Mass spectrometry analysis device error correction method

Abstract

A method for correcting the signal intensity ratio in mass spectrometry includes the following steps: measuring a calibrator containing two or more calibrators using a mass spectrometer to obtain the peak signals of each calibrator; determining the peak signal intensity ratio of the peak signal intensity of one of the two or more calibrators relative to the peak signal intensity of another calibrator; determining a correction formula based on the peak signal intensity ratio; measuring a sample containing two or more analytes using the mass spectrometer to obtain the peak signals of each analyte; determining the peak signal intensity ratio of the peak signal intensity of one of the two or more analytes relative to the peak signal intensity of another analyte; and correcting the peak signal intensity ratio of the analytes using the correction formula.

Description

Technical Field

[0001] This invention relates to a method for using peptide ratios obtained through mass spectrometry analysis. This invention also relates to a method and system for correcting the machine error of a mass spectrometry analysis device. Background Technology

[0002] When using a mass spectrometer to compare and analyze the amount of a substance, the most common method is to use the intensity ratio of two peak signals. For example, a fixed amount of internal standard material is added to the sample to be compared, and pretreatment is performed if necessary. Then, the sample is analyzed by mass spectrometry, and the values ​​obtained by calculating the intensity ratio of the target peak to the internal standard material peak are compared (Non-Patent Literature 1, 2, 3).

[0003] Examples of patent documents include international publication WO2015 / 178398 (US publication US2017 / 0184573) and international publication WO2017 / 047529 (US publication US2018 / 0238909).

[0004] Furthermore, it is possible to label target substances derived from different samples with labeled compounds of different masses due to the use of stable isotopic elements, and then perform semi-quantitative comparative analysis by calculating the intensity ratio of the peaks of target substances with different masses through mass spectrometry. The ICAT (registered trademark) and iTRAQ (registered trademark) methods used in the field of proteomics are equivalent to this method (Non-Patent Literature 4, 5).

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication WO2015 / 178398

[0008] Patent Document 2: US Publication US2017 / 0184573

[0009] Patent Document 3: International Publication WO2017 / 047529

[0010] Patent Document 4: US Publication US2018 / 0238909

[0011] Non-patent literature

[0012] Non-Patent Literature 1: Kaneko N, Nakamura A, Washimi Y, Kato T, Sakurai T, Arahata Y, Bundo M, Takeda A, Niida S, Ito K, Toba K, Tanaka K, Yanagisawa K.: Novel plasma biomarker surrogating cerebral amyloid deposition. Proc Jpn Acad Ser B Phys Biol Sci. 2014; 90(9):353-364.

[0013] Non-Patent Literature 2: Nakamura A, Kaneko N, Villemagne VL, Kato T, Doecke J, Doré V, Fowler C, Li QX, Martins R, Rowe C, Tomita T, Matsuzaki K, Ishii K, Ishii K, Arahata Y, Iwamoto S, Ito K, Tanaka K, Masters CL, Yanagisawa K.: High performance plasma amyloid-β biomarkers for Alzheimer's disease. Nature. 2018; 554(7691):249-254.

[0014] Non-patent literature 3: Nicol GR, Han M, Kim J, Birse CE, Brand E, Nguyen A, Mesri M, FitzHugh W, Kaminker P, Moore PA, Ruben SM, He T: Use of an immunoaffinity-massspectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients. Mol Cell Proteomics. 2008 Oct; 7(10):1974-82.

[0015] Non-patent literature 4: Han DK, Eng J, Zhou H, Aebersold R: Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat Biotechnol. 2001 Oct; 19(10): 946-51.

[0016] Non-patent literature 5: Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ: Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 2004 Dec; 3(12): 1154-69. Summary of the Invention

[0017] The problem the invention aims to solve

[0018] When using mass spectrometry to determine trace amounts of substances, the following phenomenon has been observed: even with the same model, different spectrometers produce different peak intensity ratios. As one method to correct for differences in peak intensity ratios between different spectrometers, the following absolute quantification methods are listed.

[0019] Prepare a standard for the target substance to be quantified and a reference substance (usually a stable isotope-labeled substance of the target substance) as a reference for the intensity ratio. A calibration curve is constructed by measuring a sample prepared by mixing a reference substance of fixed concentration with standards adjusted to different concentrations, to determine the peak intensity ratio of the standard relative to the reference substance. Absolute quantification of the target substance can be performed based on this calibration curve. Therefore, by constructing calibration curves for each apparatus and each measurement, even if there are differences in peak intensity ratios between different apparatuses, the unknown target substance present in biological samples can be quantified without being affected by these differences.

[0020] However, this calibration curve method requires standards. Calibration curves cannot be established when standards cannot be easily synthesized, when the variety of target substances is vast and it is difficult to prepare and manage all standards in terms of time and cost, or when the standards are unstable. As mentioned above, due to variations in peak intensity ratios between mass spectrometers, when calibration curves cannot be established, a single instrument must be used to measure the sample used as a comparison. Even then, detector degradation due to instrument use can occur, causing variations in peak intensity ratios and making it difficult to obtain consistent data.

[0021] In mass spectrometry, a laser is used to ionize the sample. Therefore, the data is affected by the condition of the laser in the mass spectrometry apparatus (deterioration due to factors such as the number of uses). Consequently, even when measuring the same sample, significant differences exist between different apparatuses. This results in insufficient data stability.

[0022] The purpose of this invention is to provide a method for correcting differences in mass spectrometry analysis data caused by the physical characteristics of the mass spectrometry analysis device, and a system for correcting the physical characteristics of the mass spectrometry analysis device.

[0023] Solution for solving the problem

[0024] The present invention includes the following solutions.

[0025] A method for correcting the mechanical error of the signal intensity ratio in mass spectrometry analysis includes the following steps:

[0026] The peak signals of each calibration substance are obtained by measuring the calibrator containing two or more calibration substances using a mass spectrometry analysis device.

[0027] Calculate the peak signal intensity ratio of one of the two or more calibration substances to the peak signal intensity of the other calibration substance;

[0028] The correction formula is derived based on the peak signal intensity ratio.

[0029] The peak signals of each analyte are obtained by measuring a sample containing two or more analyte substances using the mass spectrometry analysis device.

[0030] Calculate the peak signal intensity ratio of one analyte to the peak signal intensity of another analyte among the two or more analytes; and

[0031] The peak signal intensity ratio of the analyte is corrected using the aforementioned correction formula.

[0032] The present invention also includes the following solutions.

[0033] A mass spectrometer error correction system includes:

[0034] The method development department develops methods for determining calibrators used to calculate calibration values ​​for mass spectrometry analyzers; and

[0035] The correction value calculation unit analyzes the mass spectrometry analysis data obtained using the measurement method and calculates the correction value.

[0036] The effects of the invention

[0037] According to the present invention, the peak intensity ratio of the analyte can be corrected using a correction formula derived from the measurement results of a calibration substance. As a result, the same peak intensity ratio can be obtained even when the same sample is measured using different instruments. Attached Figure Description

[0038] Figure 1 The results are shown in the determination of Aβ and Aβ-related peptides by three mass spectrometers (Performance 1, 2 and 3) after immunoprecipitation (IP) of a plasma sample (Sample No. 1) incorporating an internal standard peptide (SIL-Aβ1-38). Figure 1 The vertical axis of (A) shows the intensity ratio of each peak of Aβ or Aβ-related peptide relative to SIL-Aβ1-38. Figure 1 The vertical axis of (B) shows the intensity ratios of the individual peaks of Aβ, Aβ-related peptide, or SIL-Aβ1-38 relative to APP669-711.

[0039] Figure 2 The vertical axis shows the coefficient of variation (CV) of the peak intensity ratios measured by three mass spectrometers (Performance 1, 2, and 3) after IP treatment of the plasma sample (Sample No. 1). The horizontal axis shows the average values ​​measured by the three mass spectrometers.

[0040] Figure 3The vertical axis shows the logarithmic transformation of the peak intensity ratios measured using Performance 1 (the standard) after IP treatment of the plasma sample (Sample No. 1). The horizontal axis shows the logarithmic transformation of the peak intensity ratios measured using Performance 2 or 3 for the same sample. The graph also shows the linear regression equation for the values ​​measured using Performance 2 or 3 relative to the values ​​measured using Performance 1 (the standard), and the coefficient of determination (R²). 2 ).

[0041] Figure 4 The vertical axis shows the peak intensity ratios measured using Performance 1 (the standard) after IP treatment of the plasma sample (Sample No. 1). The horizontal axis shows the peak intensity ratios measured using Performance 2 or 3 for the same sample. The figure also shows the approximate power of the measured values ​​of Performance 2 or 3 relative to the measured values ​​of Performance 1 (the standard), and the coefficient of determination (R²). 2 ).

[0042] Figure 5 This shows the value obtained by correcting the peak intensity ratio of Aβ or Aβ-related peptides measured by Performance 2 and Performance 3 relative to the internal standard peptide (SIL-Aβ1-38) to the same peak intensity ratio as Performance 1 (the standard). Performance 2 (Cal.) and Performance 3 (Cal.) refer to the calibrated value. Figure 5 The vertical axis shows the intensity ratio of each peak of Aβ or Aβ-related peptide relative to SIL-Aβ1-38. Figure 5 The values ​​(%) in the table represent the coefficient of variation (CV) of the peak intensity ratio in Performance 1 (standard), Performance 2 (Cal.), and Performance 3 (Cal.).

[0043] Figure 6 The peak intensity ratios of Aβ or Aβ-related peptide relative to SIL-Aβ1-38 are shown when measured by Performance 1, 2 and 3 after IP treatment of plasma sample (Sample No.2). Figure 6 (A) shows the peak intensity ratios of Aβ or Aβ-related peptides relative to SIL-Aβ1-38 as determined by Performance 2 and Performance 3, before correction using a correction formula. Figure 6 (B) shows the corrected peak intensity ratio. Performance 2 (Cal.) and Performance 3 (Cal.) refer to the calibrated values ​​obtained from this correction. Figure 6 The values ​​(%) in (A) and (B) represent the coefficient of variation (CV) of the peak intensity ratio in Performance 1 (standard), Performance 2 (Cal.), and Performance 3 (Cal.).

