Patent Application
20240266158
The present invention relates to a method using a peptide ratio obtained by mass spectrometry. The present invention relates to a machine difference correction method of a mass spectrometer and a machine difference correction system of a mass spectrometer.
When a comparative analysis of abundances of substances is performed using a mass spectrometer, a method using the intensity ratio of two signal peaks is most commonly used. For example, a certain amount of an internal standard substance is added to samples to be compared, the samples are optionally pretreated, and then subjected to mass spectrometry, and a comparison is made between the values of intensity ratio of the peak of an analyte to be measured relative to the peak of the internal standard substance (Non-Patent Documents 1, 2 and 3).
Examples of patent documents include WO 2015/178398 (US 2017/0184573), and WO 2017/047529 (US 2018/0238909).
In addition, a semi-quantitative comparative analysis can be performed by labeling a target substance derived from each of different samples with labeled compounds different in mass due to the use of a stable isotope element and calculating the intensity ratio of peaks of the target substance different in mass by mass spectrometry. This technique includes ICAT (registered trademark) and iTRAQ (registered trademark) used in the field of proteomics (Non-Patent Documents 4 and 5).
Patent Document 1: WO 2015/178398
Patent Document 2: US 2017/0184573
Patent Document 3: WO 2017/047529
Patent Document 4: US 2018/0238909
Non-Patent Document 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.
Non-Patent Document 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.
Non-Patent Document 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-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients. Mol Cell Proteomics. 2008 Oct; 7 (10): 1974-82.
Non-Patent Document 4: Han DK, Eng J, Zhou H, Aebersold R: Quantitative profiling of differentiationinduced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat Biotechnol. 2001 Oct; 19 (10): 946-51.
Non-Patent Document 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.
When very small amounts of substances are measured using a mass spectrometer, it is confirmed that a phenomenon occurs in which even when machines of the same type are used, a detected peak intensity ratio varies between the machines. One of means for calibrating such a difference in peak intensity ratio between different machines is the following absolute quantitation method.
A standard product of an analyte substance to be quantitated and a substance used as a reference for measuring an intensity ratio (as a reference substance, a labeled substance of the analyte substance with a stable isotope is generally used) are prepared. Samples containing the reference substance at a certain concentration and the standard product at different concentrations are measured to prepare a calibration curve of the peak intensity ratio of the standard product relative to the reference substance. The absolute quantitation of the analyte substance can be performed using this calibration curve. Therefore, even when there is a difference in peak intensity ratio between different machines, an unknown analyte substance present in a biological sample can be quantitated without the influence of such a difference by preparing a calibration curve for every machine and every measurement.
However, this method using a calibration curve requires a standard product. When a standard product cannot easily be synthesized, or when the kinds of analyte substances are very many and therefore it is difficult to prepare the standard products of all the analyte substances and control quality thereof in terms of time and cost, or when a standard product is unstable, a calibration curve cannot be prepared. As described above, the peak intensity ratio varies between different mass spectrometer machines. Therefore, when a calibration curve cannot be prepared, samples to be compared need to be measured by one machine. However, it is difficult to obtain consistent data because a detector deteriorates due to the use of the machine so that the peak intensity ratio varies.
In mass spectrometry, a sample is ionized by laser irradiation. Therefore, data is influenced by the conditions of a laser of a mass spectrometer (deterioration with, for example, an increase in the number of uses). Therefore, when the same sample is measured by different machines, there is not a little difference in data between the machines. Therefore, the data lacks in stability.
It is an object of the present invention to provide a method for collecting a difference in mass spectrometry data between mass spectrometer machines, and a system for collecting a difference between mass spectrometer machines.
The present invention includes the following aspect.
A method for calibrating a difference in signal intensity ratio between machines in mass spectrometry, the method comprising the steps of:
The present invention also includes the following aspect.
A machine difference calibration system of a mass spectrometer, the system comprising:
According to the present invention, a peak intensity ratio of analyte substances can be calibrated using a calibration formula determined from measurement results of calibration substances. Therefore, even when the same sample is measured by different machines respectively, an equivalent peak intensity ratio of the analyte substances can be obtained.
An embodiment of a method according to the present invention is a method for calibrating a difference in signal intensity ratio between machines in mass spectrometry, the method comprising the steps of:
This embodiment will be described in detail below. It should be noted that in Examples that will be described later, more specific examples will be shown with reference to
The analyte substances to be analyzed are not particularly limited, and examples thereof include peptides, glycopeptides, sugar chains, proteins, lipids, and glycolipids. The peptides, glycopeptides, sugar chains, proteins, lipids, and glycolipids include those of various kinds. More specifically, the analyte substances may be Aβ and an Aβ related peptide. The “Aβ and an Aβ related peptide” may simply collectively be called “Aβ related peptides”. The “Aβ and an Aβ related peptide” includes Aβ generated by cleaving amyloid precursor protein (APP) and peptides containing even part of the sequence of Aβ. In Examples, examples using Aβ and Aβ related peptides are shown.
Also, the peptides may be those obtained by immunoprecipitation (IP). Alternatively, the peptides may be those generated by digestion of protein with an enzyme such as peptidase, or those fractionated by chromatography.
The analyte substances may include an internal standard substance. The internal standard substance may appropriately be selected by those skilled in the art. For example, a substance labeled with a stable isotope may be used. One substance of the analyte substances may be labeled with a stable isotope. In Examples, examples using stable isotope-labeled Aβ1-38 (SIL-Aβ1-38) as an internal standard substance are shown. SIL is an abbreviation for stable isotope-labeled.
A sample containing the analyte substances is subjected to mass spectrometry. The sample to be subjected to mass spectrometry is not particularly limited, and may be, for example, a living body-derived sample. The living body-derived sample includes body fluids such as blood, cerebrospinal fluid (CSF), urine, body secreting fluid, saliva, and sputum; and feces. The blood sample includes whole blood, plasma, serum and the like. The blood sample can be prepared by appropriately treating whole blood collected from an individual. The treatment performed in the case of preparing a blood sample from collected whole blood is not particularly limited, and any treatment that is clinically acceptable may be performed. For example, centrifugal separation or the like may be performed. The blood sample to be subjected to mass spectrometry may be appropriately stored at a low temperature such as freezing in the intermediate stage of the preparation step or in the post stage of the preparation step. In the present invention, when the living body-derived sample such as a blood sample is subjected to mass spectrometry, the living body-derived sample is disposed of rather than being returned to the individual subject from which it is derived.
The sample to be subjected to mass spectrometry may be one that has been subjected to various pretreatments. For example, the sample may be one that has been subjected to immunoprecipitation (IP). The sample may be one that has been subjected to protein digestion with an enzyme such as peptidase. The sample may be one that has been subjected to chromatography. The sample to be subjected to mass spectrometry may be one to which a certain amount of an internal standard substance has been added.
