Patent Application
20240387158
The present invention relates to an imaging mass spectrometer and a method for analyzing data collected by the imaging mass spectrometer.
An imaging mass spectrometer is a device capable of visualizing a compound distribution on a sample such as a section of biological tissue. An imaging mass spectrometer disclosed in Patent Literature 1 is equipped with an ion source by a matrix assisted laser desorption/ionization (MALDI) method. The imaging mass spectrometer collects mass spectrum data over a predetermined mass-to-charge ratio (customarily referred to as “mass-to-charge ratio” or simply referred to as “m/z” in this specification) range for each micro region obtained by finely segmenting a two-dimensional measurement region on a sample.
As another method of imaging mass spectrometry, there is also known a method of acquiring mass spectrum data for each micro region by cutting out a sample piece from each micro region in a measurement region by use of a sampling method called laser microdissection, and providing a mass spectrometer with a liquid sample prepared from each sample piece (see Patent Literature 2). Here, the imaging mass spectrometer also includes a device using such a sampling method.
In any case, the imaging mass spectrometer can obtain an image showing a distribution of a specific compound by extracting, for example, a signal intensity value at an m/z value of an ion derived from the specific compound, from mass spectrum data obtained for each micro region on a sample, and creating an image in which the signal intensity value is disposed according to a two-dimensional position of each micro region on the sample. Hereinafter, such an image is referred to as mass spectrometry (MS) image.
In a typical imaging mass spectrometer, in a case in which a compound to be observed, that is, a target compound is already determined, a user specifies an m/z value corresponding to the compound. Then, an MS image at the m/z value is created and displayed on a screen of a display unit.
On the other hand, in a case in which the target compound is not specified, a user designates an appropriate mass peak while viewing an acquired mass spectrum. Then, the imaging mass spectrometer creates an MS image corresponding to the m/z value of the designated mass peak, and displays the MS image on the screen of the display unit.
In such a case, as described in Patent Literature 1, an average mass spectrum created by averaging a plurality of mass spectra obtained in all micro regions in a measurement region or a summed mass spectrum obtained by simply summing the plurality of mass spectra is often used as the mass spectrum for a user to select a mass peak. This is because it is considered that information of all the compounds existing in the measurement region is generally reflected in the average mass spectrum or the summed mass spectrum (the average mass spectrum and the summed mass spectrum are substantially the same, and thus only the average mass spectrum will be described below, but the same applies to the summed mass spectrum).
However, the above analysis method in the conventional imaging mass spectrometer has the following problems.
The above average mass spectrum is obtained by averaging so-called profile spectra created based on raw data obtained by mass spectrometry on all the micro regions in the measurement region. The width of a mass peak observed in a profile spectrum depends on the mass resolution of the mass spectrometer used for measurement.
In a time-of-flight mass spectrometer (TOFMS) generally used in an imaging mass spectrometer, the skirt of a mass peak appearing in a profile spectrum may be broad although mass resolution is generally relatively high. In such a case, if the difference in m/z value between the mass peak observed in a relatively wide region in the measurement region and the mass peak locally observed in a narrow region in the measurement region is small and the signal intensity of the latter mass peak is significantly smaller than the signal intensity of the former mass peak, there is a possibility that the mass peak having a smaller signal intensity is buried at the skirt of the mass peak having a larger signal intensity when the mass spectra are averaged or summed. In such a case, a locally observed mass peak does not appear in the average mass spectrum, and as a result, a user may overlook an important compound that is locally distributed in the measurement region.
In addition, with mass resolution of general TOFMS, mass peaks derived from different compounds having significantly close masses may not be adequately separated from each other. When an average mass spectrum is created from a plurality of profile spectra with such inadequate separation, mass peaks in which m/z values are close to each other often overlap and become almost one mass peak. An m/z value obtained from such a mass peak may be different from the m/z value corresponding to any of the plurality of overlapping compounds.
As described above, it is often seen that a mass peak derived from a compound locally contained in a trace amount on the sample cannot be observed in the average mass spectrum. In addition, an m/z value of a mass peak observed in the average mass spectrum often deviates from an m/z value corresponding to a compound actually contained in the sample. For this reason, when a mass peak observed in the average mass spectrum is selected and an MS image at the specific m/z value corresponding to the mass peak is displayed, the distribution of the compound that should be observed by the user may be inaccurate.