[0044] Figure 7 The peak intensity ratios of Aβ or Aβ-related peptide relative to SIL-Aβ1-38 are shown when measured by Performance 1, 2 and 3 after IP treatment of plasma sample (Sample No. 3). Figure 7 (A) shows the peak intensity ratios of Aβ or Aβ-related peptides relative to SIL-Aβ1-38 as determined by Performance 2 and Performance 3, before correction using a correction formula. Figure 7 (B) shows the corrected peak intensity ratio. Performance 2 (Cal.) and Performance 3 (Cal.) refer to the calibrated values ​​obtained from this correction. Figure 7 The values ​​(%) in (A) and (B) represent the coefficient of variation (CV) of the peak intensity ratio in Performance 1 (standard), Performance 2 (Cal.), and Performance 3 (Cal.).

[0045] Figure 8 The results of IP-MS analysis on plasma sample (Sample No. 1) over two separate days (Day 1 and Day 2) are shown. The vertical axis shows the logarithmic transformation of the peak intensity ratios measured using Performance 1 (the standard) for each day. Similarly, the horizontal axis shows the logarithmic transformation of the peak intensity ratios measured using Performance 2. The graph also shows the linear regression equation of the Performance 2 measurements relative to the Performance 1 measurements, and the coefficient of determination (R²). 2 ).

[0046] Figure 9The results of IP-MS analysis on plasma sample (Sample No. 1) over two separate days (Day 1 and Day 2) are shown. The vertical axis shows the logarithmic transformation of the peak intensity ratios measured using Performance 1 (the standard) for each day. Similarly, the horizontal axis shows the logarithmic transformation of the peak intensity ratios measured using Performance 3. The graph also shows the linear regression equation of the Performance 3 measurements relative to the Performance 1 measurements, and the coefficient of determination (R²). 2 ).

[0047] Figure 10 The results of IC-1 to IC-5 measurements are shown under three conditions: before and after detector replacement using Performance 1 and Performance 3. The horizontal axis shows the ratio of Aβ1-38 to SIL-Aβ1-38, and the vertical axis shows the peak intensity ratio of Aβ1-38 to SIL-Aβ1-38. The power approximation formulas are shown in the figure. Figure 10 (A) shows the results of Performance 1 (before detector replacement). Figure 10 (B) shows the results of Performance 1 (after detector replacement). Figure 10 The results of Performance 3 are shown in (C).

[0048] Figure 11 The peak intensity ratios of Aβ or Aβ-related peptides relative to SIL-Aβ1-38 obtained by IP-MS under three conditions—before and after detector replacement—on Performance 1 and Performance 3. Figure 11 (A) shows the peak intensity ratio before correction for the peak intensity ratio of Aβ or Aβ-related peptide relative to SIL-Aβ1-38 using a correction formula. Figure 11 (B) shows the corrected peak intensity ratio. Correction was performed by fixing the value of a to 1 (a = 1) and using only the value of b.

[0049] Figure 12 The peak intensity ratios of biomarkers obtained by IP-MS under three conditions—before and after detector replacement—are shown, namely the peak intensity ratios of APP669-711 / Aβ1-42 and Aβ1-40 / Aβ1-42. Figure 12 (A) shows the peak intensity ratios of the biomarkers before correction using a correction formula. Figure 12(B) shows the corrected peak intensity ratio. Correction was performed by fixing the value of a to 1 (a = 1) and using only the value of b.

[0050] Figure 13 The peak intensity ratios of Aβ or Aβ-related peptides relative to SIL-Aβ1-38 obtained by IP-MS under three conditions—before and after detector replacement—on Performance 1 and Performance 3. Figure 13 (A) shows the peak intensity ratio before correction for the peak intensity ratio of Aβ or Aβ-related peptide relative to SIL-Aβ1-38 using a correction formula. Figure 13 (B) shows the corrected peak intensity ratio. Correction was performed using the a-value and b-value method.

[0051] Figure 14 The peak intensity ratios of biomarkers obtained by IP-MS under three conditions—before and after detector replacement—are shown, namely the peak intensity ratios of APP669-711 / Aβ1-42 and Aβ1-40 / Aβ1-42. Figure 14 (A) shows the peak intensity ratio before correction using the correction formula. Figure 14 (B) shows the corrected peak intensity ratio. Correction was performed using the a-value and b-value method.

[0052] Figure 15 The comparison of the Aβ1-40 / Aβ1-42 ratio before and after b-value correction is shown. The error bars show the standard deviations of the three sets of measurements.

[0053] Figure 16 The comparison of the APP669-711 / Aβ1-42 ratio before and after b-value correction is shown. The error bars show the standard deviations in the three sets of measurements.

[0054] Figure 17 This is a graph showing the relationship between the detector voltage and the b-value of the calibration formula in Performance 4. The vertical axis represents the b-value, and the horizontal axis represents the detector voltage.

[0055] Figure 18 This is a graph showing the relationship between the detector voltage and the b-value of the calibration formula in Performance 1. The vertical axis represents the b-value, and the horizontal axis represents the detector voltage.

[0056] Figure 19This is a graph showing the relationship between the baseline level of the AD converter in Performance 1 and the b-value of the calibration formula. The vertical axis represents the b-value, and the horizontal axis represents the detector voltage. It shows the relationship between the detector voltage and the b-value when the baseline level of the AD converter is set to 181 and 179.

[0057] Figure 20 This is a schematic block diagram of one embodiment of the calibration value calculation system of the mass spectrometry analysis apparatus of the present invention.

[0058] Figure 21 This is a flowchart illustrating an example of the process for calculating the calibration values ​​of the mass spectrometry analysis apparatus in this invention.

[0059] Figure 22 This is an example of a graphical user interface (GUI) displayed on display unit 4 when a measurement method is established.

[0060] Figure 23 This is an example of a graphical user interface (GUI) displayed on display unit 4 when calculating correction values. Detailed Implementation

[0061] One embodiment of the method of the present invention is a method for correcting the mechanical error of the signal intensity ratio in mass spectrometry analysis, comprising the following steps:

[0062] The peak signals of each calibration substance are obtained by measuring the calibrator containing two or more calibration substances using a mass spectrometry analysis device.

[0063] Calculate the peak signal intensity ratio of one of the two or more calibration substances to the peak signal intensity of the other calibration substance;

[0064] The correction formula is derived based on the peak signal intensity ratio.

[0065] The peak signals of each analyte are obtained by measuring a sample containing two or more analyte substances using the mass spectrometry analysis device.

[0066] Calculate the peak signal intensity ratio of one analyte to the peak signal intensity of another analyte among the two or more analytes; and

[0067] The peak signal intensity ratio of the analyte is corrected using the aforementioned correction formula.

[0068] The following is a detailed description of this embodiment. Furthermore, in the embodiments described later, [the following is used] Figures 1 to 19 More specific examples are shown.

[0069] [1. Analyzing the object of analysis]

[0070] The analyte is not particularly limited and can include peptides, glycopeptides, glycans, proteins, lipids, glycolipids, etc. Various substances can be included among peptides, glycopeptides, glycans, proteins, lipids, and glycolipids. More specifically, it can also be Aβ and Aβ-related peptides. "Aβ and Aβ-related peptides" is sometimes simply referred to as "Aβ-related peptides." "Aβ and Aβ-related peptides" includes Aβ, generated by cleaving amyloid precursor protein (APP), and peptides containing a portion of the Aβ sequence. Examples using Aβ and Aβ-related peptides are shown in the embodiments.

[0071] Alternatively, peptides can be obtained through immunoprecipitation (IP). They can also be peptides produced by digesting proteins with enzymes such as peptidases, or peptides separated by chromatography.

[0072] Internal standard substances may also be included in the analyte. These internal standard substances can be appropriately selected by those skilled in the art. For example, substances labeled with stable isotopes may also be used. Alternatively, one substance in the analyte may use a substance labeled with a stable isotope. In the embodiments, an example is shown of using Aβ1-38 (SIL-Aβ1-38) labeled with a stable isotope as an internal standard substance. SIL stands for stable isotope-labeled.

[0073] Samples containing the analyte are used in mass spectrometry analysis. The samples used in mass spectrometry analysis are not particularly limited; for example, they can be samples derived from living organisms. Samples derived from living organisms include bodily fluids such as blood, cerebrospinal fluid (CSF), urine, bodily secretions, saliva, and sputum, as well as feces. Blood samples include whole blood, plasma, and serum. Blood samples can be prepared by appropriately processing whole blood collected from an individual. The processing performed when preparing blood samples from collected whole blood is not particularly limited; any clinically permissible processing can be performed. For example, centrifugation can be performed. Furthermore, blood samples used in mass spectrometry analysis can also be samples that have been appropriately preserved at low temperatures, such as freezing, during or after the preparation process. Moreover, in the case of using biologically derived samples such as blood samples in mass spectrometry analysis according to this invention, the biologically derived samples are discarded and not returned to the subject from whom they originated.

[0074] The sample used in mass spectrometry analysis can also be a sample that has undergone various pretreatments. For example, it can be a sample after immunoprecipitation (IP), a sample after protein digestion by enzymes such as peptidases, or a sample after chromatography. The sample used in mass spectrometry analysis can also be a sample with a fixed amount of internal standard material added.

[0075] In this embodiment, the eluent obtained by immunoprecipitation can also be used in mass spectrometry analysis after prior immunoprecipitation (IP-MS). Immunoprecipitation can also be performed using immunoglobulins with antigen-binding sites that can recognize the analyte, or using antibody immobilization carriers made from immunoglobulin fragments containing antigen-binding sites that can recognize the analyte.

[0076] Alternatively, in this embodiment, consecutive immunoprecipitation (cIP) can be performed, followed by detection of peptides in the sample using mass spectrometry (cIP-MS). By performing affinity purification twice consecutively, impurities that cannot be eliminated by a single affinity purification can be further reduced through the second affinity purification. Therefore, ionization inhibition of peptides due to impurities can be prevented, and even trace amounts of peptides in biological samples can be measured with high sensitivity using mass spectrometry.

[0077] [2. Mass Spectrometry Analysis]

[0078] Mass spectrometry is not particularly limited in method, and includes mass spectrometry based on matrix-assisted laser desorption / ionization (MALDI) mass spectrometry, electrospray ionization (ESI) mass spectrometry, etc. For example, it is possible to use MALDI-TOF (matrix-assisted laser desorption / ionization-time-of-flight) mass spectrometry devices, MALDI-IT (matrix-assisted laser desorption / ionization-ion trap) mass spectrometry devices, MALDI-IT-TOF (matrix-assisted laser desorption / ionization-ion trap-time-of-flight) mass spectrometry devices, MALDI-FTICR (matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance) mass spectrometry devices, ESI-QqQ (electrospray ionization-triple quadrupole) mass spectrometry devices, ESI-Qq-TOF (electrospray ionization-tandem quadrupole-time-of-flight) mass spectrometry devices, and ESI-FTICR (electrospray ionization-Fourier transform ion cyclotron resonance) mass spectrometry devices, etc.