In this embodiment, an eluate obtained by immunoprecipitation previously performed may be subjected to mass spectrometry (Immunoprecipitation-mass spectrometry; IP-MS). The immunoprecipitation may be performed using an antibody-immobilized carrier prepared using immunoglobulin having an antigen-binding site that can recognize an analyte substance or an immunoglobulin fraction containing an antigen-binding site that can recognize an analyte substance.
In this embodiment, consecutive immunoprecipitation (cIP) may be conducted, and then a peptide in the sample may be detected by a mass spectrometer (cIP-MS). By conducting affinity purification twice consecutively, impurities that cannot be excluded by one affinity purification can be further reduced by the second affinity purification. Therefore, it is possible to prevent the ionization suppression of polypeptide due to impurities, and it becomes possible to measure even a very small amount of polypeptide in a living body sample with high sensitivity by mass spectrometry.
The mass spectrometry is not particularly limited, and examples thereof include those for mass spectrometry such as matrix-assisted laser desorption/ionization (MALDI) mass spectrometry or electrospray ionization (ESI) mass spectrometry. For example, a MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer, a MALDI-IT (matrix-assisted laser desorption/ionization ion trap) mass spectrometer, a MALDI-IT-TOF (matrix-assisted laser desorption/ionization ion trap time-of-flight) mass spectrometer, a MALDI-FTICR (matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance) mass spectrometer, an ESI-QqQ (electrospray ionization triple quadrupole) mass spectrometer, an ESI-Qq-TOF (electrospray ionization tandem quadrupole time-of-flight) mass spectrometer, or an ESI-FTICR (electrospray ionization Fourier transform ion cyclotron resonance) mass spectrometer or the like can be used.
A matrix and a matrix solvent can be appropriately determined by a person skilled in the art depending on the analyte substance.
As the matrix, for example, α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB), sinapic acid, 3-aminoquinoline (3-AQ) or the like can be used.
The matrix solvent can be selected from the group consisting of, for example, acetonitrile (ACN), trifluoroacetic acid (TFA), methanol, ethanol and water, and used. More specifically, an ACN-TFA aqueous solution, an ACN aqueous solution, methanol-TFA aqueous solution, a methanol aqueous solution, an ethanol-TFA aqueous solution, an ethanol solution or the like can be used. The concentration of ACN in the ACN-TFA aqueous solution can be, for example, 10 to 90% by volume, the concentration of TFA can be, for example, 0.05 to 1% by volume, preferably 0.05 to 0.1% by volume.
The matrix concentration can be, for example, 0.1 to 50 mg/mL, preferably 0.1 to 20 mg/mL, or 0.3 to 20 mg/mL, further preferably 0.5 to 10 mg/mL.
In the case of employing MALDI mass spectrometry as a detecting system, a matrix additive (comatrix) is preferably used together. The matrix additive can be appropriately selected by a person skilled in the art depending on the analysis subject (poly peptides) and/or the matrix. For example, as the matrix additive, a phosphonic acid group-containing compound can be used. Specific examples of a compound containing one phosphonic acid group include phosphonic acid, methylphosphonic acid, phenylphosphonic acid, 1-naphthylmethylphosphonic acid, and the like. Specific examples of a compound containing two or more phosphonic acid groups include methylenediphosphonic acid (MDPNA), ethylenediphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid, nitrilotriphosphonic acid, ethylenediaminetetraphosphonic acid, and the like. Among the aforementioned phosphonic acid group-containing compounds, compounds having two or more, preferably two to four phosphonic acid groups in one molecule are preferred.
The use of the phosphonic acid group-containing compound is useful, for example, when metal ions of the washing solution remaining on the surface of the antibody-immobilizing carrier are contaminated into the eluate after the dissociating step. The metal ions adversely affect on the background in the mass spectrometry. The use of the phosphonic acid group-containing compound is effective for suppressing such an adverse affect.
Besides the aforementioned matrix additive, a more common additive, for example, a substance that is selected from the group consisting of ammonium salts and organic bases may be used.
The matrix additive can be prepared as a solution of 0.1 to 10 w/v %, preferably 0.2 to 4 w/v % in water or in a matrix solvent. The matrix additive solution and the matrix solution can be mixed in a volume ratio of, for example, 1:100 to 100:1, preferably 1:10 to 10:1.
In this embodiment, not less than two calibration substances are measured by a mass spectrometer. A calibration formula for the mass spectrometer is calculated using the measurement results of the calibration substances.
The calibration substances may appropriately be determined by those skilled in the art. For example, analyte substances per se may be used or substances different from analyte substances may be used. Stable isotope-labeled substances may be used. A substance labeled with a stable isotope and a substance not labeled with a stable isotope may be used. The calibration substances may be Aβ and an Aβ related peptide(s). When analyte substances are Aβ and an Aβ-related peptide(s), for example, stable isotope-labeled Aβ1-38 (SIL-Aβ1-38) may be used as one of the calibration substances.
The calibration substances may include a compound and said compound labeled with a stable isotope. In mass spectrometry, a compound is ionized for detection. The efficiency of ionization differs depending on the kind of compound, and such a difference in ionization efficiency has an influence on the measurement result of mass spectrometry. A certain compound and said compound labeled with a stable isotope are the same in ionization efficiency. Therefore, a calibration formula with higher accuracy can be obtained using them as calibration substances. For example, in Example 2, Aβ1-38 and stable isotope-labeled Aβ1-38 (SIL-Aβ1-38) are used as calibration substances.
A calibrant is a solution containing a calibration substance. In this embodiment, as a calibrant, a solution containing not less than two calibration substances is used.
As the calibrant, for example, a sample per se containing analyte substances may be used. Alternatively, the calibrant may be one obtained by adding calibration substances to a sample per se containing analyte substances. A solution containing not less than two calibration substances may be prepared and used separately from a sample per se containing analyte substances.
A calibration formula for the mass spectrometer is calculated using the measurement result of the solution containing not less than two calibration substances. In order to calculate a calibration formula, two or more pieces of data are required. For example, when a calibration formula is calculated using a signal peak intensity ratio, two or more pieces of data different in signal peak intensity ratio are required.
For example, when a calibration formula is calculated using one calibrant, the calibrant needs to contain at least three kinds of calibration substances. The ratio of, relative to the signal peak intensity of one of the calibration substances, the signal peak intensity of each of the other not less than two calibration substances may be calculated to obtain not less than two signal peak intensity ratios.
For example, when calibration substances are represented as C1, C2, and C3, C2/C1 and C3/C1 are calculated as peak intensity ratios using C1 as a reference.
Alternatively, two or more calibrants may be used which are different in the concentrations of calibration substances whose signal peak intensity ratio should be determined. In this case, at least two kinds of calibration substances need to be used. The ratio of, relative to the signal peak intensity of one of the calibration substances, the signal peak intensity of the other calibration substance may be calculated for each of the calibrants to obtain not less than two signal peak intensity ratios.