The present invention has been made to solve such problems, and an object of the present invention is to provide a mass spectrometry data analysis method and an imaging mass spectrometer capable of reliably grasping even a relatively small amount of a compound locally present in a measurement region without overlooking the presence, and to observe a proper MS image.
Another object of the present invention is to provide a mass spectrometry data analysis method and an imaging mass spectrometer capable of displaying an MS image of an m/z value accurately corresponding to a compound present in a measurement region.
One mode of a mass spectrometry data analysis method according to the present invention made to solve the above problems is a mass spectrometry data analysis method for analyzing data obtained by performing mass spectrometry for each of a plurality of micro regions in a measurement region on a sample, the mass spectrometry data analysis method including:
In addition, one mode of an imaging mass spectrometer according to the present invention made to solve the above problems is an imaging mass spectrometer including:
In the above mode of the present invention, the profile spectrum is averaged (or summed) not for the entire measurement region where the data is collected but for each analysis region having an area smaller than the entire measurement region, and the integrated peak information is obtained based on the result. Therefore, it is possible to prevent a peak derived from a compound locally present in a relatively small amount in the measurement region from being buried in the skirt of another peak having a close m/z value and high intensity, and it is possible to provide a peak list or a mass spectrum accurately reflecting such a peak to the user. Therefore, according to the above modes of the present invention, the user can accurately analyze the target compound without overlooking the presence of the compound that is locally present in a small amount in a narrow area of the measurement region.
In addition, according to the above modes of the present invention, it is possible to display a mass spectrum in which peaks having m/z values highly accurately corresponding to masses of a plurality of compounds present in the measurement region are observed while utilizing high mass accuracy of a mass spectrometer used for measurement. Thereby, the user can observe an MS image with high accuracy corresponding to each of the plurality of compounds, and can perform distribution analysis of the compound with finer and higher accuracy than before.
Hereinafter, an embodiment of an imaging mass spectrometer and a mass spectrometry data analysis method according to the present invention will be described with reference to the accompanying drawings.
The imaging mass spectrometer of the present embodiment includes an imaging mass spectrometry unit 1, a data processor 2, an input unit 3, and a display unit 4.
The imaging mass spectrometry unit 1 is a measurement unit, for example, an air pressure MALDI ion trap time-of-flight mass spectrometer (APMALDI-IT-TOFMS). However, the imaging mass spectrometry unit 1 may be a device obtained by combining a laser microdissection device and a mass spectrometer which performs mass spectrometry of a sample prepared from a minute sample piece collected from a sample by the laser microdissection device as disclosed in Patent Literature 2.
The data processor 2 mainly has a function of processing a large amount of data obtained by the imaging mass spectrometry unit 1, and includes, as functional blocks, a data storage unit 20, a region division unit 21, a profile spectrum creation unit 22, a spectrum averaging unit 23, a peak information collection unit 24, a peak information integration unit 25, a peak list display processing unit 26, a peak selection instruction reception unit 27, an MS image creation unit 28, an image display processing unit 29, and the like.
In the imaging mass spectrometer of the present embodiment, the data processor 2 usually mainly includes a personal computer or a higher-performance workstation. The data processor 2 can embody the functional blocks by executing, on the computer, a dedicated data processing software (computer program) application installed in the computer. In this case, the input unit 3 is a keyboard or a pointing device (such as a mouse) attached to the computer, and the display unit 4 is a display monitor.
The above-described computer program can be provided to a user by being stored in a non-transitory computer-readable recording medium such as a CD-ROM, a DVD-ROM, a memory card, or a USB memory (dongle). In addition, the program can be provided to the user in the form of data transfer via a communication line such as the Internet. Furthermore, the program can be pre-installed in a computer which is a part of the system (strictly, a storage device which is a part of the computer) when the user purchases the system.
An example of a data analysis process in the imaging mass spectrometer of the present embodiment will be described with reference to
An object to be measured by the imaging mass spectrometry unit 1 is, for example, a slice sample obtained by thinly slicing a biological tissue such as a brain or an internal organ of a laboratory animal. Such a sample is placed on a sample plate. A matrix for MALDI is applied to a surface of the sample. The sample is set at a predetermined position of the imaging mass spectrometry unit 1.
