Calibration method, analysis method, control device, and analysis device

Abstract

This calibration method is for calibrating a mass spectrum for mass spectrometry of a microbial sample containing a substance to be analyzed, i.e. molecules to be analyzed, said calibration method including: a step (S31) of acquiring a mass spectrum of a microbial sample to which one or more molecules having an estimated theoretical m / z smaller than that of the substance to be analyzed have been added as a first standard substance; a step (S32) of setting, as a second standard substance, one or more molecules which are derived from the microbial sample and have an estimated theoretical m / z larger than that of the substance to be analyzed; and a step (S33) of calibrating the mass spectrum on the basis of an actually measured m / z of the first standard substance and an actually measured m / z of the second standard substance as indicated by peaks on the mass spectrum, the theoretical m / z of the first standard substance, and the theoretical m / z of the second standard substance.

Description

Calibration method, analysis method, control device and analysis device

[0001] The present invention relates to a calibration method, an analysis method, a control device and an analysis device, and more particularly to a calibration method, an analysis method, a control device and an analysis device related to mass spectrometry.

[0002] When ionizing a target substance, which is a molecule to be analyzed, and analyzing its mass by mass spectrometry, it is necessary to measure the mass of the target substance with high accuracy. For example, when measuring a polymer with a molecular weight of several thousand Da, it may be necessary to distinguish between differences in mass values ​​of a few Da or less. Therefore, to improve measurement accuracy, the mass of a standard substance, the theoretical mass of which can be calculated from its composition, is measured, and the mass spectrometer is calibrated based on the difference between the measured mass and the theoretical mass of the standard substance.

[0003] Japanese Patent Application Laid-Open No. 2019-90654

[0004] When performing mass spectrometry on microorganisms, the mass spectrometer is typically calibrated using a known molecule prepared from Escherichia coli as a standard. However, since the smallest molecular weight standard commonly used has a mass-to-charge ratio (m / z) of approximately 4300, extrapolation is required when target molecules are smaller than this, resulting in reduced accuracy.

[0005] As a countermeasure, Patent Document 1 (JP 2019-90654 A) discloses a calibration method using aspartate-1-decarboxylase (m / z approximately 2800) contained in a sample derived from Escherichia coli as a standard substance. However, even with this method, aspartate-1-decarboxylase is not necessarily present in organisms other than Escherichia coli, the molecular weight is not necessarily the same, and the protein is not necessarily produced.

[0006] Therefore, in mass spectrometry of microorganisms, particularly when target substances are molecules with small molecular weights, a new calibration method has been required.

[0007] The present disclosure has been made to solve such problems, and its purpose is to improve the mass accuracy in mass spectra, thereby improving the accuracy of analysis.

[0008] A calibration method according to a first aspect of the present disclosure is a method for calibrating a mass spectrum when performing mass analysis on a microbial sample containing a target substance, which is a molecule to be analyzed, and includes the steps of: acquiring a mass spectrum of the microbial sample to which one or more molecules having a smaller estimated theoretical m / z than that of the target substance have been added as first standard substances; setting one or more molecules derived from the microbial sample having a larger estimated theoretical m / z than that of the target substance as second standard substances; and calibrating the mass spectrum based on the measured m / z of the first standard substance and the measured m / z of the second standard substance indicated by peaks on the mass spectrum, and the theoretical m / z of the first standard substance and the theoretical m / z of the second standard substance.

[0009] A control device according to a second aspect of the present disclosure is a control device that performs calibration of a mass spectrum when performing mass analysis on a microbial sample containing a target substance, which is a molecule to be analyzed, and includes a memory and a processor. The memory stores the theoretical m / z of a first standard, which is one or more molecules having a smaller estimated theoretical m / z than the target substance. The processor acquires a mass spectrum of the microbial sample to which the first standard has been added. The processor sets one or more molecules derived from the microbial sample having a larger estimated theoretical m / z than the target substance as second standard substances. The processor calibrates the mass spectrum based on the measured m / z of the first standard and the measured m / z of the second standard, which are indicated by peaks in the mass spectrum, and the theoretical m / z of the first standard and the second standard.

[0010] An analytical apparatus according to a third aspect of the present disclosure is an analytical apparatus for performing mass spectrometry on a microbial sample containing a target substance, which is a molecule to be analyzed, and includes a measurement unit, a memory, and a processor. The measurement unit acquires measurement data of the microbial sample. The memory stores theoretical m / z values ​​of first standard substances, which are one or more molecules having a smaller estimated theoretical m / z value than the target substance. The processor acquires a mass spectrum of the microbial sample to which the first standard substances have been added, based on the measurement data. The processor sets one or more molecules derived from the microbial sample having a larger estimated theoretical m / z value than the target substance as second standard substances. The processor calibrates the mass spectrum based on the measured m / z values ​​of the first standard substance and the second standard substance, which are indicated by peaks in the mass spectrum, and the theoretical m / z values ​​of the first standard substance and the second standard substance.

[0011] According to the calibration method disclosed herein, a mass spectrum can be calibrated using a first standard substance having a theoretical m / z smaller than that of the added target substance and a second standard substance having a theoretical m / z larger than that of the target substance derived from a microbial sample, so that the difference between the theoretical m / z and the measured m / z for these standard substances is small. This allows the mass spectrum in the range corresponding to the molecular weight of the target substance to be appropriately calibrated, even if the molecular weight of the target substance is small. Therefore, by improving the mass accuracy of the mass spectrum, the accuracy of the analysis can be improved.

[0012] 5 is a schematic diagram showing the configuration of an analytical device according to the present embodiment; FIG. 6 is a flowchart illustrating an analytical method according to the present embodiment; FIG. 7 is a flowchart illustrating a calibration method according to the present embodiment; FIG. 8 is a diagram showing an example of a mass spectrum before the calibration method according to the present embodiment is performed; FIG. 9 is a diagram showing an example of a mass spectrum after the calibration method according to the present embodiment is performed on the mass spectrum of FIG. 4; and FIG. 10 is a diagram showing an example of a mass spectrum after the calibration method according to the present embodiment is performed on the mass spectrum of FIG.

[0013] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the accompanying drawings. In the following description, the same or corresponding parts in the drawings are denoted by the same reference numerals, and their description will not be repeated in principle.

[0014] 1 is a schematic diagram showing the configuration of an analytical device 1 according to an embodiment of the present invention. The analytical device 1 is a mass spectrometer for performing mass analysis of substances contained in a sample, such as a MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry) device.