For example, when calibration substances are represented as C1 and C2, the peak intensity ratio of C2/C1 is calculated for each of calibrants different in concentration.
Two or more calibrants may be used which contain one calibration substance at a certain concentration and another calibration substance at different concentrations. The concentration ratios between the calibration substances, whose signal peak intensity ratio should be determined, of the two or more calibrants may be, for example, in the range of 1/4 to 4. For example, five kinds of calibrant solutions whose calibration substance concentration ratios are adjusted to 1/4, 1/2, 1, 2, and 4 may be used.
In this embodiment, the measurement results of not less than two calibration substances by a mass spectrometer are used to calculate a calibration formula for the mass spectrometer. Then, the measurement results of analyte substances are calibrated using the calculated calibration formula. The calibration method according to the present invention includes a calibration method using a calibration formula calculated using a standard machine. Further, the calibration method according to the present invention includes a method in which the measurement results of mass spectrometry are standardized using calibration substances whose abundances are known.
The calibration formula is calculated for every mass spectrometer. Even in the case of the same mass spectrometer, a new calibration formula is preferably calculated when a part such as a detector or the like is exchanged or when the setting of the machine, such as a detector voltage or the like, is changed. Alternatively, the calibration formula may regularly be calculated. This makes it possible to detect the deterioration or failure of a detector or the like of the mass spectrometer or to obtain measurement results from which the influence thereof has been removed. Therefore, it is possible to perform a comparative evaluation of abundance ratios of analyte substances between two or more samples with high accuracy irrespective of a difference between mass spectrometer machines or the machine conditions of the mass spectrometer.
More preferably, when analyte substances are measured, a calibration formula is calculated by measuring calibration substances under the same machine conditions as the measurement of the analyte substances. This makes it possible to use a calibration formula determined under the same conditions as the measurement of the analyte substances and therefore to further improve the accuracy of calibration. Therefore, it is possible to perform a comparative evaluation of abundance ratios of analyte substances between two or more samples with higher accuracy irrespective of a difference between mass spectrometer machines or the machine conditions of a mass spectrometer.
A calibrant solution containing not less than two calibration substances is measured by a standard machine to obtain a signal peak intensity of each of the calibration substances. The ratio of, relative to the signal peak intensity of one of the calibration substances, the signal peak intensity of each of the one or more other calibration substances is calculated.
As the standard machine, a mass spectrometer having high reliability is preferably used. Each user can freely set a mass spectrometer that should be used as a standard machine.
The calibrant solution containing not less than two calibration substances is measured by a mass spectrometer, for which a calibration formula should be calculated, to obtain a signal peak intensity of each of the calibration substances. The ratio of, relative to the signal peak intensity of one of the calibration substances, the signal peak intensity of each of the one or more other calibration substances is calculated.
A regression equation is calculated between the signal peak intensity ratio calculated by the standard machine and the signal peak intensity ratio calculated by the mass spectrometer for which a calibration formula should be calculated. The calculation of a regression equation may appropriately be performed using a known method such as a least-square method or the like. The calculation of a regression equation may be performed using the logarithms of the signal peak intensity ratios. The regression equation may appropriately be selected from among a linear regression equation, a multiple regression equation, an exponential regression equation, a logarithmic regression equation, and a power regression equation.
For example, a linear regression equation may be calculated using values obtained by logarithmic transformation of the signal peak intensity ratios. Alternatively, for example, a power regression equation may be calculated using the signal peak intensity ratios. The calculated regression equation is defined as a calibration formula for the said mass spectrometer.
For example, when the logarithm of the signal peak intensity ratio calculated by the mass spectrometer for which a calibration formula should be calculated is defined as x, and the logarithm of the signal peak intensity ratio calculated by the standard machine is defined as y, calibration coefficients a and b of:
a linear regression equation (y=ax+b)
can be calculated. The calculated linear regression equation (y=ax+b) can be used as a calibration formula for the said mass spectrometer.
Alternatively, when the signal peak intensity ratio calculated by the mass spectrometer for which a calibration formula should be calculated is defined as x, and the signal peak intensity ratio calculated by the standard machine is defined as y, calibration coefficients a and b of:
a power regression equation (y=axb)
may be calculated. The calculated power regression equation (y=axb) can be used as a calibration formula for the said mass spectrometer.
The calibration coefficients a and b are considered as values inherent in the said mass spectrometer. Therefore, the calibration coefficients a and b may vary with a difference between mass spectrometer machines or the machine conditions of the mass spectrometer. The calibration coefficient b is more strongly influenced by a difference between mass spectrometer machines or the machine conditions of the mass spectrometer than the calibration coefficient a. Therefore, in, for example, a power regression equation (y=axb), the value a may be fixed to 1(a=1) so that only the value b is calculated and adopted as a calibration coefficient. That is, y=xb may be used as a calibration formula instead of the power regression equation (y=axb). In such a case where only b is used as a calibration coefficient, the value b is referred to as a calibration value.
A sample containing analyte substances is measured by the mass spectrometer for which a calibration formula has been calculated in 5-1-1. to obtain a signal peak intensity of each of the analyte substances. One of the analyte substances (e.g., an internal standard substance) is used as a reference to calculate the ratio of, relative to the signal peak intensity of the analyte substance as a reference, the signal peak intensity of another analyte substance.
The calculated signal peak intensity ratio is calibrated using the calibration formula calculated above in 5-1-1. The signal peak intensity ratio after calibration is a value in which a machine difference from the standard machine is cancelled. That is, the signal peak intensity ratio after calibration is equivalent to a signal peak intensity ratio obtained by measurement using the standard machine. Therefore, the use of signal peak intensity ratios after calibration makes it possible to perform a comparative evaluation of the signal peak intensity ratios of the analyte substances, that is, the abundance ratios of the analyte substances between two or more samples irrespective of a difference between mass spectrometer machines.
A calibration formula for standardizing a signal peak intensity ratio can be calculated using two or more calibrant solutions whose concentration ratio of two calibration substances is known.
Two or more calibrant solutions (intensity ratio calibrants; ICs) whose concentration ratio of two calibration substances is known are used. Two or more solutions may be used which contain one calibration substance at a certain concentration and contain the other calibration substance at different concentrations. For example, solutions may be used which contain SIL-Aβ1-38 at a certain concentration and contain Aβ1-38 at different concentrations so that the concentration ratios of Aβ1-38 to SIL-Aβ1-38 are adjusted to 1/4, 1/2, 1, 2, and 4.
Two or more calibrant solutions (intensity ratio calibrants; ICs) whose concentration ratio of two calibration substances is known are measured by a mass spectrometer for which a calibration formula should be calculated to obtain a signal peak intensity of each of the calibration substances in each of the solutions. For each of the solutions, the ratio of, relative to the signal peak intensity of the calibration substance contained at a certain concentration, the signal peak intensity of the other calibration substance is calculated.