[0079] The matrix and matrix solvent can be appropriately determined by those skilled in the art based on the analyte.

[0080] As a matrix, for example, α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB), sinapic acid, 3-aminoquinoline (3-AQ) and the like can be used.

[0081] As a matrix solvent, it can be selected from, for example, from the group consisting of acetonitrile (ACN), trifluoroacetic acid (TFA), methanol, ethanol, and water. More specifically, aqueous solutions of ACN-TFA, aqueous solutions of ACN, aqueous solutions of methanol-TFA, aqueous solutions of methanol, aqueous solutions of ethanol-TFA, and ethanol solutions can be used. The concentration of ACN in the aqueous solution of ACN-TFA is, for example, 10 vol% to 90 vol%, and the concentration of TFA is, for example, 0.05 vol% to 1 vol%, preferably 0.05 vol% to 0.1 vol%.

[0082] The matrix concentration can be, for example, 0.1 mg / mL to 50 mg / mL, preferably 0.1 mg / mL to 20 mg / mL, or 0.3 mg / mL to 20 mg / mL, more preferably 0.5 mg / mL to 10 mg / mL.

[0083] When using a detection system based on MALDI mass spectrometry analysis, it is preferable to use a co-matrix. The co-matrix can be appropriately selected by those skilled in the art based on the analyte (peptide) and / or the matrix. For example, compounds containing phosphonic acid groups can be used as co-matrix. Specifically, examples of compounds containing one phosphonic acid group include phosphonic acid, methylphosphonic acid, phenylphosphonic acid, and 1-naphthylmethylphosphonic acid. In addition, examples of compounds containing two or more phosphonic acid groups include methylenediphosphonic acid (MDPNA), ethylenediphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid, nitrilotriphosphonic acid, and ethylenediaminetetraphosphonic acid. Among the above-mentioned phosphonic acid-containing compounds, compounds having two or more, preferably two to four phosphonic acid groups per molecule are preferred.

[0084] The use of phosphonate-containing compounds is useful, for example, in cases where metal ions from the washing solution remaining on the surface of an antibody immobilization carrier are mixed into the eluent after the dissociation process. These metal ions adversely affect the background in mass spectrometry analysis. The use of phosphonate-containing compounds can suppress this adverse effect.

[0085] In addition to the matrix additives mentioned above, more general additives may also be used, such as substances selected from the group consisting of ammonium salts and organic bases.

[0086] The matrix additive can be prepared into a solution of 0.1 w / v% to 10 w / v%, preferably 0.2 w / v% to 4 w / v%, in water or a matrix solvent. The matrix additive solution and the matrix solution can be mixed, for example, at a volume ratio of 1:100 to 100:1, preferably 1:10 to 10:1.

[0087] [3. Calibration materials]

[0088] In this embodiment, two or more calibration substances are measured using a mass spectrometry analyzer. The measurement results of the calibration substances are used to calculate the calibration formula for the mass spectrometry analyzer.

[0089] The calibration material can be appropriately determined by those skilled in the art. For example, the analyte itself can be used, or a different substance can be used. A substance labeled with a stable isotope can also be used. Both stable and unstably labeled substances can be used. Aβ and Aβ-related peptides can also be used as calibration materials. When Aβ and Aβ-related peptides are used as the analyte, for example, Aβ1-38 (SIL-Aβ1-38) labeled with a stable isotope can be used as a calibration material.

[0090] Calibration materials may also consist of a compound and a substance stably labeled with that compound. In mass spectrometry, the compound is ionized for detection. Ionization efficiency varies depending on the compound, and differences in ionization efficiency can affect the mass spectrometry results. A particular compound may have the same ionization efficiency as a substance stably labeled with that compound. Therefore, by using them as calibration materials, more accurate calibration formulas can be obtained. For example, in Example 2, Aβ1-38 and Aβ1-38 stably labeled with a stable isotope (SIL-Aβ1-38) were used as calibration materials.

[0091] [4. Calibrators]

[0092] The calibrator is a solution containing calibrating substances. In this embodiment, the calibrator is a solution containing two or more calibrating substances.

[0093] As a calibrator, the sample itself containing the analyte can be used, for example. Alternatively, a substance obtained by adding a calibrator to the sample itself containing the analyte can also be used. Solutions containing two or more calibrators can also be prepared and used separately from the sample itself containing the analyte.

[0094] The calibration formula for the mass spectrometer is calculated using the measurement results of the solution containing two or more calibration substances. Multiple data points are required to calculate the calibration formula. For example, when calculating the calibration formula using peak signal intensity ratios, multiple data points with different peak signal intensity ratios are needed.

[0095] For example, when calculating a correction formula using a single calibrator, a calibrator containing at least three calibrators is required. Alternatively, the peak signal intensity ratios of two or more calibrators can be obtained by calculating the ratio of the peak signal intensity of one calibrator to the peak signal intensity of the other two or more calibrators.

[0096] For example, when the calibration substances are described as C1, C2, and C3, the peak intensity ratios of C2 / C1 and C3 / C1 are calculated using C1 as the reference.

[0097] Alternatively, multiple calibrators with different concentrations of the calibrator for which the peak signal intensity ratio is to be calculated can be used. In this case, at least two calibrators are required. Alternatively, more than two peak signal intensity ratios can be obtained by calculating the ratio of the peak signal intensity of one calibrator to the peak signal intensity of the other calibrators for each calibrator.

[0098] For example, when calibrators are described as C1 and C2, the peak intensity ratio of C2 / C1 is calculated for multiple calibrators with different concentrations.

[0099] Multiple calibrators can also be used, where the concentration of one calibrator is fixed and the concentrations of the other calibrators are varied. The concentration ratio of the calibrator for which the peak signal intensity ratio is to be calculated among the multiple calibrators can, for example, be set to a range of 1 / 4 to 4. For example, it is also possible to use five calibrator solutions with concentration ratios adjusted to 1 / 4, 1 / 2, 1, 2, and 4.

[0100] [5. Correction of measurement results in mass spectrometry]

[0101] In this embodiment, the calibration formula for the mass spectrometry analyzer is calculated using the results obtained from measuring two or more calibration substances using a mass spectrometry analyzer. Then, the measured results of the analyte are corrected using the calculated calibration formula. The correction method in this invention includes a method of correction using a calibration formula with a standard. Additionally, it includes a method of standardizing the measured results in mass spectrometry analysis using calibration substances present in known quantities.

[0102] Calibration formulas are calculated for each mass spectrometer. Even for the same mass spectrometer, it is preferable to recalculate the calibration formula if components such as detectors are replaced, or if device settings such as detector voltage are changed. Alternatively, calibration formulas can be calculated periodically. This allows for the detection of deterioration or malfunctions in the mass spectrometer's detectors and other components, and the acquisition of measurement results after eliminating their influence. Therefore, regardless of the mass spectrometer's structure or conditions, the presence ratio of analytes among multiple samples can be compared and evaluated with high precision.

[0103] More preferably, when determining the analyte, the calibration substance is measured under the same apparatus conditions as the determination, and the calibration formula is calculated. The calibration formula can be used under the same conditions as the determination of the analyte, thereby further improving the accuracy of the calibration. Therefore, regardless of the mass spectrometry apparatus or its conditions, the presence ratio of the analyte among multiple samples can be compared and evaluated with higher precision.

[0104] [5-1. Calibration using a calibration formula with a standard]

[0105] [5-1-1. Calculation of the correction formula]

[0106] In a standard apparatus, a calibrator solution containing two or more calibrators is measured to obtain the peak signal intensity of each calibrator. The ratio of the peak signal intensity of each of the other calibrators to the peak signal intensity of one calibrator is then calculated.

[0107] As a standard, a highly reliable mass spectrometry analysis device is preferred. Each user can freely configure the mass spectrometry analysis device to be used as a standard.

[0108] In a mass spectrometry apparatus for calculating calibration formulas, a calibrator solution containing two or more calibrators is measured to obtain the peak signal intensity of each calibrator. The ratio of the peak signal intensity of one or more other calibrators to the peak signal intensity of one calibrator is then calculated.

[0109] A regression equation is calculated between the peak signal intensity ratio of the standard and the peak signal intensity ratio of the mass spectrometer to which the calibration is to be calculated. In calculating the regression equation, known methods such as the least squares method can be appropriately used. The logarithm of the peak signal intensity ratio can also be used. The regression equation can be appropriately chosen from linear, polynomial, exponential, logarithmic, or power-law equations.

[0110] For example, a linear regression equation can be calculated using the value obtained by logarithmically transforming the peak signal intensity ratio. Alternatively, a power-law regression equation can be calculated using the peak signal intensity ratio. The calculated regression equation is then used as the calibration equation for the mass spectrometry analyzer.

[0111] For example, by setting the logarithm of the peak signal intensity ratio of the mass spectrometer to be calibrated as x, and the logarithm of the peak signal intensity ratio of the standard as y, the calibration coefficients a and b of the linear regression equation (y = ax + b) can be calculated. The calculated linear regression equation (y = ax + b) can then be used as the calibration equation for the mass spectrometer.

[0112] Alternatively, the peak signal intensity ratio of the mass spectrometer to which the calibration is to be calculated can be set as x, and the peak signal intensity ratio of the standard can be set as y, and the power regression equation (y = ax) can be calculated. b The correction coefficients a and b are used to calculate the power regression equation (y = ax). b This is used as a calibration formula for the mass spectrometry analysis device.

[0113] The correction coefficients a and b are considered to be inherent values ​​of the mass spectrometry analyzer. Therefore, they can be values ​​that vary depending on the mass spectrometry analyzer itself and its operating conditions. Furthermore, since the correction coefficient b is more significantly affected by the mass spectrometry analyzer itself and its operating conditions than the correction coefficient a, it can, for example, be used in the power regression equation (y = ax). b In this equation, 'a' uses a fixed value of a = 1, while only the value of 'b' uses the calculated correction coefficient. In other words, it can also replace the power regression equation (y = ax). b And y = x b Used as a correction formula. In this case, where only b is used as the correction factor, the value of b is called the correction value.