[0015] In this embodiment, the sample is a biological sample, which is a sample derived from an organism. In one example, the sample is a sample derived from a microorganism. The sample includes a target substance, which is a molecule to be analyzed, and a standard substance (calibrant), which is a molecule used to calibrate a mass spectrum. In this embodiment, analysis by the analyzer 1 includes detecting peaks in the mass spectrum and measuring the m / z of a specific or non-specific substance contained in the sample. In one example, the substance is a protein. Analysis by the analyzer 1 may include determining whether a specific substance is contained in the sample based on the m / z indicated by the peak in the mass spectrum (hereinafter also referred to as "measured m / z"), calculating the concentration of the specific substance in the sample, and identifying the organism contained in the sample.

[0016] Referring to Fig. 1, the analytical device 1 includes a control unit 10 and a measurement unit 20. The measurement unit 20 ionizes substances (proteins) in a sample using a high voltage, separates the ions S according to a time of flight correlated with m / z, and then detects them. The measurement unit 20 includes an ionization unit 21, an ion acceleration unit 22, a mass separation unit 23, and a detection unit 24. In Fig. 1, the movement of ions S in the measurement unit 20 is schematically indicated by arrow A1.

[0017] The ionization unit 21 ionizes substances in the sample by matrix-assisted laser desorption ionization (MALDI). As an ionization method, any soft ionization method such as electrospray ionization (ESI) can be used in addition to MALDI. When ionization is performed by ESI, it is preferable that the analyzer 1 further includes a liquid chromatograph and that the ionization unit 21 ionizes substances in the sample separated by the liquid chromatograph, since this can provide high separation ability.

[0018] The ionization unit 21 includes a sample plate holder (not shown) that supports a sample plate, and an ion source that includes a laser device (not shown) that irradiates the sample plate with laser light. After the sample is placed on the sample plate, a matrix is ​​added to the sample, and the sample is dried. The sample plate is then placed in the sample plate holder in the vacuum chamber of the ionization unit 21. There are no particular limitations on the type of matrix, but from the perspective of efficiently ionizing protein samples, it is preferable to use sinapinic acid, α-cyano-4-hydroxycinnamic acid (CHCA), or the like.

[0019] The ionization unit 21 depressurizes the vacuum chamber containing the sample plate, and then irradiates each sample on the sample plate with laser light to sequentially ionize them. The type of laser device that irradiates the laser light is not particularly limited as long as it can emit light that is absorbed by the selected matrix. For example, when the matrix contains sinapinic acid or CHCA, an N2 laser (wavelength 337 nm) or the like can be suitably used. The ions S ionized in the ionization unit 21 are extracted by an electric field generated by an extraction electrode or the like (not shown) and introduced into the ion acceleration unit 22.

[0020] The ion accelerator 22 includes an acceleration electrode 221 and accelerates the introduced ions S. The flow of the accelerated ions S is appropriately focused by an ion lens (not shown) and introduced into the mass separator 23.

[0021] The mass separation unit 23 includes a flight tube 231, and separates the ions S based on the difference in flight time when each ion S flies inside the flight tube 231. While a linear type flight tube 231 is shown in FIG. 1 , a reflectron type, a multi-turn type, or the like may also be used. There are no particular limitations on the method of mass analysis as long as it is possible to separate and detect the ions S contained in the sample.

[0022] The detection unit 24 includes an ion detector such as a multi-channel plate, detects the ions S separated by the mass separation unit 23, and outputs a detection signal with an intensity corresponding to the number of ions incident on the detection unit 24. The detection signal output from the detection unit 24 is input to the processing unit 11 of the control unit 10. In Fig. 1, the flow of the detection signal of the ions S from the detection unit 24 of the measurement unit 20 is schematically shown by arrow A2.

[0023] The control unit 10 includes a processing unit 11, a storage unit 12, and an input / output unit 13. The control unit 10 corresponds to an example of a "control device" in the present disclosure.

[0024] The processing unit 11 includes a processor such as a CPU, and functions as a main unit for controlling the analysis device 1. The processing unit 11 performs various processes by executing programs stored in the storage unit 12, etc. The processing unit 11 corresponds to an example of a "processor" in the present disclosure.

[0025] The processing unit 11 includes an apparatus control unit 111 , a mass spectrum creation unit 112 , a mass spectrum analysis unit 113 , and a calibration unit 114 .

[0026] The device control unit 111 controls the operation of the measurement unit 20 based on data relating to analysis conditions input from the input unit 131, which will be described later. In Fig. 1, the control of the measurement unit 20 by the device control unit 111 is schematically shown by arrow A3.

[0027] The mass spectrum creation unit 112 converts the flight times into m / z values ​​from measurement data including the detected amount of ions detected by the detection unit 24 and the flight times of the ions, and creates a mass spectrum showing the detected amount corresponding to each m / z value.

[0028] The mass spectrum analysis unit 113 detects mass spectrum peaks in the mass spectrum. It calculates the m / z corresponding to the detected peak. The mass spectrum analysis unit 113 determines the substance to which the measured m / z indicated by the peak in the mass spectrum corresponds, based on a protein database. That is, the mass spectrum analysis unit 113 can calculate the measured m / z of a specific or non-specific substance contained in the sample. The mass spectrum analysis unit 113 may further determine whether a specific substance is contained in the sample based on the measured m / z (identification of components in the sample), calculate the concentration of a specific substance in the sample, or identify organisms contained in the sample. More generally, the mass spectrum analysis unit 113 may perform structural analysis of substances contained in the sample.

[0029] The calibration unit 114 calibrates the mass spectrum based on the measured m / z and theoretical m / z of the standard material. The theoretical m / z is generally referred to as a calculated value, theoretical value, or theoretical m / z value, and is a theoretical mass-to-charge ratio calculated taking into account the molecular weight, added ions, and charge number. Calibration in mass spectrometry involves correcting the measured m / z of the standard material so that it approaches the theoretical m / z, and applying this correction to the entire spectrum. Details of the calibration process will be described later.

[0030] The storage unit 12 includes a non-volatile storage medium and stores therein the theoretical m / z values, the mass spectrum created by the mass spectrum creation unit 112, the measurement data output from the measurement unit 20, and a program for the processing unit 11 to execute processing. The storage unit 12 corresponds to an example of "memory" in the present disclosure.

