method of Patent Document 2, imaging data including 1,000 or more three-dimensional data is generated.

The retention time and mass-to-charge ratio of the target compound are not always known, and in such a case, the user sets a set of retention time and mass-to-charge ratio, and confirms the mass spectrometric imaging image obtained by the set of the set. The work of judging suitability is repeated. In the process, each time the user inputs a set of retention time and mass-to-charge ratio, the value of the ion intensity corresponding to the set is read out from the three-dimensional data of each measurement point, and it corresponds to the position information of the measurement point. The process of generating an image is repeated, but there is a problem that it takes time to create image data from imaging data including 1,000 or more three-dimensional data.

The problem to be solved by the present invention is three-dimensional data having three axes of retention time, mass-to-charge ratio, and ionic strength obtained by performing chromatographic mass spectrometry at each of a plurality of measurement points of a sample. This is to reduce the time required to create image data from imaging data including.

The first aspect of the present invention made to solve the above problems is the retention time and mass charge obtained for each measurement point by performing chromatographic mass spectrometry at each of a plurality of measurement points of the sample. A method of processing imaging data including three-dimensional data centered on ratio and ionic strength.
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ionic strength values in the axial direction of one of the retention time and the mass-to-charge ratio in the three-dimensional data corresponding to the measurement points.
Accepts the input of the specified value, which is the other value of the retention time and the mass-to-charge ratio.
For each of the plurality of measurement points, image data is created by reading out the ionic strength value corresponding to the specified value from the two-dimensional data corresponding to the measurement point.

In the imaging data processing method according to the present invention, the imaging data including the three-dimensional data obtained for a plurality of measurement points is not used as it is, but the ion intensity value is set in the axial direction of one of the retention time and the mass-to-charge ratio. Two-dimensional data is created by reducing the dimension of the data by one by integrating or averaging. Then, the input of the specified value is accepted for the other of the holding time and the mass-to-charge ratio (that is, the one in which the ionic strength values are not integrated or averaged), and the specified value corresponds to the two-dimensional data of a plurality of measurement points. Image data is created by reading out the ionic strength value. In the imaging data processing method according to the present invention, since the image data is created by reading the image creation data file which is lightened by reducing the dimension from the three-dimensional data included in the original imaging data, the time required for the processing is reduced. Can be shortened. The specified value may be input by the user himself / herself, or may be automatically input based on the analysis result of the two-dimensional data by a program for analyzing chromatograph mass spectrometric data or the like.

A second aspect of the present invention, which has been made to solve the above problems, is a retention time obtained for each measurement point by performing chromatographic mass spectrometry at each of a plurality of measurement points of a sample. A device that processes imaging data including three-dimensional data with the mass-to-charge ratio and ionic strength as the three axes.
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ion intensity values in the axial direction of one of the retention time and the mass charge ratio in the three-dimensional data corresponding to the measurement points. Dimensional data creation department and
A parameter input receiving unit that accepts input of a specified value, which is the other value of the holding time and the mass-to-charge ratio,
For each of the plurality of measurement points, an image data creation unit is provided which reads out the ion intensity value corresponding to the designated value from the two-dimensional data corresponding to the measurement point and creates image data.

The program for imaging data processing according to the third aspect of the present invention, which is made to solve the above problems, is
Chromatograph mass spectrometry including three-dimensional data having retention time, mass-to-charge ratio, and ion intensity as three axes obtained for each measurement point by performing chromatograph mass spectrometry at each of a plurality of measurement points of a sample. A computer with a storage unit in which imaging data is stored,
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ion intensity values in the axial direction of one of the retention time and the mass charge ratio in the three-dimensional data corresponding to the measurement points. Dimensional data creation department and
A parameter input receiving unit that accepts input of a specified value, which is the other value of the holding time and the mass-to-charge ratio,
For each of the plurality of measurement points, the ion intensity value corresponding to the specified value is read out from the two-dimensional data corresponding to the measurement point, and the image data is operated as an image data creation unit.

By using the imaging data processing technique according to the present invention, the retention time, mass-to-charge ratio, and ion intensity obtained by performing chromatograph mass spectrometry at each of a plurality of measurement points of a sample are set as three axes. The time required to create image data from imaging data including three-dimensional data can be shortened.

FIG. 6 is a configuration diagram of a main part of a liquid chromatograph mass spectrometry imaging analysis system including the first embodiment of the imaging data processing apparatus according to the present invention. The flowchart of 1st Example of the imaging data processing method which concerns on this invention. The figure explaining the sampling by the hot melt laser microdissection method. Another figure illustrating sampling by the hot melt laser microdissection method. The figure explaining the 3D data and the 2D data in the 1st Example. An example of an integrated mass chromatogram in the first embodiment. FIG. 6 is a configuration diagram of a main part of a liquid chromatograph mass spectrometry imaging analysis system including the imaging data processing apparatus of the second embodiment. The flowchart of the imaging data processing method of 2nd Example.

The imaging data processing method, the imaging data processing apparatus, and the first embodiment of the imaging data processing program according to the present invention will be described below with reference to the drawings. FIG. 1 is a configuration diagram of a main part of a liquid chromatograph mass spectrometry imaging analysis system in which a control unit 4 including an imaging data processing apparatus of this embodiment is combined with a liquid chromatograph mass spectrometer.