A regression equation is calculated between the known concentration ratio of the two calibration substances in each of the calibrant solutions and the signal peak intensity ratio calculated above. The calculation of a regression equation may appropriately be performed using a known method such as a least-square method or the like. The calculation of a regression equation may be performed using the logarithms of the signal peak intensity ratios. The regression equation may appropriately be selected from among a linear regression equation, a multiple regression equation, an exponential regression equation, a logarithmic regression equation, and a power regression equation.
For example, a linear regression equation may be calculated using the logarithms of the signal peak intensity ratios. Alternatively, a power regression equation may be calculated using the signal peak intensity ratios. The calculated regression equation is defined as a calibration formula for the said mass spectrometer.
For example, when the logarithm of the known concentration ratio of the two calibration substances in each of the solutions is defined as x, and the logarithm of the signal peak intensity ratio calculated by the mass spectrometer for which a calibration formula should be calculated is defined as y, calibration coefficients a and b of:
a linear regression equation (y=ax+b)
can be calculated. The calculated linear regression equation (y=ax+b) can be used as a calibration formula for the said mass spectrometer.
Alternatively, when the known concentration ratio of the two calibration substances in each of the solutions is defined as x, and the signal peak intensity ratio calculated by the mass spectrometer for which a calibration formula should be calculated is defined as y, calibration coefficients a and b of:
a power regression equation (y=axb)
may be calculated. The calculated power regression equation (y=axb) can be used as a calibration formula for the said mass spectrometer.
The calibration coefficients a and b are considered as values inherent in the said mass spectrometer. Therefore, the calibration coefficients a and b may vary with a difference between mass spectrometer machines or the machine conditions of the mass spectrometer. The calibration coefficient b is more strongly influenced by a difference between mass spectrometer machines or the machine conditions of the mass spectrometer than the calibration coefficient a. Therefore, in, for example, a power regression equation (y=axb), the value a may be fixed to 1(a=1) so that only the value b is calculated and adopted as a calibration coefficient. That is, y=xb may be used as a calibration formula instead of the power regression equation (y=axb). In such a case where only b is used as a calibration coefficient, the value b is referred to as a calibration value.
A sample containing analyte substances is measured by the mass spectrometer for which a calibration formula has been calculated in 5-2-1. to obtain a signal peak intensity of each of the analyte substances. One of the analyte substances (e.g., an internal standard substance) is used as a reference to calculate the ratio of, relative to the signal peak intensity of the analyte substance as a reference, the signal peak intensity of another analyte substance.
The calculated signal peak intensity ratio is calibrated using the calibration formula calculated above in 5-2-1. The signal peak intensity ratio of the analyte substances is converted by calibration to a signal peak intensity ratio standardized by the calibration substances. In the obtained standardized signal peak intensity, a machine difference is cancelled by the calibration formula. Therefore, it is possible to perform a comparative evaluation of abundance ratios of the analyte substances between two or more samples irrespective of a difference between mass spectrometer machines. Specifically, it is possible to perform direct comparison with the measurement results of mass spectrometry obtained in not only Japan but also other countries such as America and France. Further, this calibration technique is a versatile technique applicable to various researches and examinations using mass spectrometry.
As described above, in this embodiment, calibration is performed by calculating a calibration formula for a mass spectrometer used for measurement of analyte substances under the same machine conditions as the measurement of the analyte substances, which makes it possible to, as described above, perform a comparative analysis of the abundance ratios of the analyte substances between two or more samples irrespective of a difference between mass spectrometer machines or the machine conditions of the mass spectrometer.
The calibration formula for intensity ratio calibration can be prepared using two or more calibrant solutions whose concentration ratio of two calibration substances is known. For example, five kinds of solutions may be used which contain SIL-Aβ1-38 at a certain concentration and contain Aβ1-38 at different concentrations so that the concentration ratios of Aβ1-38 to SIL-Aβ1-38 are adjusted to 1/4, 1/2, 1, 2, and 4. In the mass spectrum of each of the solutions, the peak of a certain amount of SIL-Aβ1-38 and the peak of Aβ1-38 that depends on the concentration thereof appear. Ideally, the peak intensity ratios are expected to correspond to the concentration ratios in the respective calibrant solutions. However, the peak intensity ratios vary depending on machine conditions, and therefore a calibration formula is calculated so that the peak intensity ratios correspond to the concentration ratios in the respective calibrant solutions, and the measurement results of analytes are calibrated. This makes it possible to cancel a machine difference.
The calibration formula varies with a difference between mass spectrometer machines. Further, even in the case of the same mass spectrometer, when a part such as a detector or the like is exchanged or a machine setting, such as a detector voltage or the like, is changed. conditions of the mass spectrometer are changed, and therefore the calibration formula varies depending on machine conditions. Further, also when machine conditions are changed due to. for example, deterioration of a detector or the like, the calibration formula may be changed.
For example, when the same sample is measured by a mass spectrometer, a signal peak intensity ratio detected by the mass spectrometer increases due to the influence of machine conditions such as deterioration of a detector or the like. The reason for this is considered to be that the signal peak of a calibrant substance whose abundance is low becomes small due to the influence of machine conditions such as deterioration of a detector or the like. Generally, when a signal peak intensity is excessively low in a mass spectrometer, measurement accuracy reduces from the viewpoint of S/N ratio. Therefore, it is preferred that the signal peak intensity is not excessively low in the mass spectrometer.
For example, in a case where a power regression equation (y=axb) is determined in 5-2-1, when a signal peak intensity detected by a mass spectrometer becomes low due to the influence of some kind of machine conditions, the calibration value b in the calibration formula increases. Therefore, by allowing the value b to fall within a certain range, the signal peak intensity is prevented from becoming excessively low, and therefore the measurement accuracy of the mass spectrometer further improves so that the accuracy of calibration further improves.
The value b can be varied by changing the detection sensitivity of the mass spectrometer. The value b can be controlled by, for example, changing a detector voltage. Alternatively, the value b may be changed by, for example, changing the baseline level of an analog digital (AD) converter.
According to this embodiment, a comparative evaluation of abundance ratios of analyte substances can be performed between two or more samples irrespective of a difference between mass spectrometer machines or the machine conditions of a mass spectrometer by calibrating the signal peak intensity ratios of the analyte substances using a calibration formula. Further, the signal peak intensity ratios of the analyte substances can more accurately be calibrated by adjusting machine conditions in such a manner that the value of a calibration coefficient in the calibration formula falls within a certain range.