[0031] The input / output unit 13 is an interface for the analysis device 1 to input and output information from and to the outside. The input / output unit 13 includes an input unit 131, an output unit 132, and a communication unit 133.

[0032] The input unit 131 is configured to include input devices such as a mouse, a keyboard, various buttons, and / or a touch panel, etc. The input unit 131 receives information necessary for controlling the operation of the measurement unit 20, information necessary for the processing performed by the processing unit 11, etc. from the user.

[0033] The output unit 132 includes a display device such as a liquid crystal monitor, a printer, etc. The output unit 132 displays information about the measurement by the measurement unit 20, the results of processing by the processing unit 11, etc. on a display device or prints them on a paper medium.

[0034] The communication unit 133 includes a communication device capable of communicating wirelessly or via a wired connection such as the Internet, etc. The communication unit 133 receives data necessary for processing by the processing unit 11, transmits data processed by the processing unit 11 such as a determination result, and transmits and receives necessary data as appropriate.

[0035] Some or all of the functions of the control unit 10 described above may be located in a computer, server, or the like that is physically separated from the measurement unit 20 .

[0036] [2. Conventional Calibration Methods] In recent years, the identification of microorganisms by mass spectrometry using MALDI (MALDI-MS) has rapidly become widespread. This is because, compared to conventional identification methods such as culture methods, MALDI-MS does not require skilled techniques and can be performed quickly and at low cost. Furthermore, conventional identification methods perform various tests based on a certain degree of "prediction" regarding the microorganism, such as its genus name. Therefore, without this "prediction," testing, i.e., even attempting identification, is impossible. In contrast, the identification of microorganisms by mass spectrometry is a groundbreaking method in that it allows identification to be attempted even without "prediction," as long as bacterial cells sufficient to prepare a sample for mass spectrometry are available. In other words, MALDI-MS allows for easier identification of microorganisms than conventional identification methods.

[0037] On the other hand, in microbial identification using MALDI-MS, bacterial cells are used as samples with little or no purification, and therefore the amount of sample and the types of substances contained in the sample are greater than in the case of ordinary MALDI-MS, which is performed after purifying proteins, which can lead to problems such as a large number of peaks scattered across the sample, making the mass spectrum complex, and large m / z errors due in part to the rise in the sample. This error can be particularly serious when analyzing which microbial molecule a certain mass spectrum peak originates from.

[0038] Mass calibration is an extremely important step in reducing such errors in mass spectrometry. Mass calibration ensures the m / z accuracy of mass spectra. This allows for the proper identification of components, microorganisms, and structural analysis using mass spectra, as described above.

[0039] As is well known, mass calibration methods can be broadly divided into internal standard methods and external standard methods (see Patent Document 1, etc.). In the internal standard method, a standard substance is mixed with a sample containing a target substance and then measured. That is, the target substance and the standard substance are measured simultaneously under the same conditions. In the resulting mass spectrum, the m / z axis of the mass spectrum is calibrated based on the difference between the theoretical m / z of the standard substance and the measured m / z value, and the measured m / z value of the target substance is also calibrated.

[0040] On the other hand, in the external standard method, a sample containing a target substance and a calibration sample containing a standard substance (hereinafter referred to as a "calibration sample") are measured separately without being mixed. First, information on the difference between the theoretical m / z and the measured m / z of the standard substance is obtained from the mass spectrum of the calibration sample. Next, this information is used to calibrate the mass spectrum of the sample containing the target substance, thereby calibrating the measured m / z value of the target substance. In such an external standard method, it is difficult to correct for the effects of variations in measurement conditions between the measurement of the sample containing the target substance and the measurement of the calibration sample containing the standard substance. Therefore, the internal standard method generally provides more accurate mass calibration than the external standard method.

[0041] As mentioned above, calibration is extremely important when performing mass spectrometry, and it is desirable to apply internal standard calibration methods whenever possible. However, in reality, due to various technical limitations, cost constraints, and other reasons, external standard calibration is generally used. On the other hand, when high accuracy is required, various solutions are being sought within these limitations and restrictions.

[0042] When performing mass spectrometry on microorganisms, calibration is generally performed using an external standard method, using a substance prepared from the well-known and well-studied Escherichia coli as a standard substance. However, even the smallest molecular weight of the standard substance derived from Escherichia coli (a ribosomal protein) is approximately m / z 4,300, and if the target molecule is smaller than this, extrapolation may be required, resulting in a decrease in accuracy.

[0043] As a countermeasure, when measuring samples derived from E. coli, one method is to use aspartate-1-decarboxylase (m / z approximately 2,800) as a new standard substance derived from E. coli (Patent Document 1). However, microorganisms other than E. coli do not necessarily have aspartate-1-decarboxylase, do not necessarily have the same amino acid sequence, and do not necessarily produce the protein.

[0044] Another method is to add a low molecular weight standard substance and a high molecular weight standard substance to the sample to be analyzed and measure the mass spectrum. However, simply adding these standards often results in high molecular weight multiply charged ions appearing in the m / z range of the target substance, which can make it difficult to obtain the mass spectrum of the target substance.

[0045] In consideration of these issues, the mass spectrometer according to this embodiment first adds one or more molecules having known molecular weights and smaller than that of the target substance to a sample as first standards, and then measures the mass spectrum of the sample. Next, one or more molecules derived from the sample and having a molecular weight greater than that of the target substance are set as second standards. The mass spectrum is then calibrated so that the measured m / z values ​​corresponding to the first and second standards shown in the sample's mass spectrum correspond to the theoretical m / z values ​​corresponding to the first and second standards. The mass accuracy of the m / z values ​​of the mass spectrum calibrated in this manner is improved, and the accuracy of analysis using these mass spectra is also improved. Therefore, the mass spectrometer according to this embodiment can improve analytical accuracy by improving mass accuracy using the above-described simple calibration method.

[0046] 3. Analysis Method According to the Present Embodiment Fig. 2 is a flowchart illustrating an analysis method according to the present embodiment. The steps shown in Fig. 2 are executed by the analysis device 1 and a user. In the figure, "S" is used as an abbreviation for "STEP."