The liquid chromatograph mass spectrometer is composed of a liquid chromatograph 1 and a mass spectrometer 2. The liquid chromatograph 1 includes a mobile phase container 10 in which a mobile phase is stored, a pump 11 that sucks the mobile phase and feeds it at a constant flow rate, and an injector 12 that injects a predetermined amount of sample liquid into the mobile phase. It is provided with a column 13 for separating various compounds contained in the sample liquid in the time direction. Further, an autosampler 14 for introducing a plurality of liquid samples one by one into the injector 12 is connected to the liquid chromatograph 1. In the imaging mass spectrometry imaging analysis system of this example, local samples are collected in advance at a plurality of measurement points of the sample by the hot melt laser microdissection (LMD) method, and a liquid sample is prepared from each of them. Then, the liquid samples corresponding to each of the plurality of measurement points are set in the autosampler 14, and they are sequentially subjected to liquid chromatograph mass spectrometry.

The mass spectrometer 2 is a first intermediate vacuum in which the degree of vacuum is gradually increased between the ionization chamber 20 having a substantially atmospheric pressure and the high vacuum analysis chamber 23 evacuated by a vacuum pump (not shown). It has a multi-stage differential exhaust system configuration including a chamber 21 and a second intermediate vacuum chamber 22. In the ionization chamber 20, an electrospray ionization probe (ESI probe) 201 that sprays the sample solution while applying an electric charge is installed. The ionization chamber 20 and the first intermediate vacuum chamber 21 in the subsequent stage communicate with each other via a small-diameter heating capillary 202. The first intermediate vacuum chamber 21 and the second intermediate vacuum chamber 22 are separated by a skimmer 212 having a small hole at the top, and ions are converged in the first intermediate vacuum chamber 21 and the second intermediate vacuum chamber 22, respectively. At the same time, ion guides 211 and 221 are installed for transporting to the subsequent stage. In the analysis chamber 23, in order from the upstream side (the side of the ionization chamber 20), the front quadrupole mass filter (Q1) 231 and the multi-pole ion guide (q2) 233 are installed inside the collision cell 232 and the rear quadrupole. A polar mass filter (Q3) 234 and an ion detector 235 are installed. Collision-Induced Dissociation (CID) gas such as argon or nitrogen is appropriately supplied to the inside of the collision cell 232 according to the measurement conditions.

The mass spectrometer 2 performs selective ion monitoring (SIM: Selected Ion Monitoring) measurement, MS / MS scan measurement (product ion scan measurement, precursor ion scan measurement), multiple reaction monitoring (MRM: Multiple Reaction Monitoring) measurement, and the like. Can be done. In SIM measurement, the front quadrupole mass filter (Q1) 231 does not select ions (does not function as a mass filter), and the mass-to-charge ratio of ions that pass through the rear quadrupole mass filter (Q3) 234 is fixed. Ions are detected.

Product ion scan In scan measurement, the mass-to-charge ratio of product ions that pass through the rear quadrupole mass filter (Q3) 234 is scanned while the mass-to-charge ratio of precursor ions that pass through the front quadrupole mass filter (Q1) 231 is fixed. While doing so, the product ions that have passed through the subsequent quadrupole mass filter (Q3) 234 are detected. In the MRM measurement, both the mass-to-charge ratio of precursor ions passing through the front quadrupole mass filter (Q1) 231 and the mass-to-charge ratio of product ion ions passing through the rear quadrupole mass filter (Q3) 234 are fixed. Product ions that have passed through the subsequent quadrupole mass filter (Q3) 234 are detected. In the precursor ion scan measurement, the mass-to-charge ratio of the product ion ion passing through the rear quadrupole mass filter (Q3) 234 while scanning the mass-to-charge ratio of the precursor ion passing through the front quadrupole mass filter (Q3) 231. Is fixed, and product ions that have passed through the subsequent quadrupole mass filter (Q3) 234 are detected. In these measurements, CID gas is supplied to the inside of the collision cell 232 in order to cleave the precursor ions to generate product ions.

The control unit 4 has a storage unit 41, and also has a measurement control unit 421, a two-dimensional data file creation unit 422, an integrated mass spectrum data creation unit 423, an integrated mass chromatogram data creation unit 424, and a first designated value as functional blocks. It includes an input reception unit 425, a peak processing unit 426, a second designated value input reception unit 427, an image data file creation unit 428, and a display image update unit 429. The substance of the control unit 4 is a personal computer, and each of the above functional blocks is embodied by executing the chromatograph mass spectrometry imaging program 42 pre-installed in the computer on the processor. Of the chromatographic mass spectrometric imaging program 42, the portion that embodies the functional blocks excluding the measurement control unit 421 corresponds to the imaging data processing program of the present invention. Further, an input unit 6 and a display unit 7 are connected to the control unit 4.

The storage unit 41 stores the three-dimensional data file 411, the two-dimensional data file 412, and the image data file 413. In addition, various data (for example, a method file describing measurement conditions in liquid chromatograph mass spectrometry) necessary for the processing described later are also stored in the storage unit 41.

Next, the procedure for analyzing the distribution of the target compound in the sample using the liquid chromatograph mass spectrometric imaging analysis system of this example will be described with reference to the flowchart of FIG.

As described above, in the imaging mass spectrometry imaging analysis system of this example, local samples are collected in advance at a plurality of measurement points of the sample by the hot melt LMD method, and a liquid sample is prepared from each (step 1).