A machine difference calibration system of a mass spectrometer according to an embodiment of the present invention comprises:
A machine difference calibration program of a mass spectrometer according to an embodiment of the present invention includes allowing a computer to execute
An embodiment of the system and program for machine difference calibration of a mass spectrometer according to the present invention will be described below with reference to the drawings. The system and program for calculating a calibration value of a mass spectrometer according to this embodiment are intended to calculate a calibration value for performing the calibration (intensity ratio calibration) for standardizing a signal peak intensity ratio described above in 5-2.
The mass analysis unit 1 is not particularly limited, and examples thereof include those for mass spectrometry such as matrix-assisted laser desorption/ionization (MALDI) mass spectrometry or electrospray ionization (ESI) mass spectrometry. For example, a MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer, a MALDI-IT (matrix-assisted laser desorption/ionization ion trap) mass spectrometer, a MALDI-IT-TOF (matrix-assisted laser desorption/ionization ion trap time-of-flight) mass spectrometer, a MALDI-FTICR (matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance) mass spectrometer, an ESI-QqQ (electrospray ionization triple quadrupole) mass spectrometer, an ESI-Qq-TOF (electrospray ionization tandem quadrupole time-of-flight) mass spectrometer, or an ESI-FTICR (electrospray ionization Fourier transform ion cyclotron resonance) mass spectrometer or the like can be used.
Examples of the data processing unit 2 actually used include a general-purpose personal computer and a higher-performance workstation. The data processing unit 2 may be a single computer or a computer system including two or more computers. This embodiment is implemented by installing a dedicated data processing program into such a computer and operating the computer. The input unit 3 is usually a keyboard or a pointing device such as a mouse or the like supplied with the computer. The display unit 4 is usually a monitor supplied with the computer.
Even when the same sample is measured by a mass spectrometer, there is a case where measurement results vary with a difference between machines or the machine conditions of the mass spectrometer. In order to cancel such a machine difference or a difference depending on machine conditions, calibration is performed as described above in 5-2.
The measuring method preparing unit 20 is intended to automatically prepare a measuring method for measuring a calibrant for calculating a calibration value. According to the measuring method prepared by the measuring method preparing unit 20, a sample is measured by the mass analysis unit 1. The measuring method preparing unit 20 further includes a setting part 201. The setting part 201 sets a laser power value used for measuring the calibrant.
The calibration value calculating unit 21 calculates a calibration value using signal peak intensity ratios by analysis of mass spectrum data measured by the mass analysis unit 1.
Hereinbelow, measurement using the system and program for calculating a calibration value of a mass spectrometer according to this embodiment will be described in detail using
A user prepares samples used to calculate a calibration value of a mass spectrometer. As the samples, five kinds of samples (IC1 to IC5) are used whose concentration ratios between an internal standard substance and a target analyte are different stepwise. For example, samples IC-1, IC-2, IC-3, IC-4 and IC-5 are used whose concentration ratios (concentration of the internal standard substance : concentration of the target analyte) are respectively 1:4, 1:2, 1:1, 1:0.5, and 1:0.25.
Then, the user allows the measuring method preparing unit to automatically prepare a measuring method.
The measuring method preparing unit 20 prepares a measuring method of the input dataset name (S2: measuring method preparing step). Specifically, a laser power value is previously calculated to set the laser power value of the mass spectrometer used in the measuring method. Further, the sample dropping position of each of the samples IC1 to IC5 is determined. The measuring method preparing unit 20 allows the GUI displayed on the display unit 4 to display the laser power, and allows the sample plate display part of the GUI to diagrammatically show the sample dropping positions of the samples IC1 to IC5.
The laser power can be calculated by various known methods.
For example, the laser power is calculated by the following method. For example, a laser power adjusting method can be used which includes:
It should be noted that the calculated laser power may vary between wells in a sample plate. For example, laser power calculated for wells (calibrant wells) into which the samples IC1 to IC5, which are measured by the present measuring method and used to calculate a calibration value, are to be dropped may be different from laser power used for wells (sample wells) used to measure a real sample to be analyzed. For example, laser power used for calibrant wells may be lower by 10 than that used for sample wells.
The sample dropping positions can be determined by various known methods.
The determined sample dropping positions are displayed on the sample plate display part as shown in
The user drops the calibrants IC1 to IC5 onto the sample dropping positions displayed on the display unit 4 to start measurement. The mass analysis unit 1 measures each of the calibrants under predetermined conditions (S3).
When the measurement ends, the user performs operation for calculating a calibration value.
The calibration value calculating unit 21 analyzes mass spectrum data measured by the mass analysis unit 1 in the selected dataset name (S4), and calculates a calibration value (S5). The calibration value calculating step includes S4 and S5. The calibration value can be calculated using, for example, the method described above in [5. Calibration of measurement results in mass spectrometer].
Specifically, the ratio of the signal peak intensity of the target analyte relative to the signal peak intensity of the internal standard substance is calculated for each of the samples IC1 to IC5. A power regression equation (y=axb) can be calculated between the concentration ratio of the target analyte relative to the internal standard substance in each of the solutions and the signal peak intensity ratio calculated above. The calibration coefficient b of the calculated regression equation is output as a calibration value b. The calibration value b is displayed on the GUI of the display unit 4.
Then, the user allows the mass analysis unit I to measure a sample to be analyzed to obtain a measurement result calibrated using the value b.
Hereinbelow; the present invention will be described more specifically with reference to examples, but is not limited to these examples.
Various kinds of peptides were measured by three mass spectrometers (all of which are of the same type: Performance) to examine differences in peak intensity ratios between these machines. As a result, it was found that the logarithms of the peak intensity ratios had a linear relation between the machines. It was confirmed that the peak intensity ratios could be calibrated by preparing a linear regression equation on the basis of logarithms of measured values between the machines. Further, it was confirmed that since the logarithms of the peak intensity ratios have a linear relation, the peak intensity ratios have an exponential (power) relation when not converted to logarithms, and therefore the peak intensity ratios could be calibrated also by preparing an exponential (power) regression equation (power approximate equation).
In order to achieve a more practical method, samples for intensity ratio calibration were prepared which contained a stable isotope-labeled substance (at a certain concentration) and the same non-labeled substance (at different concentrations). These samples were measured by the mass spectrometer, a power approximate equation was prepared from the intensity ratios and abundance ratios of the substances as a calibration formula, and the peak intensity ratios of the analyte substances were calibrated using the calibration formula.
The peak intensity ratios obtained by each of the machines were calibrated using the regression equation. The calibration made it possible to obtain equivalent peak intensity ratios even when the same sample was measured by different machines. Further, the calibration is effective at calibrating peak intensity ratios that vary due to the exchange, voltage change, or deterioration of a detector. It is possible to perform a comparative evaluation of abundances of analyte substances using one kind of stable isotope-labeled substance without preparing a stable isotope-labeled substance for each of the target analyte substances.
This method can be used not only for peptides obtained by IP but also for peptides generated by digestion of protein with an enzyme such as peptidase or the like or peptides fractionated by chromatography. Further, this method can be used not only for peptides but also for glycopeptides, sugar chains, or lipids.