[0047] In S1, the user prepares a sample by adding a first standard to the sample. The first standard is one or more molecules with an estimated theoretical m / z smaller than that of the target substance. For example, if the purpose of analysis using the analytical device 1 is to identify microorganisms contained in the sample, the m / z of the substance referenced in identifying the target microorganism is generally approximately 3,000 to 15,000. Therefore, the m / z of the first standard in this case is preferably less than approximately 3,000, and more preferably less than approximately 1,500. The first standard is, for example, a protein or peptide. A more specific example of the first standard is angiotensin 2 (molecular weight: 1,046.2) and / or angiotensin 1 (molecular weight: 1,296.5). However, the first standard substance is not limited thereto, and may include, for example, at least one of bradykinin fragment 1-7 (human) (molecular weight: approximately 757.4), P14R (synthetic peptide) (molecular weight: approximately 1533.9), ACTH fragment 18-39 (human) (molecular weight: approximately 2465.2), and oxidized insulin B chain (bovine) (molecular weight: approximately 3494.7).

[0048] In one embodiment, the theoretical m / z of the first standard is estimated by determining the molecular weight from the amino acid sequence of the first standard and calculating the theoretical m / z based on the molecular weight. The theoretical m / z is estimated simply by adding the mass of a hydrogen atom, 1.08, to the molecular weight. The theoretical m / z or molecular weight of the first standard may be obtained from a database containing theoretical m / z or molecular weights of first standard substances, if available.

[0049] In S2, the user calibrates a typical mass spectrometer using a calibration sample in the analytical device 1. The calibration sample generally contains multiple reference materials, and may be a commercially available sample for use in calibrating mass spectrometers. The calibration sample may or may not contain a first reference material and a second reference material.

[0050] The calibration using the calibration sample in S2 is a calibration that is generally performed before measuring a target substance in a mass spectrometer, but is not necessarily required if the analytical device 1 is properly adjusted and managed.

[0051] In S3, the user uses the analytical device 1 to measure the mass spectrum of the sample prepared in S1 and calibrate the mass spectrum using a first standard and a second standard derived from the sample. The second standard is derived from a microorganism contained in the sample and is one or more molecules with an estimated theoretical m / z greater than that of the target substance. The second standard is, for example, a protein. Details of the processing in S3 will be described in FIG. 3.

[0052] In S4, the user uses the analytical device 1 to analyze the target substance based on the calibrated mass spectrum.

[0053] In S5, the user stores and / or displays the analysis results in S4 in the analysis device 1. This allows the user to easily store and / or recognize the analysis results.

[0054] 3 is a flowchart illustrating a calibration method according to the present embodiment. Steps S31 to S34 shown in FIG. 3 correspond to step S3 in FIG. 2. Each step shown in FIG. 3 is performed by the processing unit 11 of the analyzer 1.

[0055] In S31, the processing unit 11 acquires a mass spectrum of the sample to which the first standard substance has been added. In S31, the user and / or the processing unit 11 may set the mass spectrum measurement conditions before starting the mass spectrum measurement. For example, the mass range for measuring the mass spectrum is set to include the m / z of the first standard substance. Other mass spectrum measurement conditions are set appropriately so as to obtain a clear spectrum.

[0056] In S32, the processing unit 11 sets a second standard substance. For example, a ribosomal protein is set as the second standard substance. Ribosomal proteins of microorganisms are often clearly observed by MALDI, and their amino acid sequences can often be found in public databases such as UniProt. It is therefore easy to calculate the molecular weight and theoretical m / z from the amino acid sequence. Therefore, if the microorganism contained in the sample is known or predicted, it is useful to set the ribosomal protein of the microorganism as the second standard substance.

[0057] The second standard substance is set by the user, for example, using the input unit 131. The user may set the second standard substance by, for example, displaying a mass spectrum on the output unit 132, and selecting a specific peak in the mass spectrum using the input unit 131, and then setting a molecule corresponding to the selected peak as the second standard substance. Alternatively, the user may set the second standard substance by, for example, using the input unit 131 to input information about a substance to be used as the second standard substance, or by selecting from options.

[0058] Note that setting the second standard substance in S32 includes estimating the theoretical m / z of the second standard substance. In one implementation example, the theoretical m / z of the second standard substance is estimated by determining the molecular weight from the amino acid sequence of the second standard substance and calculating the theoretical m / z based on the molecular weight. The theoretical m / z is estimated simply, for example, by multiplying the molecular weight by the mass of a hydrogen atom, 1.08. If a database containing theoretical m / z or molecular weights of second standard substances is available, the theoretical m / z or molecular weight of the second standard substance may be obtained from the database.

[0059] In S33, the processing unit 11 calibrates the mass spectrum based on the measured m / z of the first standard material and the measured m / z of the second standard material indicated by the peaks on the mass spectrum, and the theoretical m / z of the first standard material and the second standard material.

[0060] The processing unit 11 first detects the measured m / z of the first standard substance and the measured m / z of the second standard substance from the mass spectrum. For example, the processing unit 11 detects the measured m / z of the first standard substance by determining, in the mass spectrum, the measured m / z of a peak that falls within a predetermined error range from the theoretical m / z of the first standard substance as the measured m / z of the first standard substance. The error range is, for example, set as a predetermined numerical value. The detection of the measured m / z of the first standard substance is not limited to this. A person skilled in the art may observe the mass spectrum and determine that the measured m / z of a peak that falls within a predetermined error range from the theoretical m / z of the first standard substance is the measured m / z of the first standard substance. Similarly, the processing unit 11 detects the measured m / z of the second standard substance.

[0061] Next, the processing unit 11 accepts a selection of one or more molecules to be used for calibration from among the molecules corresponding to the first standard. This selection is performed, for example, by the user selecting a peak corresponding to the molecule in the mass spectrum using the input unit 131. Similarly, the processing unit 11 accepts a selection of one or more molecules to be used for calibration from among the molecules corresponding to the second standard. Hereinafter, the one or more molecules corresponding to the selected first standard will be referred to as "first molecules." Hereinafter, the one or more molecules corresponding to the selected second standard will be referred to as "second molecules."

[0062] The processing unit 11 then calibrates the mass spectrum based on the measured m / z of the first molecule, the measured m / z of the second molecule, and the theoretical m / z of the first molecule and the theoretical m / z of the second molecule. Calibration of the mass spectrum is performed, for example, as follows.

[0063] When the first molecule and the second molecule each contain one molecule, the measured m / z of the first molecule is calibrated to the theoretical m / z of the first molecule, and the measured m / z of the second molecule is calibrated to the theoretical m / z of the second molecule, and the m / z in the mass spectrum is calibrated linearly according to the calibration.