As illustrated in
Specific analysis operation is as follows. An ion source in the imaging mass spectrometry unit 1 irradiates one micro region 102 with a laser beam for a short time to generate ions derived from compounds present in the micro region 102. The generated ions are temporarily introduced into an ion trap, then sent into a time-of-flight mass separator, separated according to the m/z value, and detected. This detection signal is mass spectrum data corresponding to one micro region 102. As illustrated in
For each micro region 102, product ion spectrum data may be acquired by performing MS/MS analysis in which ions having a specific m/z value or included in a specific m/z range are analyzed by being dissociated by collision-induced dissociation or the like, or MSn analysis in which n is 3 or more, instead of the normal mass spectrometry.
The collection of mass spectrum data in the micro region 102 collected as described above, that is, MS imaging data for the entire measurement region 101 is transferred from the imaging mass spectrometry unit 1 to the data processor 2 and temporarily stored in the data storage unit 20. The data at this time is raw data obtained by mass spectrometry, but data obtained by performing appropriate waveform processing such as predetermined noise removal on the raw data may be stored in the data storage unit 20.
When the user instructs analysis execution from the input unit 3 in a state where the MS imaging data of the entire measurement region 101 is stored in the data storage unit 20 as described above, the data processor 2 executes the following processing.
First, in the data processor 2, the region division unit 21 performs a process of virtually dividing one measurement region 101 into a plurality of analysis regions 103 as illustrated in (A) of
Then, the profile spectrum creation unit 22 sequentially reads data corresponding to all the micro regions 102 included in the analysis region 103 from the data storage unit 20 for each analysis region 103, and creates profile spectra corresponding to the respective micro regions 102. As illustrated in
In the conventional imaging mass spectrometer, one average mass spectrum is calculated for the measurement region 101 by averaging all the profile spectra obtained in all the micro regions 102 in the measurement region 101. In contrast, in the imaging mass spectrometer of the present embodiment, the spectrum averaging unit 23 executes, for each analysis region 103, a process of averaging profile spectra in all the micro regions 102 included in one analysis region 103 (refer to (B) of
In the example shown in
In contrast, as indicated by an alternate long and short dash line in
The peak information collection unit 24 detects a peak in the average mass spectrum for each analysis region 103 according to a predetermined standard (refer to (C) of
Then, the peak information integration unit 25 collects peak lists of all the analysis regions 103 and integrates the peak information (refer to (E) of
The peak information integration unit 25 integrates all the peaks that can be integrated as described above to create an organized peak list for the entire measurement region 101 (refer to (F) of
The peak list display processing unit 26 displays the peak list for the entire measurement region 101 created as described above on the screen of the display unit 4. Alternatively, the peak list display processing unit 26 may create a mass spectrum (hereinafter, referred to as integrated mass spectrum) based on the peak list created as described above and display this integrated mass spectrum (refer to (G) of
The user checks the displayed peak list or integrated mass spectrum on the screen, and instructs a desired peak, for example, on the peak list or integrated mass spectrum with the input unit 3 for selection. The peak selection instruction reception unit 27 recognizes the instructed peak and determines the m/z value associated with the peak.
The MS image creation unit 28 reads the signal intensity value for each micro region 102 corresponding to the determined m/z value from the data storage unit 20, and creates an MS image. The image display processing unit 29 displays the created MS image on the screen of the display unit 4. For example, the MS image is displayed in the same screen as the peak list or the integrated mass spectrum, and when the user changes the peak instructed on the peak list or the integrated mass spectrum, the displayed MS image can be quickly updated in response to the change of the instruction (change in the instructed m/z value).
As described above, in the imaging mass spectrometer of the present embodiment, it is possible to present, to the user, the peak list and/or the integrated mass spectrum in which information on ion peaks derived from various compounds existing entirely or locally in the measurement region 101 is reflected without omission. This enables the user to appropriately select the peak corresponding to a target compound on the integrated mass spectrum, and check the MS image showing a distribution of the compound with high accuracy.
In the imaging mass spectrometer of the above embodiment, the region division unit 21 automatically divides the measurement region 101 into a plurality of regions according to a predetermined rule, but the rule can be appropriately set by the user. In addition, instead of dividing the entire measurement region 101, for example, only a specific range designated by the user in the measurement region 101 may be divided to define a plurality of analysis regions, and the above-described processing may be performed on the plurality of analysis regions.
Furthermore, the user may be enabled to manually designate a plurality of analysis regions in the measurement region 101.