[0114] [5-1-2. Determination and Calibration of the Analytical Substance]

[0115] In the mass spectrometer with the calibration calculated in 5-1-1, the peak signal intensity of the analyte is obtained by measuring a sample containing the analyte. Using one analyte (e.g., an internal standard) as a reference, the ratio of the peak signal intensity of the other analytes to the peak signal intensity of the reference analyte is calculated.

[0116] The calculated peak signal intensity ratio is corrected using the correction formula described in 5-1-1. above. The corrected peak signal intensity ratio becomes a value in which the physical differences between the mass spectrometer and the standard are eliminated. That is, it becomes a value equivalent to the peak signal intensity ratio obtained when measured by the standard. Therefore, as long as the corrected peak signal intensity ratio is used, the peak signal intensity ratio of the analyte among multiple samples, i.e., the presence ratio of the analyte, can be compared and evaluated regardless of the physical characteristics of the mass spectrometer.

[0117] [5-2. Standardize and correct the peak signal intensity ratio (intensity ratio calibration)]

[0118] [5-2-1. Calculation of the Correction Formula]

[0119] It is possible to calculate a correction formula that normalizes the peak signal intensity ratio using multiple calibrator solutions with known concentration ratios of two calibrators.

[0120] Multiple calibrator solutions (intensity ratio calibrators; IC) with known concentration ratios of two calibrators can be used. Alternatively, multiple solutions can be used where one calibrator is present at a fixed concentration and the concentration of another calibrator is different. For example, solutions prepared by setting SIL-Aβ1-38 to a fixed concentration and making the concentration ratio of Aβ1-38 to SIL-Aβ1-38 1 / 4, 1 / 2, 1, 2, 4 can be used.

[0121] The mass spectrometer used to calculate the calibration formula measures the peak signal intensity of each calibrator in multiple calibrator solutions with known concentration ratios of two calibrators (intensity ratio calibrator; IC) in each solution. For each solution, the ratio of the peak signal intensity of another calibrator to the peak signal intensity of a calibrator at a fixed concentration is calculated.

[0122] A regression equation is calculated between the known concentration ratio of the two calibrators in each calibrator solution and the peak signal intensity ratio calculated above. In calculating the regression equation, known methods such as the least squares method can be appropriately used. The logarithm of the peak signal intensity ratio can also be used. The regression equation can be appropriately chosen from linear, polynomial, exponential, logarithmic, or power-law types.

[0123] For example, the logarithm of the peak signal intensity ratio can be used to calculate a linear regression equation. Alternatively, the peak signal intensity ratio can be used to calculate a power-law regression equation. The calculated regression equation is then used as the calibration equation for the mass spectrometry analyzer.

[0124] For example, by setting the logarithm of the known concentration ratio of the two calibrators in each solution as x, and the logarithm of the peak signal intensity ratio of the mass spectrometer to which the calibration formula is to be calculated as y, the calibration coefficients a and b of the linear regression equation (y = ax + b) can be calculated. The calculated linear regression equation (y = ax + b) can then be used as the calibration formula for the mass spectrometer.

[0125] Alternatively, the known concentration ratio of the two calibrators in each solution can be set as x, and the peak signal intensity ratio of the mass spectrometer to which the calibration is to be calculated can be set as y, to calculate the power regression equation (y = ax). b The correction coefficients a and b are used to calculate the power regression equation (y = ax). b This is used as a calibration formula for the mass spectrometry analysis device.

[0126] The correction coefficients a and b are considered to be inherent values ​​of the mass spectrometry analyzer. Therefore, they can be values ​​that vary depending on the mass spectrometry analyzer itself and its operating conditions. Furthermore, since the correction coefficient b is more significantly affected by the mass spectrometry analyzer itself and its operating conditions than the correction coefficient a, it can, for example, be used in the power regression equation (y = ax). b In this equation, 'a' uses a fixed value of a = 1, while only the value of 'b' uses the calculated correction coefficient. In other words, it can also replace the power regression equation (y = ax). b And y = x b Used as a correction formula. In this case, where only b is used as the correction factor, the value of b is called the correction value.

[0127] [5-2-2. Determination and Calibration of the Analytical Substance]

[0128] In the mass spectrometer with the calibration calculated in 5-2-1, the peak signal intensity of the analyte is obtained by measuring a sample containing the analyte. Using one analyte (e.g., an internal standard) as a reference, the ratio of the peak signal intensity of the other analytes to the peak signal intensity of the reference analyte is calculated.

[0129] The calculated peak signal intensity ratio is corrected using the correction formula described in section 5-2-1. Through correction, the peak signal intensity ratio of the analyte is transformed into a standardized peak signal intensity ratio using calibration materials. The resulting standardized peak signal intensity ratio eliminates systemic differences through the correction formula. Therefore, regardless of the systemic characteristics of the mass spectrometry analyzer, the presence ratio of the analyte among multiple samples can be compared and evaluated. That is, it is possible to directly compare mass spectrometry analysis results not only with those from Japan, but also with those from the United States, France, and other countries. Furthermore, this correction can be applied to various studies and inspections using mass spectrometry, making it a highly versatile technique.

[0130] [5-2-3. Calibration Values ​​and Device Conditions]

[0131] As described above, in this embodiment, by calculating and correcting the calibration formula using the mass spectrometry apparatus used for the determination of the analyte and under its apparatus conditions, the presence ratio of the analyte among multiple samples can be compared and evaluated regardless of the physical or apparatus conditions of the mass spectrometry apparatus.

[0132] In establishing the calibration formula for intensity ratio calibration, multiple calibrator solutions with known concentration ratios of two calibrators can be used. For example, five solutions can be prepared by setting SIL-Aβ1-38 to a fixed concentration and adjusting the concentration ratio of Aβ1-38 relative to SIL-Aβ1-38 to 1 / 4, 1 / 2, 1, 2, and 4. A peak of a fixed amount of SIL-Aβ1-38 and a peak of Aβ1-38 corresponding to its concentration appear in the mass spectrum of each solution. Ideally, this should correspond to the peak intensity ratio in each calibrator solution. However, since this peak intensity ratio varies depending on the device condition, a calibration formula is calculated to ensure that the peak intensity ratio is consistent with the concentration ratio in each calibrator solution to correct the test sample measurement results. This eliminates device error.

[0133] The calibration formula varies depending on the mass spectrometer itself. Furthermore, even with the same mass spectrometer, the state of the instrument can differ if components such as the detector are replaced, or if settings such as the detector voltage are changed; therefore, the calibration formula will vary depending on the instrument's condition. Additionally, the calibration formula may change even if the instrument's state changes due to factors such as detector deterioration.

[0134] For example, due to the influence of device conditions such as detector deterioration, the peak signal intensity ratio detected by a mass spectrometer may increase when measuring the same sample. This is believed to be because the peak signal of a small amount of calibrator substance decreases due to device conditions such as detector deterioration. In a mass spectrometer, if the peak signal intensity becomes too low, the measurement accuracy will generally decrease from the point of view of the signal-to-noise ratio (S / N ratio). Therefore, in a mass spectrometer, it is preferable that the peak signal intensity does not become too low.

[0135] For example, in 5-2-1, the power regression equation (y = ax) is derived. b In cases where the peak signal intensity detected by the mass spectrometer decreases due to device conditions, the correction value b in the calibration formula increases. Therefore, if this b value is kept within a certain fixed range, the peak signal intensity will not be too low, further improving the measurement accuracy of the mass spectrometer and the accuracy of the calibration.

[0136] The b-value can be varied by changing the detection sensitivity of the mass spectrometer. The b-value can be controlled, for example, by changing the detector voltage. Alternatively, the b-value can also be varied, for example, by changing the baseline level of the analog-to-digital (AD) converter.

[0137] According to this embodiment, by using a calibration formula to correct the peak signal intensity ratio of the analyte, the presence ratio of the analyte among multiple samples can be compared and evaluated regardless of the physical or instrumental conditions of the mass spectrometer. Furthermore, by adjusting the instrumental conditions to keep the values ​​of the correction coefficients in the calibration formula within a fixed range, the peak signal intensity ratio of the analyte can be corrected with even greater precision.

[0138] [6. Calibration value calculation system and program for mass spectrometry analysis device]

[0139] An embodiment of the mass spectrometry analysis apparatus of the present invention includes a mechanical error correction system comprising:

[0140] The method development department develops methods for determining calibrators used to calculate calibration values ​​for mass spectrometry analyzers; and

[0141] The correction value calculation unit analyzes the mass spectrometry analysis data obtained using the measurement method and calculates the correction value.

[0142] Furthermore, the error correction program for the mass spectrometry analysis apparatus according to one embodiment of the present invention includes having a computer perform the following steps:

[0143] The steps for developing a measurement method include: developing a measurement method for measuring samples used to calculate calibration values ​​for a mass spectrometry analyzer; and...

[0144] The correction value calculation step involves analyzing the mass spectrometry analysis data obtained using the measurement method and calculating the correction value.

[0145] Hereinafter, an embodiment of the error correction system and error correction program of the mass spectrometry analysis apparatus of the present invention will be described with reference to the accompanying drawings. The correction value calculation system and correction value calculation program of the mass spectrometry analysis apparatus of this embodiment calculate the correction value for performing the correction (intensity ratio calibration) of standardizing the peak signal intensity ratio as described in 5-2 above.

[0146] Figure 20 This is a schematic block diagram of the calibration value calculation system of the mass spectrometry analysis apparatus of this embodiment. Figure 20 As shown, this system includes a mass spectrometry analysis unit 1 for performing measurements on samples, a data processing unit 2 for processing data before and after the measurement, and an input unit 3 and a display unit 4 serving as user interfaces. The data processing unit 2 includes, as a functional module, a measurement method formulation unit 20, which formulates a measurement method for measuring samples used to calculate correction values ​​for the mass spectrometry analysis unit 1; and a correction value calculation unit 21, which analyzes the mass spectrometry data obtained from the mass spectrometry analysis unit 1 and calculates correction values.