[0064] When at least one of the first molecule and the second molecule contains a plurality of molecules, the measured m / z of each molecule is calibrated to approach the theoretical m / z of the corresponding molecule. As an example, under the condition that the m / z in the mass spectrum is linearly calibrated, the mass spectrum is calibrated so that the sum of the squares of the errors between the measured m / z and the theoretical m / z for each of these molecules is minimized. That is, the mass spectrum is calibrated so that the sum of the squares of the difference between the measured m / z and the theoretical m / z for the first standard and the squares of the difference between the measured m / z and the theoretical m / z for the second standard is small. As another example, the measured m / z of each of these molecules is calibrated to the respective theoretical m / z, and the measured m / z values ​​between these molecules are also linearly calibrated.

[0065] The method of calibrating a mass spectrum based on a first standard material and a second standard material is not limited to the above example, and may be any method that calibrates the measured m / z of the first standard material so that it approaches the theoretical m / z of the first standard material and the measured m / z of the second standard material so that it approaches the theoretical m / z of the second standard material, and applies the calibration to the entire spectrum. That is, the mass spectrum may be calibrated so that the difference between the measured m / z and the theoretical m / z of the first standard material and the difference between the measured m / z and the theoretical m / z of the second standard material are both small. For example, any one of the methods already known as calibration methods using standard materials, or a combination of several methods, may be used.

[0066] In S34, the processing unit 11 stores the calibration results in the memory unit 12 and / or outputs them from the output unit 132, as necessary. The calibration results include, for example, at least one of the mass spectrum after calibration, the measured m / z value after calibration for a specific substance, and the error value of the measured m / z value relative to the theoretical m / z value after calibration for the specific substance (see, for example, FIGS. 6 and 7, which will be described later). This allows the user to easily save and / or recognize the calibration results. Note that if the calibration results include the above-mentioned indices before calibration, the effects before and after calibration can be compared (see, for example, FIGS. 4 and 5, which will be described later).

[0067] According to the calibration method of this embodiment, as shown in FIG. 3 , a first standard substance having a theoretical m / z smaller than that of the added target substance and a second standard substance having a theoretical m / z larger than that of the target substance derived from a biological sample are used to perform calibration so that the difference between the measured m / z and the theoretical m / z is small for each standard substance. Then, according to this calibration, the measured m / z of the target substance having a theoretical m / z between that of the first standard substance and that of the second standard substance is also calibrated. Therefore, by improving the mass accuracy of the mass spectrometer, the accuracy of the analysis can be improved.

[0068] This calibration method uses a first standard substance with a smaller theoretical m / z than the external standard method, which uses a substance prepared from general Escherichia coli as a standard substance, and therefore can appropriately calibrate a smaller m / z range. Therefore, even when the target substance is a small molecule, accurate analysis can be performed.

[0069] Furthermore, in a typical external standard method, it is necessary to set the conditions for measuring a sample in accordance with the conditions for measuring a calibration sample containing a reference material, but the calibration method according to this embodiment uses an internal standard method, so there is no such restriction, thereby reducing the burden on the user when setting measurement conditions.

[0070] Furthermore, in S32, the theoretical m / z of the second standard substance may be estimated based on the measured m / z of the second standard substance measured in advance using the internal standard method, instead of being calculated from the amino acid sequence. Specifically, the measured m / z of the second standard substance measured in advance using the internal standard method may be used as the theoretical m / z of the second standard substance in this embodiment. The measured m / z measured using the internal standard method is often sufficiently accurate compared to the measured m / z measured using the external standard method. Therefore, even in this embodiment, the theoretical m / z of the second standard substance is often sufficiently accurate. This method is useful when the theoretical m / z cannot be calculated, such as when the amino acid sequence is unknown. Naturally, this method is extremely useful because it can also be applied to molecules other than proteins and molecules that have undergone post-translational modifications.

[0071] 3, the calibration method according to this embodiment can be performed by acquiring a mass spectrum of a biological sample to which a first standard substance has been added. Therefore, the measurement unit 20 is not essential to the present analytical device 1, and the calibration method according to this embodiment may be performed by the control unit 10 based on the mass spectrum acquired from an external source.

[0072] 2 and 3 may also be appropriately automated so that the operations and settings performed by the user are performed by the analytical apparatus 1. For example, when the user places the sample prepared by the user in S1 on the analytical apparatus 1, the user may input information that allows the user to estimate the theoretical m / z value of the target substance, information that allows the user to estimate the theoretical m / z value of the first standard substance, and information that allows the user to estimate the theoretical m / z value of the second standard substance into the analytical apparatus 1, and then input an instruction to execute the calibration method according to this embodiment, so that the subsequent steps are automatically performed by the analytical apparatus 1.

[0073] Furthermore, among the processes described in Figures 2 and 3, processes whose effect does not change even if they are performed in a different order may be performed in a different order, and processes whose effect does not change even if they are performed simultaneously may be performed simultaneously.

[0074] 5. Examples Next, examples performed to verify the effects of the present invention will be described.

[0075] (5-1. Setting the Target Substance) In this example, an Acid Shock protein fragment from Erwinia persicina was used as the target substance. Most microorganisms belonging to the order Enterobacteriaceae possess the gene for Acid Shock protein. However, its amino acid sequence varies greatly depending on the genus and species. Furthermore, it is believed that the protein is cleaved and fragmented at a specific sequence, but this is still under investigation and has not been confirmed. Furthermore, the inventor's research has revealed that Acid Shock protein from Enterobacteriaceae is produced when a medium containing sugar is used. When the target of analysis is thus diverse or uncertain, the accuracy of the hypothesis can be improved by minimizing the error between the theoretical m / z value estimated from the amino acid sequence of the fragment and the actual m / z value. In other words, the actual m / z value of the mass spectrum peak corresponding to the target substance must be obtained with higher accuracy by calibration or other means.

[0076] (5-2. Culture Conditions) To increase the production of Acid Shock Protein, Erwinia persicina was cultured overnight (18 hours) in IFO804 medium (0.5% hypopeptone, 0.5% yeast extract, 0.5% glucose, 0.1% magnesium sulfate). One mL of the suspension in which Erwinia persicina had grown was centrifuged, and the supernatant was discarded to obtain a microbial sediment. The sediment was then resuspended in purified water, centrifuged again, and the supernatant was discarded to obtain a sediment that had been washed with purified water. A 1% trifluoroacetic acid solution was added to this sediment to lyse the bacteria.