Hereinbelow, the examples will be described in more detail.
In this example, Aβ and Aβ related peptides as analyte substances were prepared in the following manner.
A blood plasma sample (Sample No. 1) was subjected to immunoprecipitation-mass spectrometry (IP-MS) using SIL-Aβ1-38 as an internal standard peptide.
First, 0.5 μL of a matrix solution (0.5 mg/mL α-cyano-4-hydroxycinnamic acid: CHCA, 0.2% (w/v) Methanediphosphonic acid: MDPNA) was added to wells of a μFocus MALDI plate™ 900 μm and dried.
IP was performed in the following manner. Antibody-immobilized beads obtained by immobilizing anti-Aβ monoclonal antibodies (clones 6E10) and 4G8) to magnetic beads were washed twice with an OTG-glycine buffer (1% n-Octyl-β-D-thioglucoside (OTG), 50 mM glycine, pH 2.8) and washed three times with 100 μL of a washing buffer. Then, 250 μL of the blood plasma sample was mixed with 250 μL of a binding buffer (0.2% (w/v) n-Dodecyl-β-D-maltoside (DDM), 0.2% (w/v) n-Nonyl-β-D-thiomaltoside (NTM), 800 mM GlcNAc, 100 mM Tris-HCl, 300 mM NaCl, pH 7.4) containing 10 pM SIL-Aβ1-38 (AnaSpec, San Jose, CA, USA), the antibody-immobilized beads were then added thereto, and the resultant was incubated at 4° C. for 1 hour to capture Aβ and Aβ related peptides. Then, the resultant was washed once with a washing buffer (500 μL or 100 μL), washed four times with 100 μL of a washing buffer, and washed twice with 50 mM ammonium acetate (50 μL or 20 μL). Further, the resultant was washed once with H2O (30 μL or 20 μL), and then the Aβ and Aβ related peptides captured by the antibody-immobilized beads were eluted with 5 μL of 70% acetonitrile containing 5 mM hydrochloric acid. Then, 1 μL of the eluate was dropped into each of 4 wells in the μFocus MALDI plate™ 900 μm to which the matrix had been added. The resultant was measured using three machines of AXIMA Performance (Shimadzu/KRATOS, Manchester, UK) by Linear TOF in a positive ion mode. A mass spectrum was obtained by integrating 40 shots per each of the points of 400 spots in a raster mode. As a quantitative value, an average of peak intensity ratios of each of the Aβ and Aβ related peptides relative to the internal standard peptide (SIL-Aβ1-38) in spectrums obtained by measuring the 4 wells was used. A detection limit was an S/N ratio of 3, and peaks of not more than the detection limit were regarded as not detectable. The amino acid sequences of the measured Aβ and Aβ related peptides are shown in Table 1.
Further, also in the case of the peak intensity ratio of each of the Aβ, Aβ related peptides, and SIL-Aβ1-38 relative to APP669-711, the difference between the three machines tended to be smaller when the peak intensity ratios were closer to 1, and the difference between the three machines tended to be larger when the peak intensity ratios were farther from 1 (
The present inventors analyzed whether there was a rule in the difference in peak intensity ratio between the three machines, and as a result, values obtained by logarithmic transformation of the peak intensity ratios had a linear relation between the machines (
The linear regression equation between Performance 1 and Performance 2:
wherein x is a value obtained by logarithmic transformation of the signal peak intensity ratio measured by Performance 2, and y is a value obtained by logarithmic transformation of the signal peak intensity ratio measured by Performance 1.
The linear regression equation between Performance 1 and Performance 3:
wherein x is a value obtained by logarithmic transformation of the signal peak intensity ratio measured by Performance 3, and y is a value obtained by logarithmic transformation of the signal peak intensity ratio measured by Performance 1.
Although exactly the same as above, when logarithmic transformation is not performed, the relationship between the machines is represented by a power approximate equation (
The power approximate equation between Performance 1 and Performance 2:
wherein x is a value of the signal peak intensity ratio measured by Performance 2, and y is a value of the signal peak intensity ratio measured by Performance 1.
The power approximate equation between Performance 1 and Performance 3:
wherein x is a value of the signal peak intensity ratio measured by Performance 3, and y is a value of the signal peak intensity ratio measured by Performance 1.
The present inventors examined whether, when Performance 1 was used as a standard machine, the peak intensity ratios of the Aβ and Aβ related peptides relative to the internal standard peptide (SIL-Aβ1-38) measured by Performance 2 and Performance 3 could be calibrated to be equivalent to the peak intensity ratios measured by the standard machine (Performance 1) by using these regression equations. The peak intensity ratios measured by Performance 2 and Performance 3 were calibrated by plugging in them for x in the above regression equations and determining y (
This result indicates that even when the same sample is measured by different mass spectrometers, the same peak intensity ratios can be obtained by calibrating peak intensity ratios using a calibration formula. In other words, this result indicates that the calibration formula makes it possible to cancel errors (machine difference) of peak intensity ratios caused by a difference between mass spectrometer machines.
The reason why logarithms of the peak intensity ratios have a linear relationship between the machines is considered to be that a secondary electron multiplier (SEM) used as a detector for mass spectrometry exponentially amplifies the electric signals of ions so that large signals are obtained.
In order to examine whether the calibration formulas prepared in 1-2 were applicable to other samples, blood plasma samples (Sample No. 2 and Sample No. 3) different from the sample used above in 1-1 were subjected to IP-MS, and peak intensity ratios before and after calibration were compared. The IP-MS was performed in the same manner in 1-1, and linear regression equations calculated in 1-2, that is, linear regression equations calculated using logarithmically transformed values of the peak intensity ratios were used as calibration formulas. As a result, it was confirmed that in the cases of both Sample No. 2 and Sample No. 3, the peak intensity ratios obtained by the three machines became equivalent by calibration (
These results indicate that when any of the samples (Samples No. 1, No. 2, and No. 3) is measured by the three machines, equivalent peak intensity ratios can be derived by performing calibration using the calibration formulas prepared in 1-2, and demonstrate the validity of the calibration formulas.
The reproducibility of the calibration formulas prepared in 1-2 was verified. IP-MS was performed in the same manner as in 1-1 in a day different from the day when 1-1 was performed to prepare calibration formulas (linear regression equations using logarithmically transformed values) (
Analysis of covariance (ANCOVA) was performed to verify whether the calibration formulas between the machines obtained in Day 1 and Day 2 were the same. First, in the test of ANCOVA between 4 groups, analysis was performed using a statistical significance of p<0.05. As a result, the slope was p<0.0001. This demonstrated that the four groups were statistically significantly different from each other.