[0147] The mass spectrometry analysis section 1 is not particularly limited, and includes mass spectrometry analysis methods based on matrix-assisted laser desorption / ionization (MALDI) mass spectrometry, electrospray ionization (ESI) mass spectrometry, etc. For example, MALDI-TOF (matrix-assisted laser desorption / ionization-time-of-flight) mass spectrometry devices, MALDI-IT (matrix-assisted laser desorption / ionization-ion trap) mass spectrometry devices, MALDI-IT-TOF (matrix-assisted laser desorption / ionization-ion trap-time-of-flight) mass spectrometry devices, MALDI-FTICR (matrix-assisted laser desorption / ionization-Fourier transform ion cyclotron resonance) mass spectrometry devices, ESI-QqQ (electrospray ionization-triple quadrupole) mass spectrometry devices, ESI-Qq-TOF (electrospray ionization-tandem quadrupole-time-of-flight) mass spectrometry devices, and ESI-FTICR (electrospray ionization-Fourier transform ion cyclotron resonance) mass spectrometry devices can be used.

[0148] The data processing unit 2 is, for example, a general-purpose personal computer or a higher-performance workstation. It can be a single unit or a computer system consisting of multiple units. This embodiment is implemented by installing a dedicated data processing program in such a computer and making the computer operational. Furthermore, the input unit 3 is typically a pointing device such as a keyboard and mouse attached to the computer. Additionally, the display unit 4 is typically a monitor attached to the computer.

[0149] In mass spectrometry, even when measuring the same sample, the results can sometimes differ depending on the equipment and apparatus conditions. To eliminate these differences, corrections are performed as described in section 5-2.

[0150] The measurement method formulation unit 20 automatically formulates a measurement method for the calibrator used to calculate the correction value. The mass spectrometry analysis unit 1 measures the sample according to the measurement method formulated by the measurement method formulation unit 20. The measurement method formulation unit 20 also includes a setting unit 201. The setting unit 201 sets the laser power value used in the measurement of the calibrator.

[0151] The correction value calculation unit 21 analyzes the mass spectrometry data measured by the mass spectrometry analysis unit 1 and calculates the correction value using the peak signal intensity ratio.

[0152] Next, use Figure 20 and Figure 21 The measurement performed using the calibration value calculation system of the mass spectrometry analysis apparatus of this embodiment and the calibration value calculation program is described in detail. Figure 21 This is a flowchart illustrating the process for calculating calibration values ​​for a mass spectrometry analyzer.

[0153] The user prepares samples for use in the calibration calculation of the mass spectrometer. Five samples (IC1 to IC5) with progressively different concentration ratios of the internal standard to the target substance are used as samples. For example, samples with the following concentration ratios (internal standard concentration: target substance concentration) are used: 1:4 in IC1, 1:2 in IC2, 1:1 in IC3, 1:0.5 in IC4, and 1:0.25 in IC5.

[0154] The user then automatically sets the measurement method. Figure 22 The diagram shows an example of a graphical user interface (GUI) displayed on display unit 4 when a measurement method is defined. The GUI includes a dataset name input section and a sample plate display section. The user operates via input unit 3 to input the dataset name and press the file creation button (S1).

[0155] The measurement method formulation unit 20 formulates the measurement method for the input dataset name (S2, measurement method formulation step). Specifically, in order to set the laser power value of the mass spectrometry analysis device used in the measurement method, the laser power value is calculated in advance. Furthermore, the sample drop positions for each sample IC1 to IC5 are determined. The measurement method formulation unit 20 displays the laser power on the GUI displayed on the display unit 4, and also displays the sample drop positions for IC1 to IC5 on the sample plate display section of the GUI.

[0156] Laser power can be calculated using various well-known methods.

[0157] For example, this can be done using the following method. For example, a laser power adjustment method with the following steps can be used: a measurement step, in which the laser power is varied in n levels (n is an integer greater than or equal to 3) for the same sample, and the signal intensity of ions originating from a specific component in the sample is obtained; and a processing step, in a two-axis curve obtained by plotting the n signal intensities obtained in the measurement step or as a relationship between the signal value and the SN ratio calculated based on the signal intensity and the laser power, the slope of the straight line connecting two adjacent plotted points on the laser power axis is calculated, and for each plotted point, an index value reflecting the ratio of the slope of the straight line in front of it to the slope of the straight line behind it is obtained, and an appropriate laser power is selected using this index value.

[0158] Furthermore, the calculated laser power can be a value that varies depending on the holes in the sample plate. For example, the laser power calculated for the holes (calibrator holes) of the sample IC1 to IC5 used in the calculation of the correction value after being dropped using this method can be a different value from the laser power used in the holes (sample holes) used in the measurement of the actual sample to be analyzed. For example, the laser power used in the calibrator holes can be 10% lower than the laser power used in the sample holes.

[0159] The sample drop position can be determined using various well-known methods.

[0160] The determined sample drop position is as follows: Figure 22 As illustrated, it is displayed on the sample plate display section. Figure 22 For example, it can be determined that IC-1, IC-2, IC-3, IC-4, and IC-5 are added starting from the top right end.

[0161] The user adds each calibrator IC1 to IC5 to the sample addition position shown on the display unit 4 and begins the measurement. The mass spectrometry analysis unit 1 measures each calibrator under specified conditions (S3).

[0162] At the end of the measurement, the user calculates the correction value. Figure 23 The image shows an example of a graphical user interface (GUI) displayed on display unit 4 when calculating correction values. The user selects the dataset name and presses the parsing button via input unit 5.

[0163] The correction value calculation unit 21 analyzes the mass spectrometry data measured by the mass spectrometry analysis unit 1 under the selected dataset name (S4) and calculates the correction value (S5). The correction value calculation steps include S4 and S5. In the calculation of the correction value, for example, the method described in [5. Correction of measurement results in mass spectrometry analysis apparatus] can be used.

[0164] Specifically, the ratio of the peak signal intensity of the target substance to the peak signal intensity of the internal standard substance in each sample (IC1–IC5) is calculated. A power-law regression equation (y = ax) can then be calculated between the concentration ratio of the target substance to the internal standard substance in each solution and the calculated peak signal intensity ratio. b The calculated correction coefficient b of the regression equation is output as the correction value b. The correction value b is displayed on the GUI of display unit 4.

[0165] The user then uses the mass spectrometry analysis unit 1 to measure the sample to be analyzed and obtain the measurement results after correction using the b-value.

[0166] Example

[0167] The following embodiments are shown to illustrate the invention in detail, but the invention is not limited to these embodiments.

[0168] The differences in peak intensity ratios among organisms were investigated by measuring multiple types of peptides using three identical mass spectrometers (all of model name: Performance). The results showed a linear relationship between the logarithms of the peak intensity ratios across the different instruments. It was confirmed that a linear regression equation could be established based on the logarithms of the measurements obtained across organisms to correct for the peak intensity ratios. Furthermore, it was confirmed that since the logarithms of the peak intensity ratios are linear, they also exhibit a power-law relationship without logarithmic transformation; therefore, a power-law regression equation (the power approximation) can also be established to correct for the peak intensity ratios.

[0169] To make the method more practical, samples were prepared for intensity ratio calibration, containing both stable isotope-labeled substances (at a fixed concentration) and unlabeled identical substances (at multiple different concentrations). These samples were measured using a mass spectrometer, and a correction formula based on a power approximation of their intensity and mass ratios was established. This correction formula was then used to correct the peak intensity ratio of the target substance.

[0170] The peak intensity ratios obtained for each organism were corrected using regression equations. This correction ensures that the same peak intensity ratio can be obtained even when measuring the same sample using different organisms. Furthermore, it also corrects for peak intensity ratios that may vary due to detector changes, voltage variations, or degradation. There is no need to prepare stable isotopes for each target substance; comparative evaluation of the substance's quality can be performed using only one stable isotope.

[0171] This method is not limited to peptides obtained through IP, but can also be used for peptides generated by protein digestion with enzymes such as peptidases, and peptides separated by chromatography. Furthermore, it is not limited to peptides, but can also be used for glycopeptides, glycans, and lipids.

[0172] The following shows a more detailed description of the embodiments.

[0173] [Example 1: A correction method for ensuring consistent peak intensity ratios across a single device]

[0174] [1-1 Determination of Aβ and Aβ-related peptide in plasma]

[0175] In this embodiment, Aβ and Aβ-related peptides, which are the substances to be analyzed, are prepared as follows.

[0176] For the plasma sample (Sample No. 1), immunoprecipitation-mass spectrometry (IP-MS) was performed using SIL-Aβ1-38 as the internal standard peptide.

[0177] First, 0.5 μL of matrix solution (0.5 mg / mL α-cyano-4-hydroxycinnamic acid: CHCA, 0.2% (w / v) methylene diphosphonic acid: MDPNA) was added to the μFocus MALDI plate. TM It is dried on 900μm pores.

[0178] Perform IP as follows. Wash the antibody-immobilized magnetic beads, which have been immobilized with anti-Aβ monoclonal antibodies (clones 6E10 and 4G8), twice with OTG-glycine buffer (1% n-octyl-β-D-thioglucosinolate (OTG), 50 mM glycine, pH 2.8), and wash three times with 100 μL of washing buffer. 250 μL of binding buffer containing 10 pM SIL-Aβ1-38 (AnaSpec, San Jose, CA, USA) (0.2% (w / v) n-dodecyl-β-D-maltodextrin (DDM), 0.2% (w / v) n-nonyl-β-D-thiomaltodextrin (NTM), 800 mM GlcNAc, 100 mM Tris-HCl, 300 mM NaCl, pH 7.4) was mixed with 250 μL of plasma sample, followed by the addition of previously immobilized antibody beads. The mixture was incubated at 4°C for 1 hour to capture Aβ and Aβ-related peptides. The sample was then washed once with washing buffer (500 μL or 100 μL), four times with 100 μL of washing buffer, and finally twice with 50 mM ammonium acetate (50 μL or 20 μL). After washing once with H2O (30 μL or 20 μL), elute the Aβ and Aβ-related peptide captured by the antibody-immobilized magnetic beads with 5 μL of 70% acetonitrile containing 5 mM hydrochloric acid. Pour the solution into a μFocus MALDI plate containing the matrix. TM 1 μL of eluent was added to each of the four wells at 900 μm. Mass spectra were determined using three AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) linear TOF in positive ion mode. Mass spectra were acquired by accumulating 40 runs at each of 400 locations using grating mode. Quantitative values ​​were obtained by averaging the peak intensities of each Aβ and Aβ-related peptide relative to the internal standard peptide (SIL-Aβ1-38) in the spectra determined in the four wells. The detection limit was set at S / N = 3, and peaks below the detection limit were considered undetectable. The amino acid sequences of the determined Aβ and Aβ-related peptides are shown in Table 1.