The procedure for collecting a sample by the hot melt LMD method will be described with reference to FIGS. 3 and 4. First, a section-shaped sample 101 to be measured is attached to one surface of the sample holding slide glass 100, and a heat-soluble film 103 is attached to one surface of another sampling slide glass 102. Prepare a sample. Then, both are set so that the sample 101 and the heat-soluble film 103 face each other (procedure 1 in FIG. 3).

Next, the two sample holding slide glasses 100 and the sample collecting slide glass 102 are brought close to each other to bring the heat-soluble film 103 into close contact with the sample 101 (procedure 2 in FIG. 3). Then, a laser beam (near infrared laser beam) 104 is formed so as to be substantially orthogonal to the surface of the sample collection slide glass 102 from the side of the sample collection slide glass 102 opposite to the surface to which the heat-soluble film 103 is attached. (Procedure 3 in FIG. 3). The laser beam 104 is irradiated to a range corresponding to one measurement point set on the sample 101.

The irradiated laser beam 104 passes through the sample-collecting slide glass 102 and heats the heat-soluble film 103. As a result, the heat-soluble film 103 near the area irradiated with the laser beam 104 is melted and locally penetrates into the measurement point of the sample 101. After that, the sample holding slide glass 100 and the sample collecting slide glass 102 are separated from each other to separate the heat-soluble film 103 from the sample 101. Then, the local sample 105, which is a part of the sample 101, is collected on the surface of the heat-soluble film 103 (procedure 4 in FIG. 4). By using a near-infrared laser beam as the laser beam 104 and appropriately adjusting the wavelength and intensity of the laser beam, the local area in a very small range is not affected by the components contained in the collected biological tissue. The sample 105 can be collected on the heat-soluble film 103.

The above process is an operation of collecting a local sample 105 at one measurement point on the sample 101. By repeating the same operation while moving the relative positions of the sample holding slide glass 100 and the sample collecting slide glass 102 in the plane direction thereof, as shown in FIG. 4, in the analysis target region 1011 of the sample 101. Local samples 105 near a plurality of measurement points 1012 can be collected on the heat-soluble film 103, respectively. At this time, the distance between the measurement points 1012 on the sample 101 corresponds to the spatial resolution in the mass spectrometric imaging image, and is very narrow, for example, 1 μm, but the distance between the local samples 105 collected on the thermosoluble film 103 is For example, the sampling position is controlled so as to be as wide as several mm. The two-dimensional positional relationship between the plurality of measurement points on the sample 101 and the two-dimensional positional relationship of the local sample 105 collected on the thermosoluble film 103 need not necessarily be maintained. It suffices that the correspondence between the position of the measurement point on the sample 101 (the position defined by the address information in the X direction and the Y direction) and the position of the local sample 105 on the thermosoluble film 103 is determined.

Next, a liquid sample is prepared from the individual local samples 105 collected on the heat-soluble film 103. For example, as shown in FIG. 4, a microtiter plate (MTP) 110 having a large number of wells 1101 is used, and a predetermined extract for extracting components in the local sample 105 is previously applied to each well 1101 of the MTP 110. Inject. The slide glass 102 for sampling is attached to the upper surface of the MTP 110 (the surface on the side where each well 1101 is open) so that the local sample 105 on the thermosoluble film 103 is located inside each well 1101. In that state, for example, by turning the entire MTP 110 upside down, the local sample 105 is immersed in the extract in each well 1101 to prepare a liquid sample in which the components in the local sample 105 are dissolved. As described above, if the correspondence between the position of the measurement point on the sample 101 and the position of the local sample 105 on the thermosoluble film 103 is determined, the position of each prepared liquid sample and the measurement point on the sample 101 is determined. The relationship with is also uniquely determined. In this way, liquid samples are prepared from each of the plurality of measurement points and set in the autosampler 14.

Note that FIG. 4 is a simplification for the sake of explanation, and usually, the number of measurement points on the sample 101 is much larger than that shown in the figure (typically at least 1,000 points or more), and one sample 101 Prepares a liquid sample contained in each well 1101 of a large number of MTP 110s.

After setting the liquid sample obtained from a plurality of measurement points of the sample in the auto sampler 14, when the user instructs the start of measurement, the measurement control unit 421 reads out the method file previously stored in the storage unit 41, and there. Based on the measurement conditions described in the above, a plurality of liquid samples are introduced into a liquid chromatograph mass analyzer in a predetermined order for measurement. In this embodiment, the liquid sample is introduced from the autosampler 14 into the injector 12 of the liquid chromatograph, and the sample liquid after the components are separated by the column 13 is repeatedly MS / at a predetermined timing (t1, t2,…, tm). MS scan measurement (product ion scan measurement). In this way, as shown in the upper part of FIG. 5, mass spectrum data (product ion spectrum data) can be obtained for each of the plurality of measurement points at each of the plurality of holding times (bands). Then, by integrating these plurality of mass spectrum data, three-dimensional data having the retention time, the mass-to-charge ratio, and the ionic strength as the three axes can be obtained (step 2). The three-dimensional data is associated with the position information of the measurement point of the sample, and is sequentially stored in the storage unit 41.

When the liquid chromatograph mass analysis is completed for all the liquid samples and the three-dimensional data is stored in the storage unit 41, the two-dimensional data file creation unit 422 displays the ions in the retention time axis direction in the three-dimensional data of each measurement point. Intensity values are integrated (or averaged) (step 3) to create two-dimensional data (mass spectrum data). Then, a two-dimensional data file in which the position information of the measurement point and the two-dimensional data are associated with each other is created (step 4) and stored in the storage unit 41.