Then, a pairwise comparison between the 4 groups was performed by ANCOVA (Table 3). In this case, the test needed to be performed four times, and therefore a statistical significance of p<0.0125 was set by Bonferroni correction. First, the slopes of the regression equations were tested, and when significant differences were not observed, the intercepts of the regression equations were tested. As a result, a comparison between Performance 2 (Day 1) and Performance 3 (Day 1) and a comparison between Performance 2 (Day 2) and Performance 3 (Day 2) demonstrated that the regression equations were different; and a comparison between Performance 2 (Day 1) and Performance 2 (Day 2) and a comparison between Performance 3 (Day 1) and Performance 3 (Day 2) demonstrated that the regression equations were the same. These results indicate that the machines have their respective own regression equations as calibration formulas, and the regression equations do not vary according to a measurement day unless the conditions of the machines do not change due to, for example, the deterioration of a detector or the like.
In this example, Aβ and Aβ related peptides as analyte substances were prepared in the following manner.
Aβ related peptides in human blood plasma were measured using IP-MS in which immunoprecipitation (IP) using an anti-Aβ monoclonal antibody and mass spectrometry (MS) were performed in combination. In the IP, antibody beads were used which were prepared by covalently bonding an anti-Aβ antibody clone 6E10 (BioLegend) to Dynabeads Epoxy (Thermo Fisher Scientific) as magnetic beads. Two hundred and fifty microliters (250 μL) of blood plasma and 250 μL of a reaction solution containing an internal standard peptide were mixed, and the antibody beads were added thereto to perform an antigen-antibody reaction at 4° C. for 1 hour (1st IP). As the internal standard peptide, 11 pM of stable isotope-labeled (SIL) Aβ1-38 was used. After the antigen-antibody reaction, the antibody beads were washed, and the Aβ related peptides were eluted using a 1st IP eluent (glycine buffer containing DDM (pH 2.8)). The eluate was neutralized with a tris buffer containing DDM, an antigen-antibody reaction was then again performed between the antibody beads and the Aβ related peptides (2nd IP), the antibody beads were washed, and the Aβ related peptides were then eluted with a 2nd IP eluent (5 mM HCl, 0.1 mM Methionine, 70% (v/v) acetonitrile). In advance, 0.5 μL of 0.5 mg/mL CHCA/0.2% (w/v) MDPNA was dropped into each well of a μFocus MALDI plate™ 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ) and dried. The eluate after IP was dropped into four wells of the μFocus MALDI plate™ 900 μm and dried.
Mass spectrum data was obtained using AXIMA Performance (Shimadzu/KRATOS, Manchester, UK) by Linear TOF in a positive ion mode. The m/z value of Linear TOF was indicated by the average mass of peaks. The m/z value was calibrated using, as external standards, human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, and bovine insulin. A mass spectrum was obtained by integrating 40 shots per each of the points of 400 spots in a raster mode. As a quantitative value, an average of peak intensity ratios of each of the Aβ and Aβ related peptides relative to the internal standard peptide (SIL-Aβ1-38) in spectrums obtained by measuring the 4 wells was used. A detection limit was an S/N ratio of 3, and peaks of not more than the detection limit were regarded as not detectable.
Five kinds of concentrated solutions of intensity ratio calibrant (IC) reagents for calibrating the peak intensity ratios of the Aβ and Aβ-related peptides relative to SIL-Aβ1-38 obtained by IP-MS were prepared to have compositions shown in Table 4 and preserved by freezing. Before MS measurement, the concentrated solutions IC-1 to IC-5 were thawed and diluted 10-fold with the 2nd IP eluent (5 mM HCl, 0.1 mM Methionine, 70% (v/v) acetonitrile) used in 2-1 to prepare IC-1 to IC-5. In advance, 0.5 μL of 0.5 mg/mL CHCA/0.2% (w/v) MDPNA was dropped into each well of a μFocus MALDI plate™ 900 μm and dried. Each of the IC-1 to IC-5 was dropped into four wells in an amount of 1 μL per well and dried. The protein/peptide compositions of the IC-1 to IC-5 are shown in Table 5. The abundance ratios of Aβ1-38 to SIL-Aβ1-38 in the IC-1 to IC-5 were set to 4, 2, 1, 1/2, and 1/4, respectively.
The mass spectrum data of each of the IC-1 to IC-5 was obtained in the same manner as in 2-1.
IP-MS of human blood plasma and measurements of the IC-1 to IC-5 were performed under three conditions of before and after exchange of a detector of Performance 1, and after exchange of a detector of Performance 3.
Power approximate equations were prepared from the intensity ratios and the abundance ratios of Aβ1-38 relative to SIL-Aβ1-38 in the mass spectra of the IC-1 to IC-5 (
Power approximate equation: y=axb,
In
In
In
As shown in
The peak intensity ratios of the Aβ and Aβ related peptides relative to SIL-Aβ1-38 were calculated from the mass spectrum of IP-MS, and calibrated by being plugged into an IC curve, that is, into the power approximate equation calculated using the measurement results of the IC-1 to IC-5. Specifically, the power approximate equations were standardized to y=x by plugging in the peak intensity ratios obtained by IP-MS for y to determine x. The averages of the quadruple measurement data (n=4) of the peak intensity ratios were calculated and used for analysis.
Further, APP669-711/Aβ1-42 and Aβ1-40/Aβ1-42 functioning as biomarkers were also evaluated. In the case of APP-669-711/Aβ1-42, differences in peak intensity between them before calibration were small and CV was also sufficiently small because the intensity ratios were close to 1, and therefore the effect of calibration was not observed. On the other hand, in the case of Aβ1-40/Aβ1-42, the intensity ratios were large, and therefore CV was reduced from 41.1% (before calibration) to 8.0% (after calibration) (
Furthermore, it was observed that also in the case of the method in which calibration was performed using the value a and the value b, CVs of the peak intensity ratios of the Aβ and Aβ related peptides relative to SIL-Aβ1-38 between the three conditions were effectively reduced, and the results of the biomarkers were exactly the same as the results obtained by the method in which a=1 (
The above results indicated that these calibration methods were effective at reducing variations in peak intensity ratios between machines. Further, the above results indicated that the value a did not change depending on machine conditions, and therefore even when only the value b was used as a calibration value, variations in peak intensity ratios between machines were effectively reduced. The method of Example 2 standardizes peak intensity ratios by using power approximate equations, and therefore can be applied without the necessity of using a certain machine as a standard machine.
A sample obtained by spiking standard blood plasma with three kinds of Aβ peptides (Aβ1-40, Aβ1-42, and APP669-766) and an internal standard peptide was subjected to IP treatment, and the sample and IC reagents were measured by three machines of AXIMA-Performance. The machines used for measurement, detector voltages, and values b calculated from the results of IC measurement are shown in Table 6.
It should be noted that there is a correlation between the value b and the detector voltage, and therefore the value b can be adjusted by the detector voltage.