[0179] [Table 1]

[0180]

[0181] [Analysis of the comparison of peak intensity ratios between devices 1 and 2]

[0182] exist Figure 1Figure (A) shows the peak intensity ratios of Aβ or Aβ-related peptides relative to the internal standard peptide (SIL-Aβ1-38) obtained by IP-MS for plasma sample (Sample 1). The same sample was measured using three identical mass spectrometers (Performance 1–3), but the peak intensity ratios of most Aβ and Aβ-related peptides differed among the three instruments. The coefficient of variation (CV) for Aβ1-40 was 48.7%, and the CV for Aβ1-42 was 54.0%, confirming a particularly large difference among the three instruments. On the other hand, the CV for Aβ6-40 was 3.8%, yielding roughly the same results among the three instruments. The peak intensity ratio of Aβ6-40 was closer to 1, but the peak intensity ratios of Aβ1-40 and Aβ1-42 were farther from 1. This indicates a tendency that the closer the peak intensity ratio is to 1, the smaller the difference among the three instruments, and the farther it is from 1 (i.e., the more smaller or larger than 1), the greater the difference among the three instruments.

[0183] Furthermore, the peak intensity ratios of Aβ, Aβ-related peptide, or SIL-Aβ1-38 relative to APP669-711 also exhibit the following tendency: the closer the peak intensity ratio is to 1, the smaller the difference between the three organisms; the farther it is from 1, the greater the difference between the three organisms. Figure 1 (B)

[0184] exist Figure 2 The figure shows the peak intensity ratios of Aβ or Aβ-related peptides relative to the internal standard peptide (SIL-Aβ1-38), the average peak intensity ratios of Aβ, Aβ-related peptides, or SIL-Aβ1-38 relative to APP669-711, and the CVs, measured by three mass spectrometers. As clearly shown in the figure, the closer the peak intensity ratio is to 1, the smaller the CV among the three spectrometers; the farther the peak intensity ratio is from 1, the larger the CV among the three spectrometers.

[0185] When analyzing whether there are any rules governing the differences in peak intensity ratios among the three systems, it was shown that the values ​​obtained by transforming the peak intensity ratios to logarithms exhibit a linear relationship among the systems. Figure 3 The coefficients of determination for the regression lines between Performance 1 and Performance 2, and between Performance 1 and Performance 3, are both R. 2 The linearity was ≥0.985, thus confirming good linearity. The linear regression equations between the organisms are as follows.

[0186] The linear regression equation between Performance 1 and Performance 2 is as follows:

[0187] y = 1.282x + 0.045.

[0188] Here, x is the value obtained by logarithmic transformation of the peak signal intensity ratio in Performance 2, and y is the value obtained by logarithmic transformation of the peak signal intensity ratio in Performance 1.

[0189] The linear regression equation between Performance 1 and Performance 3 is as follows:

[0190] y = 1.981x + 0.081.

[0191] Here, x is the value obtained by logarithmic transformation of the peak signal intensity ratio in Performance 3, and y is the value obtained by logarithmic transformation of the peak signal intensity ratio in Performance 1.

[0192] Furthermore, although they are exactly the same, if the values ​​have not undergone logarithmic transformation, the relationship between the organisms is represented by a power approximation. Figure 4 ).

[0193] The approximate formula for the power of Performance 1 and Performance 2 is:

[0194] y = 1.108 × 1.282 .

[0195] Here, x is the peak signal intensity ratio in Performance 2, and y is the peak signal intensity ratio in Performance 1.

[0196] The approximate formula for the power of Performance 1 and Performance 3 is:

[0197] y = 1.204 × 1.981 .

[0198] Here, x is the peak signal intensity ratio in Performance 3, and y is the peak signal intensity ratio in Performance 1.

[0199] This study investigated whether, using Performance 1 as a standard, the peak intensity ratios of Aβ or Aβ-related peptides measured by Performance 2 and Performance 3 relative to the internal standard peptide (SIL-Aβ1-38) could be corrected to be equivalent to those of the standard (Performance 1) through these regression equations. Correction was performed by substituting the peak intensity ratios measured by Performance 2 and Performance 3 into x in the aforementioned linear regression equation to obtain y. Figure 5The corrected values ​​are denoted as Performance 2 (Cal.) and Performance 3 (Cal.), respectively. The result is that the differences among all peptides across the three levels are smaller, and the CV is also lower. The corrected values ​​are the same regardless of whether the linear regression equation with logarithmic transformation values ​​is used or the power approximation is used.

[0200] The results show that even when the same sample is measured by different mass spectrometry devices, the same peak intensity ratio can be obtained by correcting the peak intensity ratio using the correction formula. In other words, this indicates that the correction formula can eliminate the error (mechanical difference) in peak intensity ratio caused by the differences in the mass spectrometry devices.

[0201] The reason why the logarithm of the peak intensity ratio has a linear relationship between different organisms is believed to be that the secondary electron multiplier (SEM) used in mass spectrometry amplifies the electrical signal of ions exponentially, thus obtaining a large signal.

[0202] [Verification of the appropriateness of the 1-3 correction formula]

[0203] To investigate whether the correction formula established in 1-2 could also be applied to other samples, IP-MS was performed on plasma samples (Sample No. 2 and No. 3) different from those used in 1-1 above, and the peak intensity ratios before and after correction were compared. IP-MS was performed using the same method as in 1-1, with the correction formula employing a linear regression equation for the logarithmic transformation of the peak intensity ratio calculated in 1-2. The results confirmed that Sample No. 2 and No. 3, after correction, achieved equivalent peak intensity ratios across the three samples. Figure 6 , Figure 7 There are also peptides whose detection sensitivity is insufficient depending on the individual organism. Data below the detection limit in such cases are indicated as ND (Not detectable).

[0204] These results demonstrate the appropriateness of the correction formula established in 1-2, which yields identical peak intensity ratios in measurements performed on the three separate instruments for all samples (Sample No.1, No.2, and No.3).

[0205] [Reproducibility verification of 1-4 correction formulas]

[0206] The reproducibility of the correction formula established in 1-2 was verified. On a different day than 1-1, IP-MS was performed using the same procedure as in 1-1 to establish the correction formula (a linear regression equation for logarithmic transformation values). Figure 8 , Figure 9 The day on which step 1-1 was implemented is called Day 1, and the day on which the regression equation for correction (a linear regression equation for the logarithmic transformation value) is re-established is called Day 2. The correction equations between the organisms obtained on Day 1 and Day 2 are shown in Table 2.

[0207] [Table 2]

[0208]

[0209] We used analysis of covariance (ANCOVA) to verify whether the correction formulas obtained for each organism on Day 1 and Day 2 were the same. First, in the ANCOVA four-group test, we set p < 0.05 as statistically significant and performed analysis; the slope showed p < 0.0001. This demonstrates that the four groups were statistically significantly different.

[0210] Next, pairwise comparisons of the four groups were performed using ANCOVA (Table 3). In this case, four tests were conducted, therefore Bonferroni adjustments were made, and p < 0.0125 was set for statistical significance. First, the slope of the regression equation was tested; if no significant difference was found, the intercept of the regression equation was then tested. The results showed that the regression equations were different in the comparisons of Performance 2 (Day 1) vs. Performance 3 (Day 1) and Performance 2 (Day 2) vs. Performance 3 (Day 2), while the regression equations were the same in the comparisons of Performance 2 (Day 1) vs. Performance 2 (Day 2) and Performance 3 (Day 1) vs. Performance 3 (Day 2). These results indicate that the correction has an inherent regression equation based on the organism, and as long as the state of the device does not change due to detector degradation, the regression equation will not change based on the measurement date.

[0211] [Table 3]

[0212]

[0213] [Example 2: Method for correction by standardizing peak intensity ratio]

[0214] [2-1 Determination of Aβ and Aβ-related peptide in plasma]

[0215] In this embodiment, Aβ and Aβ-related peptides, which are the substances to be analyzed, are prepared as follows.

[0216] Immunoprecipitation (IP) with anti-Aβ monoclonal antibodies was combined with mass spectrometry (MS) to determine Aβ-related peptides in human plasma. In the IP, anti-Aβ antibody clone 6E10 (BioLegend) was covalently bound to DynabeadsEpoxy (Thermo Fisher Scientific) as magnetic beads, thus utilizing antibody magnetic beads. 250 μL of plasma was mixed with 250 μL of reaction solution containing internal standard peptides, and then mixed with the antibody magnetic beads. The antigen-antibody reaction was performed at 4°C for 1 hour (1st IP). The internal standard peptide was labeled with 11 pM stable isotope labeling (SIL) Aβ1-38. After the antigen-antibody reaction, the antibody magnetic beads were washed, and the Aβ-related peptides were eluted using 1st IP elution buffer (glycine buffer containing DDM, pH 2.8). After restoring to neutral with Tris buffer containing DDM, the antibody-associated peptide was reacted with the antibody magnetic beads again (2nd IP). After washing, the Aβ-related peptide was eluted with 2nd IP elution buffer (5 mM HCl, 0.1 mM methionine, 70% (v / v) acetonitrile). A μFocus MALDI plate, pre-filled with 0.5 μL of 0.5 mg / mL CHCA / 0.2% (w / v) MDPNA and dried, was then used. TM The elution buffer after IP was added to four wells of a 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ) and then dried.

[0217] Mass spectrometry data were acquired using a linear Time-of-Flight (TOF) in positive ion mode on an AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) microscope. The m / z values ​​for the linear TOF are expressed as the average mass of the peaks. The m / z values ​​were obtained by calibration using human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, and bovine insulin as external standards. Mass spectra were acquired by accumulating 40 measurements at each of 400 locations using grating mode. Quantitative values ​​were obtained by averaging the peak intensities of each Aβ and Aβ-related peptide in the spectra measured in four wells relative to the internal standard peptide (SIL-Aβ1-38). The detection limit was set at S / N = 3; peaks below the detection limit were considered undetectable.

[0218] [2-2 Determination of Intensity Ratio Calibrator (IC)]

[0219] Five concentrated solutions of intensity ratio calibrators (ICs) were prepared according to the composition in Table 4 to correct the peak intensity ratios of each Aβ and Aβ-related peptide obtained by IP-MS relative to SIL-Aβ1-38, and stored frozen. Before MS analysis, the IC-1 to IC-5 concentrates were thawed and diluted 10-fold with 2nd IP elution buffer (5 mM HCl, 0.1 mM methionine, 70% (v / v) acetonitrile) to prepare IC-1 to IC-5. These were then added to a dried μFocus MALDI plate pre-filled with 0.5 μL of 0.5 mg / mL CHCA / 0.2% (w / v) MDPNA. TM 1 μL of each of IC-1 to IC-5 was added to four wells on a 900 μm plate and then dried. The protein / peptide composition of IC-1 to IC-5 is shown in Table 5. The ratio of Aβ1-38 to SIL-Aβ1-38 in IC-1 to IC-5 was set to 4, 2, 1, 1 / 2, and 1 / 4, respectively.