Subsequently, the integrated mass spectrum data creation unit 423 creates integrated mass spectrum data by integrating (or averaging) the ion intensity values of the two-dimensional data of a plurality of measurement points (step 5), and displays the integrated mass spectrum. Display on the screen of 7 (step 6). This results in a single integrated mass spectrum that reflects the total intensity (or average intensity) of the ions produced from the various compounds present at multiple measurement points of the sample.

When the integrated mass spectrum is displayed on the screen of the display unit 7, the first designated value input receiving unit 425 receives the input of the mass-to-charge ratio value (or range) by the user. This is done, for example, by clicking on the peak of the integrated mass spectrum displayed on the screen or by specifying the range with the mouse (step 7).

When the value (or range) of the mass-to-charge ratio is input by the user, the integrated mass chromatogram data creation unit 424 determines the input mass-to-charge ratio from each of the three-dimensional data obtained at the plurality of measurement points. Mass chromatogram data is extracted based on the value, and one integrated mass chromatogram data is created by integrating the mass chromatogram data of a plurality of measurement points, and the integrated mass chromatogram is displayed on the screen of the display unit 7. (Step 8). Note that this process may be performed in the order of first integrating the three-dimensional data to create the integrated three-dimensional data, and then extracting the integrated mass chromatogram data based on the input mass-to-charge ratio value. ..

When the integrated mass chromatogram is displayed on the screen of the display unit 7, the peak processing unit 426 performs peak picking of the integrated mass chromatogram based on a predetermined algorithm to extract the peak (step 9), and the peak top thereof. Retention time (or peak retention time range) is calculated. For peak picking, an appropriate method may be used from various conventionally proposed methods (for example, Patent Document 3).

If there is only one peak extracted by peak picking (NO in step 10), the value of the peak top holding time (or the peak holding time range) of that peak is determined as the second specified value.

On the other hand, when a plurality of peaks are extracted by peak picking (YES in step 10), a display specifying the peak position on the integrated mass chromatogram (for example, passing through the peak top as shown by a broken line in FIG. 6). (Lines, etc.) are superimposed to let the user select one of the peaks. When the user selects one of the peaks (step 11), the value of the peak top holding time (or the peak holding time range) of the peak is determined as the second designated value.

The second designated value input receiving unit 427 accepts the input of the second designated value determined as described above (step 12). Here, peak picking was performed based on a predetermined algorithm to extract the peak, but the user confirmed the integrated mass chromatogram to identify the peak, and the retention time of the peak top (or the retention time of the peak). Range) may be entered.

When the second designated value is input, the image data file creation unit 428 corresponds to the first designated value (mass-to-charge ratio) and the second designated value (holding time) from the three-dimensional data of each of the plurality of measurement points. The ion intensity to be measured is read out, image data in which the ion intensity of each measurement point is mapped is created (step 13), and the image is displayed on the screen of the display unit 7.

This image shows the intensity of ions having the first specified value (mass-to-charge ratio) in the mass spectrometer 2 produced from the compound flowing out from the column 13 of the liquid chromatograph 1 at the second specified value (holding time). It was done. Therefore, the user confirms whether or not this image correctly represents the distribution of the target compound. For example, when it is considered that the distributions of compounds other than the target compound appear to overlap, the first designated value (mass-to-charge ratio) or the second designated value (holding time) is changed (YES in step 14). When the first designated value (mass-to-charge ratio) or the second designated value (holding time) is updated, the display image updating unit 429 causes each of the above units to execute the above processing, and the updated first specified value ( Image data in which the ionic strength of each measurement point is mapped corresponding to the mass-to-charge ratio) and the second designated value (holding time) is created, and the image is displayed on the screen of the display unit 7.

When the user determines that the target compound is correctly mapped, the user instructs the end of the analysis by a predetermined input operation such as clicking the enter button with the mouse (no update of the specified value. NO in step 14). When the end of analysis is instructed, the image data file creation unit 428 uses the image data corresponding to the image displayed on the display unit 7 as the first specified value (mass charge ratio) used for creating the image data and the image data. An image data file associated with the second specified value (holding time) is created (step 15), and the image data file is stored in the storage unit 41.

In the conventional imaging data processing method, in order to know the distribution of the target compound, it was necessary to specify one measurement point and further specify a set of retention time and mass-to-charge ratio at a time. It is not always easy to identify the measurement point where the target compound is surely present, or to specify both the retention time and the mass-to-charge ratio at the same time and accurately. , The selection of measurement points, and the input of the set of retention time and mass-to-charge ratio had to be determined by trial and error many times.

On the other hand, in the imaging data processing method of this example, one integrated mass chromatogram data obtained by integrating the ionic strengths derived from the target compound distributed in the analysis target range of the sample is confirmed, and the mass-to-charge ratio (specified value) is first determined. You can specify it. Then, after specifying the mass-to-charge ratio, the integrated mass chromatogram may be confirmed and the holding time (second designated value) may be specified. Therefore, even those unfamiliar with the analysis can easily obtain image data showing the distribution of the target compound.