For example, the region division unit 21 displays the optical microscopic image of the entire measurement region 101, and receives designation of a plurality of analysis regions by the user on the display screen. In the example of
In the imaging mass spectrometer of the embodiment described above, the imaging mass spectrometry unit 1 uses the time-of-flight mass separator as a mass separator. However, the imaging mass spectrometry unit 1 is not limited to the time-of-flight mass separator, and may use a mass separator having a mass resolution equal to or higher than that of the time-of-flight mass separator. Specifically, in addition to various time-of-flight mass spectrometers, a Fourier transform mass spectrometer using a Fourier transform ion cyclotron resonance mass separator, an Orbitrap mass separator, or the like is useful. In addition, the ion source in the imaging mass spectrometry unit 1 is not limited to the air pressure MALDI ion source, and an ion source by an optional ionization method can be used.
In addition, instead of performing mass spectrometry while sequentially scanning the micro regions 102 set in the measurement region 101 on the sample 100, the imaging mass spectrometry unit 1 may perform stigmatic (or projection-type) imaging mass spectrometry for simultaneously performing mass spectrometry on a large number of micro regions in parallel. Furthermore, the imaging mass spectrometry unit 1 may physically cut out a minute sample piece from each of the micro regions 102 in the measurement region 101 by a method such as laser microdissection, and perform mass spectrometry on a liquid sample prepared from each sample piece to acquire data. That is, the measurement method may be any method as long as the mass spectrum data in a predetermined m/z range in each micro region 102 in the measurement region 101 can be obtained.
In addition, the embodiment and the modification described above are merely examples of the present invention, and it is a matter of course that modifications, corrections, additions, and the like appropriately made within the scope of the gist of the present invention are included in the claims of the present application.
A person skilled in the art can understand that the previously described illustrative embodiments are specific examples of the following modes of the present invention.
(Clause 1) One mode of a mass spectrometry data analysis method according to the present invention is a mass spectrometry data analysis method for analyzing data obtained by performing mass spectrometry for each of a plurality of micro regions in a measurement region on a sample, the mass spectrometry data analysis method including:
(Clause 4) One mode of an imaging mass spectrometer according to the present invention is an imaging mass spectrometer including:
In the mass spectrometry data analysis method according to clause 1 and the imaging mass spectrometer according to clause 4, the profile spectrum is averaged or summed not for the entire measurement region where the data is collected but for each analysis region having an area smaller than the entire measurement region, and the integrated peak information is obtained based on the result. Therefore, it is possible to prevent a peak derived from a compound locally present in a relatively small amount in the measurement region from being buried in the skirt of another peak having a close m/z value and high intensity, and it is possible to provide a peak list or a mass spectrum accurately reflecting such a peak to the user. Therefore, according to the above modes of the present invention, the user can accurately analyze the target compound without overlooking the presence of the compound that is locally present in a small amount in a narrow area of the measurement region.
In addition, with the mass spectrometry data analysis method described in clause 1 and the imaging mass spectrometer described in clause 4, it is possible to display a mass spectrum in which peaks having m/z values highly accurately corresponding to masses of a plurality of compounds present in the measurement region are observed while utilizing high mass accuracy of a mass spectrometer used for measurement. Thereby, the user can observe an MS image with high accuracy corresponding to each of the plurality of compounds, and can perform distribution analysis of the compound with finer and higher accuracy than before.
(Clause 2) The mass spectrometry data analysis method according to clause 1 may further include:
In the peak list or the mass spectrum displayed by the display step in the mass spectrometry data analysis method described in clause 1, a peak with high accuracy corresponding to each of a plurality of compounds present in the measurement region or m/z value information of the peak is obtained. With the mass spectrometry data analysis method described in clause 2, the user can display the MS image by designating a specific peak based on the peak list or the mass spectrum, and thus it is possible to display the MS image reflecting the distribution of a target compound with high accuracy. In addition, the user can observe the distribution of the compound by the MS image without overlooking the compound locally present in a slight amount in the measurement region.
(Clause 3) In the mass spectrometry data analysis method according to clause 1 or 2, the peak information integration step may perform a process of determining whether mass-to-charge ratio values of a plurality of peaks are substantially the same according to a standard corresponding to mass accuracy of a device used for mass spectrometry.
In the mass spectrometry data analysis method according to clause 3, information on peaks derived from a plurality of different compounds having m/z values significantly close to each other can be appropriately provided to the user as different peaks by sufficiently utilizing the high mass accuracy of the device. In addition, peaks estimated to be derived from the same compound although having different m/z values can be integrated and provided to the user as information of one peak, and unnecessary and redundant information can be avoided from being provided to the user.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/037385 | 10/8/2021 | WO |