The intensity ratios of Aβ1-40, Aβ1-42, and APP669-766 relative to the internal standard were read from obtained mass spectra, and biomarkers (APP669-711/Aβ1-42 and Aβ1-40/Aβ1-42) were compared between when the intensity ratios were calibrated by the value b and when the intensity ratios were not calibrated.
The following Table 7 shows CVs of the Aβ peptide ratios calculated on the basis of data classified by focusing the value b.
1) three conditions of P1 2850 V (0.9560), P3 2750 V (0.9547), P4 2850 V (0.9371)
2) three conditions of P1 2800 V (1.0917), P3 2775 V (0.9141), P4 2850 V (0.9371)
“Total” represents CVs calculated using all the results measured under six conditions.
“Detector voltage change in one machine” represents CVs calculated using the results measured under three conditions achieved by changing the detector voltage in the machine P1.
“Small variations in value b between three machines” represents CVs calculated using the results measured by the three machines under the condition where the values b are close to 0.95.
“Large variations in value b between three machines” represents CVs calculated using the results measured by the three machines under three conditions where the values b are different.
In the case of “Small variations in value b between three machines”, the CVs are originally low, and therefore there is no large difference between before and after calibration, but in other cases, it is clear that the CVs are greatly reduced by calibration using the value b. This indicates that calibration using the value b is effective in both cases where a machine setting is changed in one machine and where machine conditions are different between different machines.
As described in 2-4 in Example 2, since there is a correlation between the value b and the detector voltage, the value b can be adjusted by the detector voltage. Hereinbelow, the results of examination of the relationship between the value b and the detector voltage will be shown.
IC measurements were performed using, as calibration substances, normal Aβ1-38 and stable isotope-labeled Aβ1-38 (SIL-Aβ1-38) which were the same in ionization efficiency. As IC1 to IC5, the same IC1 to IC5 as used in 2-2 in Example 2 were used.
The IC measurements were performed by the mass spectrometer (Performance 4) by setting the detector voltage to 2700 V, 2750 V, 2800 V, 2850 V, 2900 V, and 2950 V, respectively. As in the case of 2-3 in Example 2, a calibration formula (power approximate equation: y=axb) was determined using measurement results at different detector voltages.
The IC measurements were performed by the mass spectrometer (Performance 1) by setting the detector voltage to 2700 V, 2725 V, 2750 V, 2775 V, 2800 V, and 2825 V, respectively. As in the case of 2-3 in Example 2, a calibration formula (power approximate equation: y=axb) was determined using measurement results at different detector voltages.
As shown in
When the value b increases due to, for example, deterioration of the detector, the value b can be adjusted to fall within a certain range by increasing the detector voltage by using such a relationship between the detector voltage and the value b. By adjusting the value b to fall within a certain range, the signal peak intensity ratios of analyte substances can more accurately be calibrated. For example, the value b may be adjusted to fall within a range of 0.9 to 1.1.
As another means for adjusting the value b, the baseline level of an analog-digital converter (AD converter) may be adjusted. Hereinbelow, the results of examination of a relationship among the value b, the baseline level setting of the AD converter, and the detector voltage will be shown.
IC measurements were performed using, as calibration substances, normal Aβ1-38 and stable isotope-labeled Aβ1-38 (SIL-Aβ1-38) which were the same in ionization efficiency. As IC1 to IC5, the same IC1 to IC5 as used in 2-2 in Example 2 were used.
The IC measurements were performed by the mass spectrometer (Performance 1) by setting the detector voltage to 2700 V, 2725 V, 2750 V, 2775 V, 2800 V, and 2825 V, respectively. Two baseline levels of the AD converter were set: one was a normal setting of 181, and the other was a state where the baseline level setting was reduced therefrom (179) so that a noise level was increased. Data was obtained at each of the two baseline levels. As in the case of 2-3 in Example 2, a calibration formula (power approximate equation: y=axb) was determined using measurement results at different detector voltages.
As shown in
When the value b increases due to, for example, deterioration of the detector, the value b can be adjusted to fall within a certain range by reducing the baseline setting by using such a relationship between the baseline setting of the AD converter and the value b. By adjusting the value b to fall within a certain range, the signal peak intensity ratios of analyte substances can more accurately be calibrated. For example, the value b may be adjusted to fall within a range of 0.9 to 1.1.
The present invention includes, for example, the following aspects.
(1)
A method for calibrating a difference in signal intensity ratio between machines in mass spectrometry, the method comprising the steps of:
The method according to the above (1), wherein the calibration substances include a substance labeled with a stable isotope and a substance not labeled with a stable isotope.
(3)
The method according to the above (1) or (2), wherein the analyte substances are selected from the group consisting of peptides, glycopeptides, sugar chains, lipids, and glycolipids.
(4)
The method according to any one of the above (1) to (3), wherein the calibration substances are Aβ related peptides.
(5)
The method according to any one of the above (1) to (4), wherein the calibration substances are Aβ1-38 and stable isotope-labeled Aβ1-38.
(6)
The method according to any one of the above (1) to (5), wherein the calibrant contains not less than three kinds of calibration substances.
(7)
The method according to any one of the above (1) to (6), wherein a number of the calibrants is two or more, and
The method according to the above (7), wherein a ratio of, relative to a concentration of one of the calibration substances, a concentration of another calibration substance is in a range of 1/4 to 4.
(9)
The method according to any one of the above (1) to (8), wherein in the step of determining a calibration formula, a calibration formula is determined using a value obtained by logarithmic transformation of the signal peak intensity ratio.
(10)
The method according to any one of the above (1) to (9), wherein the calibration formula is a linear, polynomial, exponential, logarithmic, or power calibration formula.
(11)
The method according to any one of the above (1) to (10), wherein a coefficient in the calibration formula is within a predetermined range.
(12)
The method according to the above (11), wherein the coefficient in the calibration formula is adjusted to fall within the predetermined range by adjusting a detector voltage and/or a baseline level in an AD converter of the mass spectrometer.
(13)
The method according to any one of the above (1) to (12), wherein the calibration formula is represented by a power approximate equation:
A machine difference calibration system of a mass spectrometer, the system comprising:
The machine difference calibration system according to the above (14), wherein the measuring method preparing unit includes a setting part that sets a previously-calculated laser power value as a laser power value used to measure the calibrant.
(16)
The machine difference calibration system according to the above (14) or (15), which includes a sample plate display part that displays a sample plate of the mass spectrometer.
(17)
The machine difference calibration system according to the above (16), wherein the sample plate display part shows sample dropping positions in the measuring method.
(18)
The machine difference calibration system according to any one of the above (15) to (17), wherein the setting part sets a laser power value different between sample wells and calibrant wells.
(19)
A machine difference calibration program of a mass spectrometer, the program including allowing a computer to execute
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/000235 | 1/6/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63062677 | Aug 2020 | US |