Further, in the imaging data processing method of this embodiment, the imaging data including the three-dimensional data obtained for a plurality of measurement points is not used as it is, but the ion intensity in the axial direction of one of the retention time and the mass-to-charge ratio. Two-dimensional data is created by reducing the dimension of the data by one by integrating or averaging the values. Then, the input of the specified value is accepted for the other of the holding time and the mass-to-charge ratio (that is, the one in which the ionic strength values are not integrated or averaged), and the specified value corresponds to the two-dimensional data of a plurality of measurement points. Image data is created by reading out the ionic strength value. That is, in the imaging data processing method of the present embodiment, the image data is created by reading the image creation data file which is lightened by reducing the dimension from the three-dimensional data included in the original imaging data, so that the processing is required. You can save time.

Next, a second embodiment of the imaging data processing method, the imaging data processing apparatus, and the imaging data processing program according to the present invention will be described. The liquid chromatograph mass spectrometric system including the imaging data processing apparatus of the second embodiment has a control unit 5 shown in FIG. 7 in place of the control unit 4 of the first embodiment. In the following description, the description of the configuration and the process common to the first embodiment will be omitted as appropriate.

The control unit 5 has a storage unit 51, and also has a measurement control unit 521, a two-dimensional data file creation unit 522, an integrated chromatogram data creation unit 523, an integrated mass chromatogram data creation unit 524, and a first designated value as functional blocks. It includes an input reception unit 525, a peak processing unit 526, a second designated value input reception unit 527, an image data file creation unit 528, and a display image update unit 529. The substance of the control unit 5 is a personal computer, and each of the above functional blocks is embodied by executing the chromatograph mass spectrometry imaging program 52 pre-installed in the computer on the processor. Of the chromatographic mass spectrometric imaging program 52, the portion that embodies the functional blocks excluding the measurement control unit 521 corresponds to the imaging data processing program of the present invention. Further, as in the first embodiment, the input unit 6 and the display unit 7 are connected to the control unit 5.

Next, the procedure for analyzing the distribution of the target compound in the sample using the liquid chromatograph mass spectrometric imaging analysis system of this example will be described with reference to the flowchart of FIG. Since the collection and preparation of the sample by the hot melt LMD method and the acquisition of the three-dimensional data by the liquid chromatograph mass spectrometry are the same as those in the first embodiment, the description thereof will be omitted.

When the liquid chromatograph mass spectrometry is completed for all the liquid samples and the three-dimensional data is obtained (step 2), the two-dimensional data file creation unit 522 indicates the ions in the mass-to-charge ratio axial direction in the three-dimensional data of each measurement point. The intensity values are integrated (or averaged) (step 23) to create two-dimensional data (total ion current chromatogram data). Then, a two-dimensional data file in which the position information of the measurement point and the two-dimensional data are associated with each other is created (step 24) and stored in the storage unit 51.

Subsequently, the integrated chromatogram data creation unit 523 creates integrated chromatogram data by integrating (or averaging) the ionic strength values of a plurality of two-dimensional data (step 25), and displays the integrated chromatogram on the screen of the display unit 7. Is displayed in (step 26). This results in a single integrated chromatogram that reflects the total intensity (or average intensity) of the ions produced from the various compounds present at multiple measurement points on the sample.

When the integrated chromatogram is displayed on the screen of the display unit 7, the peak processing unit 526 performs peak picking of the integrated chromatogram based on a predetermined algorithm to extract the peak (step 27), and holds the peak top. Find the time (or peak retention time range).

If there is only one peak extracted by peak picking (NO in step 28), the value of the peak top holding time (or the peak holding time range) of that peak is determined as the specified value.

On the other hand, when a plurality of peaks are extracted by peak picking (YES in step 28), a code for specifying the peak position (such as a line passing through the peak top) is superimposed and displayed on the integrated chromatogram, and eventually the user is informed. Let the peak be selected. When the user selects one of the peaks (step 29), the value of the peak top holding time (or the peak holding time range) of the peak is determined as the specified value.

The first designated value input receiving unit 525 accepts the input of the designated value determined as described above (step 30). Here, peak picking was performed based on a predetermined algorithm to extract peaks, but the user confirmed the integrated chromatogram to identify the peak, and the retention time (or peak retention time range) of the peak top was identified. ) May be entered as the specified value.

Next, the integrated mass chromatogram data creation unit 524 extracts mass chromatogram data from each of the three-dimensional data obtained at the plurality of measurement points based on the input retention time value, and further extracts the mass chromatogram data from the plurality of measurement points. One integrated mass chromatogram data is created by integrating the mass chromatogram data of the above, and the integrated mass chromatogram is displayed on the screen of the display unit 7 (step 31).

Subsequently, the second designated value input receiving unit 527 causes the user to input the value (or range) of the mass-to-charge ratio, which is the second designated value, by an operation such as selecting a peak on the integrated mass chromatogram (step). 32).

When the second specified value is input, the image data file creation unit receives the ion intensity corresponding to the specified value (holding time) and the second specified value (mass-to-charge ratio) from the three-dimensional data of each of the plurality of measurement points. Is read out, image data whose intensity is mapped is created (step 33), and the image is displayed on the screen of the display unit 7. Subsequent steps 14 and 15 are the same as those in the first embodiment, and thus the description thereof will be omitted.

The first embodiment and the second embodiment are both examples, and can be appropriately changed according to the gist of the present invention.

In the above embodiment, the two-dimensional data of all measurement points are integrated to create an integrated mass spectrum or an integrated chromatogram, but it is not always necessary to integrate the two-dimensional data of all measurement points. For example, when one or more measurement points where the target compound exists are known by observing the sample with an observation device or the like, the two-dimensional data of those one or more measurement points may be integrated.