[0220] [Table 4]

[0221]

[0222] [Table 5]

[0223]

[0224] The mass spectrometry data for IC-1 to IC-5 were obtained using the same method as in 2-1.

[0225] [2-3 Strength Ratio Correction]

[0226] Under the three conditions of Performance 1 and Performance 3 before and after detector replacement, IP-MS and IC-1 to IC-5 assays of human plasma were performed.

[0227] Based on the intensity and quantity ratios of Aβ1-38 relative to SIL-Aβ1-38 in the mass spectra of IC-1 to IC-5, a power approximation was established. Figure 10 ).like Figure 10 As shown, depending on the state of the organism, even with the same quantity ratio, the peak intensity ratio will differ, and therefore the power approximation will also differ. Correction is performed by aligning each power approximation with y = x.

[0228] Power approximation: y = ax b

[0229] Here, x is the ratio of Aβ1-38 / SIL-Aβ1-38, y is the peak intensity ratio of Aβ1-38 / SIL-Aβ1-38, and a and b are coefficients in the approximation.

[0230] exist Figure 10 In (A),

[0231] y = 1.0032x 1.0402 .

[0232] exist Figure 10 In (B),

[0233] y = 1.0413 × 0.8182 .

[0234] exist Figure 10 In (C),

[0235] y = 1.0353 × 0.714 .

[0236] like Figure 4 , Figure 10 As shown, the value of a in the exponentiation approximation does not change according to the state of the organism, but the value of b is greatly affected by the conditions of the organism. Therefore, two methods are used for evaluation: one is to correct using the a and b values ​​obtained by IC measurement, and the other is to fix the a value at 1 (a=1) and only use the b value for correction.

[0237] The peak intensity ratios of Aβ or Aβ-related peptides relative to SIL-Aβ1-38 were calculated based on IP-MS mass spectrometry. These intensity ratios were then applied to the IC curves, specifically using a power-law approximation calculated from the IC-1 to IC-5 measurements for correction. More precisely, x was calculated by substituting the IP-MS peak intensity ratios into y, and this was used for operations consistent with y = x. The average of four peak intensity ratio measurements (n = 4) was calculated and used in the analysis.

[0238] exist Figure 11 The figure shows the results obtained by using a method that fixes the value of 'a' in the power approximation to 1 (a = 1) and corrects using only the value of 'b'. The peak intensity ratio of Aβ or Aβ-related peptide relative to SIL-Aβ1-38 reduced the difference between the three conditions through correction based on the power approximation, with the coefficient of variation (CV) decreasing from 16.8%–21.5% before correction to 3.9%–13.6% after correction.

[0239] In addition, APP669-711 / Aβ1-42 and Aβ1-40 / Aβ1-42, which function as biomarkers, were also evaluated. In the uncorrected stage, no effect was observed in APP669-711 / Aβ1-42, where the difference between the two peak intensities was small, the intensity ratio was close to 1, and therefore the CV was very small. However, in Aβ1-40 / Aβ1-42, which had a larger intensity ratio, the CV before correction was 41.1%, which decreased to 8.0% after correction. Figure 12 ).

[0240] Furthermore, even in the method using a and b values ​​for correction, the effect of decreasing the CV of the peak intensity ratio of Aβ or Aβ-related peptide relative to SIL-Aβ1-38 across the three conditions was confirmed, yielding identical results for biomarkers as with the method setting a=1. Figure 13 , Figure 14 ).

[0241] The above results demonstrate that this correction method effectively reduces the deviation in peak intensity ratio between devices. Furthermore, it shows that since the value of 'a' does not change based on the state of the device, even when only the value of 'b' is used as the correction value, the deviation in peak intensity ratio between devices can still be reduced. The method in Example 2 standardizes the peak intensity ratio using a power approximation, thus eliminating the need for a specific device as a standard.

[0242] [Verification of Strength Ratio Correction 2-4]

[0243] Samples obtained by incorporating three Aβ peptides (Aβ1-40, Aβ1-42, and APP669-711) and an internal standard peptide into standard plasma were subjected to IP treatment and measured using three AXIMA-Performance analyzers along with IC reagents. The in vivo and detector voltages used for the measurements, as well as the b-values ​​calculated from the IC measurement results, are shown in Table 6.

[0244] [Table 6]

[0245]

[0246] Furthermore, since the b-value is correlated with the detector voltage, the b-value can be adjusted using the detector voltage.

[0247] The intensity ratios of Aβ1-40, Aβ1-42, and APP669-711 relative to the internal standard were read from the obtained mass spectra. The biomarkers (APP669-711 / Aβ1-42 and Aβ1-40 / Aβ1-42) were compared without correcting the intensity ratios for the b-value.

[0248] Figure 15 This is a comparison of the Aβ1-40 / Aβ1-42 ratio before and after correction. The data before correction showed large deviations across the three units, and also large deviations in one unit when the detector voltage was changed. It was confirmed that by using the b-value for correction, the deviations across the three units were suppressed, and the peptide ratio remained constant even when the detector voltage was changed in one unit.

[0249] Figure 16 This is a comparison of the APP669-711 / Aβ1-42 ratio before and after calibration. The intensity difference of the APP669-711 / Aβ1-42 ratio is small, even before calibration, with small deviations between the three units and with small deviations when the detector voltage is changed in one unit. However, it can be confirmed that the deviation is further suppressed by using the b-value for calibration.

[0250] Table 7 below is derived by classifying the data based on the b-value and calculating the CV of each Aβ peptide ratio.

[0251] [Table 7]

[0252]

[0253] "Overall" shows the CV calculated using all six conditions.

[0254] "Changing the detector voltage within a single machine" shows the CV values ​​obtained under three conditions by changing the detector voltage within machine P1.

[0255] "Small deviation of b-values ​​in 3 organisms" indicates the CV obtained based on the measurement results under the condition that the b-values ​​in 3 organisms are close to 0.95.

[0256] "Large deviation of b values ​​in 3 organisms" indicates the CV obtained based on the measurement results under 3 conditions where the b values ​​are different in 3 organisms.

[0257] It can be seen that when the deviation of the b-value among the three mechs is small, the CV is already low and there is no significant difference before and after correction. However, in other cases, the CV is significantly reduced through b-value-based correction. This indicates that b-value-based correction is effective whether the device settings are changed in one mech or the device states are different in different mechs.

[0258] [Example 3: Adjustment of the correction value b 1]

[0259] As described in 2-4 of Example 2, since the b-value is correlated with the detector voltage, the b-value can be adjusted using the detector voltage. The results obtained from investigating the relationship between the b-value and the detector voltage are shown below.

[0260] As calibration materials, conventional Aβ1-38 with equal ionization efficiency and SIL-Aβ1-38 with stable isotope labeling were used for IC determination. IC1 to IC5 were the same as those used in 2-2 of Example 2.

[0261] In the Performance 4 mass spectrometer, IC measurements were performed with detector voltages set to 2700V, 2750V, 2800V, 2850V, 2900V, and 2950V. Using the measurement results at each detector voltage, the correction formula (exponentiation approximation: y = ax) was calculated as described in steps 2-3 of Example 2. b ). Figure 17 This is a graph showing the relationship between the detector voltage and the b-value of the calibration formula. The vertical axis represents the b-value, and the horizontal axis represents the detector voltage (V).

[0262] In the Performance 1 mass spectrometer, IC measurements were performed with detector voltages set to 2700V, 2725V, 2750V, 2775V, 2800V, and 2825V. Using the measurement results at each detector voltage, the correction formula (exponentiation approximation: y = ax) was calculated as described in steps 2-3 of Example 2. b ). Figure 18 This is a graph showing the relationship between the detector voltage and the b-value of the calibration formula. The vertical axis represents the b-value, and the horizontal axis represents the detector voltage.

[0263] like Figure 17 and Figure 18 As shown, the following trend was observed: the b-value decreases when the detector voltage is increased. When the b-value is above 1.1, the b-value changes by about 0.15 when the detector voltage is increased by 25V. When the b-value is below 1.1, the b-value changes by about 0.03 to 0.07 when the detector voltage is increased by 25V.

[0264] By using this relationship between detector voltage and b-value, it is possible to adjust the b-value to a fixed range by increasing the detector voltage when the b-value increases due to detector degradation or other reasons. By adjusting the b-value to a fixed range, the peak signal intensity ratio of the analyte can be corrected with higher precision. For example, the range of the b-value can be adjusted to 0.9–1.1.

[0265] [Example 4: Adjustment of the correction value b 2]

[0266] Another method for adjusting the b-value is to adjust the baseline level of the analog-to-digital converter (AD converter). The results of investigating the relationship between the b-value and the baseline level setting of the AD converter, as well as the detector voltage, are shown below.

[0267] As calibration materials, conventional Aβ1-38 with equal ionization efficiency and SIL-Aβ1-38 with stable isotope labeling were used for IC determination. IC1 to IC5 were the same as those used in 2-2 of Example 2.

[0268] In the mass spectrometer Performance 1 unit, IC measurements were performed with detector voltages set to 2700V, 2725V, 2750V, 2775V, 2800V, and 2825V. The AD converter baseline level was set to two conditions: the normally set 181, and a state after lowering the baseline level and increasing the noise level (179). Data were acquired at both baseline levels. Using the measurement results at each detector voltage, the correction formula (exponentiation approximation: y = ax) was calculated as in Examples 2-3. b ). Figure 19 This is a graph showing the relationship between the detector voltage and the b-value of the calibration formula. The vertical axis represents the b-value, and the horizontal axis represents the detector voltage (V). Circular data points represent baseline setting 181, and square data points represent baseline setting 179.

[0269] like Figure 19As shown, the following trend was observed: even at the same detector voltage, decreasing the baseline level setting results in a decrease in the b-value. When the b-value is above 1.1, decreasing the baseline setting by 2 results in a decrease in the b-value of approximately 0.2 to 0.35. When the b-value is below 1.1, decreasing the baseline setting by 2 results in a decrease in the b-value of approximately 0.15.