[0077] (5-3. Sample Preparation) As a first standard substance having a theoretical m / z smaller than the theoretical m / z of the target substance, which is one of the features of the analytical method according to the present embodiment, angiotensin 1 (molecular weight: 1296.5, theoretical m / z: 1297.5) and angiotensin 2 (molecular weight: 1046.2, theoretical m / z: 1047.2) were added to the lysate to a final concentration of 0.1 pmol / μL.

[0078] The lysate containing the first standard was mixed with an equal volume of 10 mg / mL α-cyano-4-hydroxycinnamic acid (CHCA), and 1 μL of the mixture was dropped onto a MALDI target plate and dried to prepare a sample for mass spectrometry.

[0079] (5-4. Obtaining Mass Spectrum) The sample was placed in a mass spectrometer (Shimadzu Corporation, MALDI-8020), and the measurement range was set to m / z 900 to 15,000, and measurement was performed in positive linear mode to obtain the mass spectrum shown in FIG.

[0080] FIG. 4 shows an example of a mass spectrum before performing the calibration method according to this embodiment. The lower part of FIG. 4 shows a mass spectrum ranging from about m / z 1,000 to about 6,000. The upper part of FIG. 4 shows an enlarged view of the mass spectrum ranging from about m / z 1,800 to about 2,000. In FIG. 4, the horizontal axis corresponds to m / z, and the vertical axis corresponds to the signal intensity ratio. The numbers above each peak indicate the m / z corresponding to the peak. P11 and P12 indicate peaks corresponding to angiotensin 2 and angiotensin 1, respectively. P13 indicates a peak corresponding to 50S ribosomal protein L34 of Erwinia persicina, the second standard material described below. P1 to P7 indicate peaks corresponding to seven fragments of acid shock protein described below.

[0081] FIG. 5 is a table showing the m / z error of the target substance shown in the mass spectrum of FIG. 4 . FIG. 5 was used to calculate the m / z error of the target substance. Based on the inventors' research, it was predicted that the Acid Shock Protein of Erwinia persicina would consist of seven fragments. These seven fragments were assigned "fragment numbers" 1 to 7, and the amino acid sequences corresponding to fragments 1 to 7 are shown in the "Amino Acid Sequence" column. The theoretical m / z values ​​of fragments 1 to 7 estimated from the amino acid sequences are entered in the "Theoretical m / z" column. Then, the peak numbers P1 to P7 determined to correspond to fragments 1 to 7 in the mass spectrum of FIG. 4 are entered in the "Peak Number" column. The m / z values ​​corresponding to peaks P1 to P7 are entered in the "Measured m / z" column. Furthermore, the error between the measured m / z value and the theoretical m / z value is entered in Da and ppm in the "Error" column.

[0082] (5-5. Calibration Based on First and Second Standards) Next, calibration based on the first and second standard materials, another feature of the analytical method according to this embodiment, was performed. Specifically, for a peak with an actual m / z of 1048.217 determined to correspond to angiotensin 2, 1047.2, the theoretical m / z of angiotensin 2, was input as the set value. The set value is a target value to which the actual m / z is to be approximated during calibration. In this example, the analytical device 1 was used to calibrate the m / z of the mass spectrum so that the actual m / z of the standard material would be the set value during calibration. Similarly, for a peak with an actual m / z of 1298.804 determined to correspond to angiotensin 1, 1297.5, the theoretical m / z, was input as the set value.

[0083] Then, as a peak of a second standard substance derived from the sample having a theoretical m / z greater than the theoretical m / z of the target substance, a peak with an actual m / z of 5414.934 determined to correspond to 50S ribosomal protein L34 of Erwinia persicina was selected. The theoretical m / z of the selected peak, 5,410.39, was then input as the set value. The mass spectrum was then calibrated using the analytical apparatus 1 based on the set values ​​of the first standard substance and the second standard substance, and the calibrated mass spectrum shown in FIG. 6 was obtained.

[0084] FIG. 6 shows an example of a mass spectrum obtained after performing the calibration method according to the present embodiment. Similar to FIG. 4 , the lower part of FIG. 6 shows a mass spectrum ranging from about m / z 1,000 to about 6,000. The upper part of FIG. 6 shows an enlarged view of the mass spectrum ranging from about m / z 1,800 to about 2,000. In FIG. 6 , the horizontal axis corresponds to m / z, and the vertical axis corresponds to the signal intensity ratio. The numbers above each peak indicate the m / z corresponding to the peak. P11A and P12A indicate peaks corresponding to angiotensin 2 and angiotensin 1, respectively. P13A indicates a peak corresponding to 50S ribosomal protein L34 of Erwinia persicina, the second standard material. P1A to P7A indicate peaks corresponding to seven fragments of acid shock protein.

[0085] FIG. 7 is a table showing the m / z error of the target substance shown in the mass spectrum of FIG. 6. FIG. 7 was used to calculate the m / z error of the target substance. As in FIG. 5, information corresponding to fragments 1 to 7 of the Acid Shock Protein of Erwinia persicina was entered. Specifically, the same values ​​as in FIG. 5 were entered in the "Amino Acid Sequence," "Theoretical m / z," and "Peak Number" columns of FIG. 7. The calibrated measured m / z value was entered in the "Measured m / z" column of FIG. 7. The error between the calibrated measured m / z and the theoretical m / z was entered in the "Error" column.

[0086] (5-6. Verification of the Effect of the Calibration Method According to the Present Embodiment) The mass spectrum in Figure 4 and the table in Figure 5 are from before the calibration based on the first and second standard materials (Figure 3) was performed, and can be considered as a case where the calibration method according to the present embodiment was not applied. In this case, the error between the predicted theoretical m / z of Acid Shock Protein fragments 1 to 7 and the measured m / z was 1.4 Da (860 ppm) or more.

[0087] In contrast, the mass spectrum in Figure 6 and the table in Figure 7 are obtained after calibration based on the first and second standard materials (Figure 3), and can be considered to be the results obtained when the calibration method according to the embodiment is applied. In this case, the error between the measured m / z and the predicted theoretical m / z for Acid Shock Protein Fragments 1 to 7 was 0.43 Da (220 ppm) or less. In other words, the error in the m / z for the target substance was significantly reduced in the mass spectrum after calibration.