Further, in the above embodiment, three-dimensional data was acquired using a liquid chromatograph mass spectrometer, but a gas chromatograph mass spectrometer can also be used. In that case, the prepared liquid sample may be vaporized and introduced into a gas chromatograph. In addition, when a compound is fragmented at the time of ionization, such as the electron ionization (EI) method, which is an ionization method widely used in gas chromatograph mass spectrometers, three-dimensional data is acquired by performing MS scan measurement. You may. In addition, it is not always necessary to perform scan measurement, and a pseudo-mass spectrum is acquired by performing SIM measurement or MRM measurement using (a set of) one or more mass-to-charge ratios, and the pseudo-mass spectrum is combined with the retention time in three dimensions. The data may be analyzed.

Further, in the above embodiment, after creating the integrated mass chromatogram data, the second designated value is input, and the ionic strength corresponding to both the designated value and the second designated value is read out to create the image data. 2 The specified value does not necessarily have to be entered. In that case, the image data may be created from the integrated value (or average value) of the ionic strength corresponding to the specified value. This corresponds to the peak intensity of the total ion chromatogram, for example, when the specified value is the retention time.

[Aspect]
It will be understood by those skilled in the art that the plurality of exemplary embodiments described above are specific examples of the following embodiments.

(Section 1)
One aspect of the present invention is a three-dimensional structure having a retention time, a mass-to-charge ratio, and an ionic strength as three axes, which are obtained for each measurement point by performing chromatographic mass spectrometry at each of a plurality of measurement points of a sample. A method of processing imaging data, including data.
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ionic strength values in the axial direction of one of the retention time and the mass-to-charge ratio in the three-dimensional data corresponding to the measurement points.
Accepts the input of the specified value, which is the other value of the retention time and the mass-to-charge ratio.
For each of the plurality of measurement points, image data is created by reading out the ionic strength value corresponding to the designated value from the two-dimensional data corresponding to the measurement point.

(Section 16)
Another aspect of the present invention has three axes of retention time, mass-to-charge ratio, and ionic strength obtained for each measurement point by performing chromatographic mass spectrometry at each of the plurality of measurement points of the sample. A device that processes imaging data including three-dimensional data.
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ion intensity values in the axial direction of one of the retention time and the mass charge ratio in the three-dimensional data corresponding to the measurement points. Dimensional data creation department and
A designated value input receiving unit that accepts input of a designated value that is the other value or range of the holding time and the mass-to-charge ratio, and
For each of the plurality of measurement points, an image data creation unit is provided which reads out the ion intensity value corresponding to the designated value from the two-dimensional data corresponding to the measurement point and creates image data.

(Section 17)
The imaging data processing program according to still another aspect of the present invention is
Chromatograph mass spectrometry including three-dimensional data having retention time, mass-to-charge ratio, and ion intensity as three axes obtained for each measurement point by performing chromatograph mass spectrometry at each of a plurality of measurement points of a sample. A computer with a storage unit in which imaging data is stored,
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ion intensity values in the axial direction of one of the retention time and the mass charge ratio in the three-dimensional data corresponding to the measurement points. Dimensional data creation department and
A parameter input receiving unit that accepts input of a specified value, which is the other value of the holding time and the mass-to-charge ratio,
For each of the plurality of measurement points, the ion intensity value corresponding to the specified value is read out from the two-dimensional data corresponding to the measurement point, and the image data is operated as an image data creation unit.

The imaging data processing method according to paragraph 1, the imaging data processing apparatus according to paragraph 16, and the imaging data processing program according to paragraph 17 include imaging including three-dimensional data obtained for a plurality of measurement points. Instead of using the data as it is, two-dimensional data is created by reducing the dimension of the data by one by integrating or averaging the ion intensity values in the axial direction of one of the retention time and the mass charge ratio. Then, the input of the specified value is accepted for the other of the holding time and the mass-to-charge ratio (that is, the one in which the ionic strength values are not integrated or averaged), and the specified value corresponds to the two-dimensional data of a plurality of measurement points. Image data is created by reading out the ionic strength value. In the imaging data processing method according to the present invention, since the image data is created by reading the image creation data file which is lightened by reducing the dimension from the three-dimensional data included in the original imaging data, the time required for the processing is reduced. Can be shortened.

(Section 2)
In the imaging data processing method described in paragraph 1,
One of the above is the holding time, and the specified value is the value or range of the mass-to-charge ratio.

In the imaging data processing method described in item 2, image data representing the distribution of the target compound can be obtained based on the mass-to-charge ratio of ions generated from the target compound.

(Section 3)
In the imaging data processing method described in Section 2,
The three-dimensional data is obtained by performing a scan measurement in the chromatograph mass spectrometry.

In the imaging data processing method described in item 3, it is possible to select peaks of the mass-to-charge ratio of ions characteristic of the target compound from a large number of peaks appearing on the mass spectrum and obtain image data showing the distribution of the target compound. it can.

(Section 4)
In the imaging data processing method according to the second or third paragraph, further
Integrated mass spectrum data is created by integrating or averaging the ionic strength values of some or all of the two-dimensional data of the plurality of measurement points.

In the imaging data processing method described in item 4, the user can easily determine the specified value by confirming only one integrated mass spectrum data. Further, in the imaging data processing method described in item 4, a characteristic portion is designated as a region of interest based on an image of the sample surface obtained by an observation device, and two measurement points located within the region of interest are designated. By integrating or averaging the ion intensity values of the dimensional data, integrated mass spectrum data with more emphasized sample characteristics can be obtained.