[0270] By using the relationship between the baseline setting and the b-value of such an AD converter, the b-value can be adjusted to a fixed range by lowering the baseline setting when the b-value increases due to detector degradation or other factors. By adjusting the b-value to a fixed range, the peak signal intensity ratio of the analyte can be corrected with higher precision. For example, the range of the b-value can be adjusted to 0.9 to 1.1.

[0271] In this invention, for example, the following methods are included. (1)

[0273] A method for correcting the mechanical error of the signal intensity ratio in mass spectrometry analysis includes the following steps:

[0274] The peak signals of each calibration substance are obtained by measuring the calibrator containing two or more calibration substances using a mass spectrometry analysis device.

[0275] Calculate the peak signal intensity ratio of one of the two or more calibration substances to the peak signal intensity of the other calibration substance;

[0276] The correction formula is derived based on the peak signal intensity ratio.

[0277] The peak signals of each analyte are obtained by measuring a sample containing two or more analyte substances using the mass spectrometry analysis device.

[0278] Calculate the peak signal intensity ratio of one analyte to the peak signal intensity of another analyte among the two or more analytes; and

[0279] The peak signal intensity ratio of the analyte is corrected using the aforementioned correction formula. (2)

[0281] According to the calibration method described in (1) above, the calibration material includes a material labeled with a stable isotope and a material not labeled with a stable isotope. (3)

[0283] According to the correction method described in (1) or (2) above, the analyte is selected from substances belonging to the group consisting of peptides, glycopeptides, glycans, lipids and glycolipids. (4)

[0285] According to any one of the calibration methods described in (1) to (3) above, wherein the calibration substance is an Aβ-related peptide. (5)

[0287] According to any one of the calibration methods described in (1) to (4) above, wherein the calibration substance is Aβ1-38 and Aβ1-38 with stable isotope labeling. (6)

[0289] According to any one of the calibration methods described in (1) to (5) above, wherein the calibrator contains three or more calibration substances. (7)

[0291] According to any of the correction methods described in (1) to (6) above, wherein,

[0292] There are multiple calibrators.

[0293] Among the plurality of calibrators, the concentration of at least one of the calibrators for which the peak signal intensity ratio is to be determined is different. (8)

[0295] According to the calibration method described in (7) above, the ratio of the concentration of one calibrator to the concentration of another calibrator is in the range of 1 / 4 to 4. (9)

[0297] According to any one of the correction methods described in (1) to (8) above, in the step of obtaining the correction formula, the value obtained by logarithmically transforming the peak signal intensity ratio is used to obtain the correction formula. (10)

[0299] According to any one of the correction methods described in (1) to (9) above, wherein the correction formula is a linear, polynomial, exponential, logarithmic, or power-law correction formula. (11)

[0301] According to any one of the correction methods described in (1) to (10) above, the coefficients in the correction formula are within a specified range of values. (12)

[0303] According to the correction method described in (11) above, the coefficients in the correction formula are adjusted to the range of the specified values ​​by adjusting the detector voltage and / or the baseline level of the AD converter of the mass spectrometer. (13)

[0305] According to any of the correction methods described in (1) to (12) above, wherein,

[0306] The correction formula is expressed by the following power approximation:

[0307] y = ax b

[0308] (Where, x is the peak signal intensity ratio or concentration ratio used as a reference, y is the peak signal intensity ratio obtained in the mass spectrometry analysis device that requires the calibration formula, a is a coefficient in the approximation formula, and b is a coefficient in the approximation formula and is also a calibration value). (14)

[0310] A mass spectrometer error correction system includes:

[0311] The method development department develops methods for determining calibrators used to calculate calibration values ​​for mass spectrometry analyzers; and

[0312] The correction value calculation unit analyzes the mass spectrometry analysis data obtained using the measurement method and calculates the correction value. (15)

[0314] According to the machine error correction system described in (14) above, the measurement method formulation unit includes a setting unit that sets the laser power value used in the measurement of the calibrator to a pre-calculated laser power value. (16)

[0316] According to the machine error correction system described in (14) or (15) above, there is a sample plate display unit that displays the sample plate of the mass spectrometry analysis device. (17)

[0318] According to the machine error correction system described in (16) above, the sample plate display shows the sample drop position in the measurement method. (18)

[0320] According to any of the implementations (15) to (17) of the machine error correction system, the setting unit sets different laser power values ​​for the sample hole and the calibrator hole. (19)

[0322] A procedure for correcting machine error in a mass spectrometry analyzer includes having a computer perform the following steps:

[0323] The steps for developing a measurement method include: developing a measurement method for measuring samples used to calculate calibration values ​​for a mass spectrometry analyzer; and...

[0324] The correction value calculation step involves analyzing the mass spectrometry analysis data obtained using the measurement method and calculating the correction value.

[0325] Explanation of reference numerals in the attached figures

[0326] 1: Mass spectrometry analysis unit; 2: Data processing unit; 20: Measurement method formulation unit; 201: Setting unit; 21: Calibration value calculation unit; 3: Input unit; 4: Display unit. sequence list <110> SHIMADZU CORPORATION <120> Mass spectrometry analysis device error correction method <130> SP20200444 <150> US 63 / 062,677 <151> 2020-08-07 <160> 10 <210> 1 <211> 33 <212> PRT <213> Homo sapiens <400> 1 His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala 1 5 10 15 Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly 20 25 30 Gly <210> 2 <211> 33 <212> PRT <213> Homo sapiens <400> 2 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly <210> 3 <211> 35 <212> PRT <213> Homo Sapiens <400> 3 His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala 1 5 10 15 Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly 20 25 30 Gly Val Val 35 <210> 4 <211> 35 <212> PRT <213> Homo Sapiens <400> 4 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met 35 <210> 5 <211> 37 <212> PRT <213> Homo Sapiens <400> 5 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly 35 <210> 6<000082[10><211> 38 <212> PRT <213> Homo Sapiens <400> 6 Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val 1 5 10 15 Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu 20 25 30 Met Val Gly Gly Val Val 35 <210> 7 <211> 40 <212> PRT <213> Homo Sapiens<00[00834><400> 7 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val 35 40 <210> 8 <211> 40 <212> PRT <213> Homo Sapiens <400> 8 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val 35 40 <210> 9 <211> 42 <212> PRT <213> Homo Sapiens <400> 9 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 <210> 10 <211> 43 <212> PRT <213> Homo Sapiens <400> 10 Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His 1 5 10 15 His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly 20 25 30 Ala Ile Ile Gly Leu Met Val Gly Gly Val Val 35 40

Claims

1. A method for correcting the mechanical error of the signal intensity ratio in mass spectrometry analysis, comprising the following steps: The peak signals of each calibration substance are obtained by measuring the calibrator containing two or more calibration substances using a mass spectrometry analysis device. Calculate the peak signal intensity ratio of one of the two or more calibration substances to the peak signal intensity of the other calibration substance; The correction formula is derived based on the peak signal intensity ratio. The peak signals of each analyte are obtained by measuring a sample containing two or more analyte substances using the mass spectrometry analysis device. Calculate the peak signal intensity ratio of one analyte to the peak signal intensity of another analyte among the two or more analytes; and The peak signal intensity ratio of the analyte is corrected using the aforementioned correction formula. in, The calibration materials include substances labeled with stable isotopes and substances not labeled with stable isotopes.

2. The correction method according to claim 1, wherein, The substances to be analyzed are selected from those belonging to the group consisting of peptides, glycopeptides, glycans, lipids, and glycolipids.

3. The correction method according to claim 1, wherein, The calibration substance is an Aβ-related peptide.

4. The correction method according to claim 1, wherein, The calibration material is Aβ1-38 and Aβ1-38 with stable isotope labeling.

5. The correction method according to claim 1, wherein, The calibrator contains three or more calibrating substances.

6. The correction method according to claim 1, wherein, There are multiple calibrators. Among the plurality of calibrators, the concentration of at least one of the calibrators for which the peak signal intensity ratio is to be determined is different.

7. The correction method according to claim 6, wherein, The ratio of the concentration of one calibrator to the concentration of another calibrator is in the range of 1 / 4 to 4.

8. The correction method according to claim 1, wherein, In the process of deriving the correction formula, the value obtained by performing a logarithmic transformation on the peak signal intensity ratio is used to derive the correction formula.

9. The correction method according to claim 1, wherein, The correction formula can be a linear, polynomial, exponential, logarithmic, or power-law correction formula.

10. The correction method according to claim 1, wherein, The coefficients in the correction formula are within the specified range of values.

11. The correction method according to claim 10, wherein, The coefficients in the correction formula are adjusted to the range of the specified values ​​by adjusting the detector voltage and / or the baseline level of the AD converter of the mass spectrometer.

12. The correction method according to claim 1, wherein, The correction formula is expressed by the following power approximation: Where x is the peak signal intensity ratio or concentration ratio used as a reference, y is the peak signal intensity ratio obtained in the mass spectrometry analysis device that requires the calibration formula, a is a coefficient in the approximation formula, and b is a coefficient in the approximation formula and is also a calibration value.

13. A mechanical error correction system for a mass spectrometry analysis apparatus used in the correction method according to any one of claims 1-12, comprising: The Measurement Method Development Department develops measurement methods for calibrators used to calculate calibration values ​​for mass spectrometry analyzers. as well as The correction value calculation unit analyzes the mass spectrometry data obtained using the measurement method and calculates the correction value. The mass spectrometry analysis device's error correction system includes a sample plate display unit that displays the sample plate of the mass spectrometry analysis device. The sample plate display shows the sample drop position in the measurement method.

14. The machine error correction system according to claim 13, wherein, The measurement method formulation unit includes a setting unit that sets the laser power value used in the measurement of the calibrator to a pre-calculated laser power value.

15. The machine error correction system according to claim 14, wherein, The setting unit sets different laser power values ​​for the sample hole and the calibration hole.

16. A program product for error correction of a mass spectrometry analysis apparatus for the correction method according to any one of claims 1-12, comprising causing a computer to perform the following steps: The steps for developing a measurement method include: developing a measurement method for measuring calibrators used to calculate calibration values ​​for a mass spectrometry analyzer; and... The correction value calculation step involves parsing the mass spectrometry analysis data obtained using the aforementioned measurement method and calculating the correction value. The calibrator contains two or more calibrating substances. The calibration materials include substances labeled with stable isotopes and substances not labeled with stable isotopes.

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