[0088] As described above, the accuracy of the assumption was verified with higher accuracy by performing the calibration method according to the embodiment on the assumed Acid Shock Protein fragment of Erwinia persicina and improving the mass accuracy. In other words, by performing the calibration method according to the present embodiment, analysis of microbial molecules using mass spectrometry could be performed easily and with higher accuracy.

[0089] Aspects It will be understood by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0090] (Item 1) A calibration method according to one embodiment is a method for calibrating a mass spectrum when performing mass analysis on a microbial sample containing a target substance, which is a molecule to be analyzed, and includes the steps of: acquiring a mass spectrum of the microbial sample to which one or more molecules having a smaller estimated theoretical m / z than the target substance have been added as first standard substances; setting one or more molecules derived from the microbial sample having a larger estimated theoretical m / z than the target substance as second standard substances; and calibrating the mass spectrum based on the measured m / z of the first standard substance and the measured m / z of the second standard substance indicated by peaks on the mass spectrum, and the theoretical m / z of the first standard substance and the theoretical m / z of the second standard substance.

[0091] According to the calibration method described in paragraph 1, a mass spectrum can be calibrated using a first standard substance having a theoretical m / z smaller than that of the added target substance and a second standard substance having a theoretical m / z larger than that of the target substance derived from a microbial sample, so that the difference between the theoretical m / z and the measured m / z for these standard substances is small. This allows the mass spectrum in the range corresponding to the molecular weight of the target substance to be appropriately calibrated, even if the molecular weight of the target substance is small. Therefore, by improving the mass accuracy of the mass spectrum, the accuracy of the analysis can be improved.

[0092] (Item 2) In the calibration method described in item 1, the calibrating step includes a step of calibrating the mass spectrum so that the sum of the square of the difference between the measured m / z and the theoretical m / z in the first standard material and the square of the difference in error between the measured m / z and the theoretical m / z in the second standard material is small.

[0093] According to the calibration method described in paragraph 2, the mass spectrum can be calibrated by simple calculation so that the measured m / z values ​​for both the first and second standard materials approach the theoretical m / z values. In other words, the m / z range in the mass spectrum corresponding to the target substance can also be appropriately calibrated by simple calculation.

[0094] (Item 3) In the calibration method according to item 1 or 2, the first standard is a protein or peptide.

[0095] According to the calibration method described in paragraph 3, one of proteins or peptides with a known molecular weight can be selected as the first standard substance depending on the target substance, and the calibration method described in paragraph 1 can be carried out.

[0096] (Item 4) In the calibration method described in any one of Items 1 to 3, the first standard substance comprises at least one of angiotensin 1, angiotensin 2, bradykinin fragment (1-7), P14R, ACTH fragment (18-39), and oxidized insulin B chain.

[0097] According to the calibration method described in paragraph 4, at least one of the molecules with relatively small molecular weights as described above can be selected as the first standard substance depending on the target substance, and the calibration method described in paragraph 1 can be carried out.

[0098] (Item 5) In the calibration method according to any one of Items 1 to 4, the second standard substance comprises a ribosomal protein.

[0099] Ribosomal proteins of microorganisms are often clearly observed by MALDI, and their amino acid sequences can often be found in public databases such as UniProt, making it easy to calculate the molecular weight and theoretical m / z from the amino acid sequence. Therefore, according to the calibration method described in Section 5, a second standard substance can be easily established from these characteristics of ribosomal proteins, allowing for easy calculation of the theoretical m / z.

[0100] (Item 6) In the calibration method according to any one of Items 1 to 5, the theoretical m / z of the second standard is estimated from the amino acid sequence of the second standard.

[0101] According to the calibration method described in Section 6, the theoretical m / z of the second standard can be easily calculated based on the amino acid sequence.

[0102] (Item 7) In the calibration method described in any one of Items 1 to 5, the theoretical m / z of the second standard is estimated based on the measured m / z of the second standard that is measured in advance using an internal standard method.

[0103] According to the calibration method described in Section 7, the actual m / z measured using the internal standard method is often sufficiently accurate, so even if the theoretical m / z of the second standard is set in this way, its accuracy is often sufficient. This method is particularly useful when the theoretical m / z cannot be calculated, such as when the amino acid sequence is unknown.

[0104] (Item 8) An analytical method further comprising the step of analyzing a target substance based on a mass spectrum calibrated using the calibration method according to any one of items 1 to 7.

[0105] According to the calibration method described in paragraph 8, the target substance can be analyzed by an analytical method with improved analytical accuracy, thereby obtaining more accurate analytical results.

[0106] (Item 9) A control device according to another aspect performs mass spectral calibration when performing mass analysis on a microbial sample containing a target substance, which is a molecule to be analyzed. The control device includes a memory and a processor. The memory stores the theoretical m / z of a first standard, which is one or more molecules having a smaller estimated theoretical m / z than the target substance. The processor acquires a mass spectrum of the microbial sample to which the first standard has been added. The processor sets one or more molecules derived from the microbial sample having a larger estimated theoretical m / z than the target substance as second standard substances. The processor calibrates the mass spectrum based on the measured m / z of the first standard and the measured m / z of the second standard, which are indicated by peaks in the mass spectrum, and the theoretical m / z of the first standard and the second standard.

[0107] According to the control device described in paragraph 9, a mass spectrum can be calibrated using a first standard substance having a theoretical m / z smaller than that of the added target substance and a second standard substance having a theoretical m / z larger than that of the target substance derived from the microbial sample, so that the difference between the theoretical m / z and the measured m / z for these standard substances is small. This allows the mass spectrum in the range corresponding to the molecular weight of the target substance to be appropriately calibrated, even if the molecular weight of the target substance is small. Therefore, improving mass accuracy can improve analytical accuracy.

[0108] (Item 10) An analytical device according to another aspect performs mass analysis on a microbial sample containing a target substance, which is a molecule to be analyzed, and includes a measurement unit, a memory, and a processor. The measurement unit acquires measurement data of the microbial sample. The memory stores theoretical m / z values ​​of first standards, which are one or more molecules having a smaller estimated theoretical m / z value than the target substance. The processor acquires a mass spectrum of the microbial sample to which the first standards have been added, based on the measurement data. The processor sets one or more molecules derived from the microbial sample having a larger estimated theoretical m / z value than the target substance as second standards. The processor calibrates the mass spectrum based on the measured m / z values ​​of the first and second standards, which are indicated by peaks in the mass spectrum, and the theoretical m / z values ​​of the first and second standards.