(Section 5)
In the imaging data processing method according to any one of paragraphs 2 to 4, further
An integrated mass chromatogram data corresponding to the specified value in the integrated three-dimensional data in which the three-dimensional data of the plurality of measurement points are integrated is created.
Accepting the input of the second designated value which is the value or range of the retention time in the integrated mass chromatogram data,
At each of the plurality of measurement points, the ionic strength corresponding to the designated value and the second designated value in the three-dimensional data is read out to create the image data.

In the imaging data processing method described in item 5, both the mass-to-charge ratio value or range corresponding to the target compound and the retention time value or range are simply specified to obtain image data representing the distribution of the target compound. Can be done.

(Section 6)
In the imaging data processing method described in Section 5, further
A peak included in the integrated mass chromatogram data is extracted based on a predetermined algorithm, and a value or range of the retention time of the peak is input as the second designated value.

In the imaging data processing method described in item 6, the second specified value can be input even if the user is not familiar with chromatographic mass spectrometry.

(Section 7)
In the imaging data processing method according to any one of paragraphs 1 to 6, further
Every time the designated value or the second designated value is input, the image is updated and displayed.

In the imaging data processing method described in item 7, since the image displayed on the display unit is updated every time the specified value or the second specified value is changed, it is easy to determine the suitability of the combination of the specified value and the second specified value. And it can be confirmed immediately.

(Section 8)
In the imaging data processing method described in paragraph 1,
One of the above is the mass-to-charge ratio, and the specified value is the value or range of the holding time.

With the imaging data processing method described in item 8, image data representing the distribution of the target compound can be obtained based on the retention time of the target compound in the chromatograph.

(Section 9)
In the imaging data processing method described in item 8,
The three-dimensional data is obtained by performing a scan measurement in the chromatograph mass spectrometry.

In the imaging data processing method described in item 9, an image showing the distribution of the target compound from a chromatogram obtained by integrating the intensities of all the ions generated from the sample within the mass-to-charge ratio range obtained by scanning measurement. You can get the data.

(Section 10)
In the imaging data processing method according to paragraph 8 or 9, further
Integrated chromatogram data is created by integrating or averaging the ionic strength values of some or all of the two-dimensional data of the plurality of measurement points.

In the imaging data processing method described in paragraph 10, the user can easily determine a specified value by confirming only one integrated chromatogram data. Further, in the imaging data processing method described in item 10, a characteristic portion is designated as a region of interest based on an image of the sample surface obtained by the observation device, and two measurement points located within the region of interest are designated. By integrating or averaging the ion intensity values of the dimensional data, integrated chromatogram data emphasizing the characteristics of the sample can be obtained.

(Section 11)
In the imaging data processing method according to paragraph 10, further
A peak included in the integrated chromatogram data is extracted based on a predetermined algorithm, and a value or range of the retention time of the peak is input as the specified value.

In the imaging data processing method described in paragraph 11, the specified value can be determined even if the person is not familiar with the analysis of the chromatogram.

(Section 12)
In the imaging data processing method according to any one of items 8 to 11, further
An integrated mass chromatogram data corresponding to the specified value in the integrated three-dimensional data in which the three-dimensional data of the plurality of measurement points are integrated is created.
Accepting the input of the second designated value which is the value or range of the mass-to-charge ratio in the integrated mass spectrum data,
At each of the plurality of measurement points, the ionic strength corresponding to the designated value and the second designated value in the three-dimensional data is read out to create the image data.

In the imaging data processing method according to Item 12, both the value or range of the mass-to-charge ratio corresponding to the target compound and the value or range of the retention time are simply specified to obtain image data representing the distribution of the target compound. Can be done.

(Section 13)
In the imaging data processing method according to the eighth to twelfth items, further
Every time the designated value or the second designated value is input, the image data is updated and displayed.

In the imaging data processing method described in item 13, since the image displayed on the display unit is updated every time the specified value or the second specified value is changed, it is easy to determine the suitability of the combination of the specified value and the second specified value. And it can be confirmed immediately.

(Section 14)
In the imaging data processing method described in paragraph 1, further
In addition to the three-dimensional data, a data file for creating image data in which the two-dimensional data is described is created.

In the imaging data processing method described in paragraph 14, it is possible to obtain image data showing the distribution of the target compound at an arbitrary timing by using the image data in which the two-dimensional data is described.

(Section 15)
In the imaging processing method described in paragraph 1, the three-dimensional data is
Local samples are taken from each of the multiple measurement points of the sample using the laser microdissection method.
Liquid samples are individually prepared from the local samples collected at the plurality of measurement points.
It was obtained by performing chromatographic mass spectrometry on the prepared sample.

In the imaging data processing method described in paragraph 15, ion suppression can be suppressed, and chromatographic mass spectrometric data having high sensitivity and quantification can be acquired and analyzed.

1 ... Liquid chromatograph 2 ...

Mass analyzer

4, 5 ...

Control unit

41, 51 ...

Storage unit

411, 511 ... Three-dimensional data file 412, 512 ... Two-dimensional data file 413, 513 ... Image data file 42, 52 ... Chromato Graph mass analysis Imaging program 421 ...

Measurement control unit

422, 522 ... Two-dimensional data file creation unit 423 ... Integrated mass spectrum data creation unit 523 ... Integrated chromatogram

data creation unit

424, 524 ... Integrated mass chromatogram

data creation unit

425, 525 ... 1st designated value

input reception unit

426, 526 ...

Peak processing unit

427, 527 ... 2nd designated value

input reception unit

428, 528 ... Image data file creation unit 429, 259 ... Display image update unit 6 ... Input unit 7 ... Display unit 100 ... Sample holding slide glass 101 ... Sample 1011 ... Analysis target area 1012 ... Measurement point 102 ... Sample sampling slide glass 103 ... Thermolytic film 104 ... Laser light 105 ... Local sample

Claims

Processing imaging data, which is three-dimensional data centered on retention time, mass-to-charge ratio, and ionic strength, obtained for each measurement point by performing chromatographic mass spectrometry at each of the multiple measurement points of the sample. How to do
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ionic strength values in the axial direction of one of the retention time and the mass-to-charge ratio in the three-dimensional data corresponding to the measurement points.
Accepts the input of the specified value, which is the other value of the retention time and the mass-to-charge ratio.
An imaging data processing method for creating image data by reading out an ionic strength value corresponding to the specified value from two-dimensional data corresponding to the measurement point for each of the plurality of measurement points.
The imaging data processing method according to claim 1, wherein one of the above is a holding time and the specified value is a value or a range of a mass-to-charge ratio.
The imaging data processing method according to claim 2, wherein the three-dimensional data is obtained by performing a scan measurement in the chromatographic mass spectrometry.
further,
The imaging data processing method according to claim 2, wherein integrated mass spectrum data is created by integrating or averaging the ionic strength values of some or all of the two-dimensional data of the plurality of measurement points.
further,
An integrated mass chromatogram data corresponding to the specified value in the integrated three-dimensional data in which the three-dimensional data of the plurality of measurement points are integrated is created.
Accepting the input of the second designated value which is the value or range of the retention time in the integrated mass chromatogram data,
The imaging data processing method according to claim 2, wherein the image data is created by reading out the ionic strength corresponding to the designated value and the second designated value in the three-dimensional data at each of the plurality of measurement points.
further,
The imaging data processing method according to claim 5, wherein a peak included in the integrated mass chromatogram data is extracted based on a predetermined algorithm, and a value or range of the retention time of the peak is input as the second designated value.
further,
The imaging data processing method according to claim 1, wherein the image data is updated and displayed each time the designated value or the second designated value is input.
The imaging data processing method according to claim 1, wherein one of them is a mass-to-charge ratio and the specified value is a value or range of holding time.
further,
The imaging data processing method according to claim 8, wherein a peak included in the two-dimensional data is extracted based on a predetermined algorithm, and a value or range of the holding time of the peak is input as the specified value.
The imaging data processing method according to claim 8, wherein the three-dimensional data is obtained by performing a scan measurement in the chromatographic mass spectrometry.
further,
The imaging data processing method according to claim 8, wherein integrated chromatogram data is created by integrating or averaging the ionic strength values of some or all of the two-dimensional data of the plurality of measurement points.
further,
An integrated mass spectrum data corresponding to the specified value in the integrated three-dimensional data in which the three-dimensional data of the plurality of measurement points are integrated is created.
Accepting the input of the second designated value which is the value or range of the mass-to-charge ratio in the integrated mass spectrum data,
The imaging data processing method according to claim 8, wherein the image data is created by reading out the ionic strength corresponding to the designated value and the second designated value in the three-dimensional data at each of the plurality of measurement points.
further,
The imaging data processing method according to claim 8, wherein the image data is updated and displayed each time the designated value or the second designated value is input.
further,
The imaging data processing method according to claim 1, wherein a data file for creating image data in which the two-dimensional data is described is created separately from the three-dimensional data.
The three-dimensional data is
Local samples are taken from each of the multiple measurement points of the sample using the laser microdissection method.
Liquid samples are individually prepared from the local samples collected at the plurality of measurement points.
The imaging data processing method according to claim 1, which is obtained by performing chromatographic mass spectrometry on the prepared sample.
Processing imaging data, including three-dimensional data centered on retention time, mass-to-charge ratio, and ionic strength, obtained for each measurement point by performing chromatographic mass spectrometry at each of the multiple measurement points of the sample. It is a device that
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ion intensity values in the axial direction of one of the retention time and the mass charge ratio in the three-dimensional data corresponding to the measurement points. Dimensional data creation department and
A designated value input receiving unit that accepts input of a designated value that is the other value or range of the holding time and the mass-to-charge ratio, and
An imaging data processing apparatus including an image data creation unit that reads out an ion intensity value corresponding to the specified value from two-dimensional data corresponding to the measurement point and creates image data for each of the plurality of measurement points.
Chromatograph mass spectrometry including three-dimensional data having retention time, mass-to-charge ratio, and ion intensity as three axes obtained for each measurement point by performing chromatograph mass spectrometry at each of a plurality of measurement points of a sample. A computer with a storage unit in which imaging data is stored,
For each of the plurality of measurement points, two-dimensional data is created by integrating or averaging the ion intensity values in the axial direction of one of the retention time and the mass charge ratio in the three-dimensional data corresponding to the measurement points. Dimensional data creation department and
A parameter input receiving unit that accepts input of a specified value, which is the other value of the holding time and the mass-to-charge ratio,
An imaging data processing program that operates as an image data creation unit that creates image data by reading out the ion intensity value corresponding to the specified value from the two-dimensional data corresponding to the measurement point for each of the plurality of measurement points. ..