[0109] According to the analytical device described in paragraph 9, a mass spectrum can be calibrated using a first standard substance having a theoretical m / z smaller than that of the added target substance and a second standard substance having a theoretical m / z larger than that of the target substance derived from the microbial sample, so that the difference between the theoretical m / z and the measured m / z for these standard substances is small. This allows the mass spectrum in the range corresponding to the molecular weight of the target substance to be appropriately calibrated, even if the molecular weight of the target substance is small. Therefore, by improving the mass accuracy of the mass spectrum, the analytical accuracy of the analytical device can be improved.

[0110] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims.

[0111] 1 analytical device, 10 control unit, 11 processing unit, 12 memory unit, 13 input / output unit, 18 fragment, 20 measurement unit, 21 ionization unit, 22 ion acceleration unit, 23 mass separation unit, 24 detection unit, 111 device control unit, 112 mass spectrum creation unit, 113 mass spectrum analysis unit, 114 calibration unit, 131 input unit, 132 output unit, 133 communication unit, 221 acceleration electrode, 231 flight tube, S ion.

Claims

1. A method for calibrating a mass spectrum when performing mass spectrometry on a microorganism sample containing a target substance that is a molecule to be analyzed, comprising the steps of: Acquiring a mass spectrum of the microbial sample to which one or more molecules having a theoretical m / z smaller than that of the target substance have been added as a first standard substance; setting one or more molecules derived from the microbial sample and having a theoretical m / z larger than that of the target substance as a second standard substance; and calibrating the mass spectrum based on the measured m / z of the first standard material and the measured m / z of the second standard material indicated by peaks on the mass spectrum, and the theoretical m / z of the first standard material and the theoretical m / z of the second standard material.

2. 2. The calibration method of claim 1, wherein the calibrating step includes a step of calibrating the mass spectrum so that a sum of the square of the difference between the measured m / z and the theoretical m / z in the first standard material and the square of the difference between the measured m / z and the theoretical m / z in the second standard material is small.

3. 3. The calibration method according to claim 1, wherein the first standard is a protein or a peptide.

4. 3. The calibration method according to claim 1, wherein the first standard comprises at least one of angiotensin 1, angiotensin 2, bradykinin fragment (1-7), P14R, ACTH fragment (18-39), and oxidized insulin B chain.

5. The calibration method according to claim 1 or 2, wherein the second standard comprises a ribosomal protein.

6. The calibration method according to claim 1 or 2, wherein the theoretical m / z of the second standard is calculated from an amino acid sequence of the second standard.

7. 3. The calibration method according to claim 1, wherein the theoretical m / z of the second standard substance is an actual m / z of the second standard substance that is actually measured in advance using an internal standard method.

8. An analytical method comprising the step of analyzing the target substance based on a mass spectrum calibrated using the calibration method according to claim 1 or 2.

9. A control device for performing mass spectrum calibration when performing mass spectrometry on a microorganism sample containing a target substance that is a molecule to be analyzed, comprising: A memory that stores a theoretical m / z of a first standard substance, which is one or more molecules having a theoretical m / z smaller than that of the target substance; a processor; The processor, Obtaining a mass spectrum of the microbial sample to which the first standard has been added; One or more molecules derived from the microbial sample and having a theoretical m / z larger than that of the target substance are set as a second standard substance; A control device that calibrates the mass spectrum based on the measured m / z of the first standard material and the measured m / z of the second standard material indicated by peaks on the mass spectrum, and the theoretical m / z of the first standard material and the theoretical m / z of the second standard material.

10. An analytical apparatus for performing mass spectrometry on a microbial sample containing a target substance that is a molecule to be analyzed, comprising: A measurement unit for acquiring measurement data of the microbial sample; A memory that stores a theoretical m / z of a first standard substance, which is one or more molecules having a theoretical m / z smaller than that of the target substance; a processor; The processor, obtaining a mass spectrum of the microbial sample to which the first standard substance has been added based on the measurement data; One or more molecules derived from the microbial sample and having a theoretical m / z larger than that of the target substance are set as a second standard substance; An analytical apparatus that calibrates the mass spectrum based on the measured m / z of the first standard material and the measured m / z of the second standard material indicated by peaks on the mass spectrum, and the theoretical m / z of the first standard material and the theoretical m / z of the second standard material.

11. A method for calibrating a mass spectrum when performing mass spectrometry on a microbial sample containing a target substance that is a molecule to be analyzed, comprising the steps of: Acquiring a mass spectrum of the microbial sample to which one or more molecules having a theoretical m / z smaller than that of the target substance have been added as a standard substance; and calibrating the mass spectrum based on an actual m / z of the standard material indicated by a peak on the mass spectrum and a theoretical m / z of the standard material.

12. The calibration method of claim 11, wherein the standard substance is a protein or a peptide.

13. The calibration method according to claim 11 or 12, wherein the standard substance comprises at least one of angiotensin 1, angiotensin 2, bradykinin fragment (1-7), P14R, ACTH fragment (18-39), and oxidized insulin B chain.

14. An analytical method comprising a step of analyzing the target substance based on a mass spectrum calibrated using the calibration method described in claim 11 or 12.

15. A control device for performing mass spectral calibration when performing mass spectrometry on a microbial sample containing a target substance that is a molecule to be analyzed, comprising: A memory that stores theoretical m / z values ​​of a standard substance, which is one or more molecules having a theoretical m / z value smaller than that of the target substance; a processor; The processor, Obtaining a mass spectrum of the microbial sample to which the standard substance has been added; A control device that calibrates the mass spectrum based on an actual m / z of the standard material indicated by a peak on the mass spectrum and a theoretical m / z of the standard material.

16. An analytical apparatus for performing mass spectrometry on a microbial sample containing a target substance that is a molecule to be analyzed, comprising: A measurement unit for acquiring measurement data of the microbial sample; A memory that stores theoretical m / z values ​​of a standard substance, which is one or more molecules having a theoretical m / z value smaller than that of the target substance; a processor; The processor, A mass spectrum of the microbial sample to which the standard substance has been added is obtained based on the measurement data; An analytical apparatus that calibrates the mass spectrum based on an actual m / z of the standard material indicated by a peak on the mass spectrum and a theoretical m / z of the standard material.