Standard solutions for quality control in peptide measurements and quality control in peptide measurements

Abstract

The present invention provides: a quality control standard solution used in assay for peptides (including proteins), and quality control of assay for peptides (including proteins). A QC standard sample for assay of a target peptide of the present invention comprises a blood sample containing said peptide and a peptide that is labelled with a stable isotope and that is used to spike the blood sample. The peptide that is the target of the assay includes, for example, at least one selected from the group consisting of Aβ 1-42 (SEQ ID NO: 9), Aβ 1-38 (SEQ ID NO: 11), Aβ 1-40 (SEQ ID NO: 7), and APP 669-711 (SEQ ID NO: 10). The peptide labelled with the stable isotope includes, for example, at least one selected from the group consisting of SIL-Aβ 1-42, SIL-Aβ 1-38, SIL-Aβ 1-40, and SIL-APP 669-711.

Description

[Technical Field]

[0001] The present invention belongs to the fields of biological sample analysis and quantitative analysis of peptides (including proteins), and relates to a standard solution for quality control in peptide measurement and quality control of peptide measurement. [Background technology]

[0002] Analysis of various biological samples and quantitative analysis of peptides (including proteins) are performed using immunoprecipitation (IP)-mass spectrometry (IP-MS), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS), among others.

[0003] When measuring peptides in multiple samples using these analytical instruments, quality control of the analytical data obtained is important. That is, when measuring peptides in multiple samples, it is necessary to obtain accurate analytical data based on certain standards for each sample, regardless of the timing of the sample measurement.

[0004] For example, Non-Patent Document 1 discloses a procedure for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled with mass spectrometry, and discloses on page 1073, paragraph 19, that in GC-TOF-MS analysis, a QC sample is injected at the start of each analytical batch. Also, on page 1074, Box 1, in UPLC-TOF-MS analysis, a QC sample is injected at the start of each analytical batch.

[0005] In order to determine whether the analytical data obtained should be accepted or not, it is considered to measure quality control standard solutions before and after the measurement of multiple consecutive samples in normal measurements.

[0006] On the other hand, in recent years, various peptides (including proteins) that are useful as biomarkers have been discovered, and accurate quantitative analysis of them is required.

[0007] For example, International Publication WO2015 / 178398 (its family U.S. publication US2017 / 0184573) and International Publication WO2017 / 047529 (its family U.S. publication US2018 / 0238909) disclose surrogate biomarkers for assessing the state of amyloid beta peptide (Aβ) accumulation in the brain and methods for analyzing them. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication WO2015 / 178398 [Patent Document 2] US Publication US2017 / 0184573 [Patent Document 3] International Publication WO2017 / 047529 [Patent Document 4] US Publication US2018 / 0238909 [Non-patent literature]

[0009] [Non-Patent Document 1] NATURE PROTOCOLS, 2011, VOL.6, NO.7, 1060-1083, "Procedures for large-scale metabolic profiling of serum plasma and using gas chromatography and liquid chromatography coupled to mass spectrometry" Summary of the Invention [Problem to be solved by the invention]

[0010] In measuring biomarker peptides in multiple samples, it is necessary to obtain accurate analytical data for each sample based on a certain standard, regardless of the timing of the sample measurement. However, the above patent documents make no mention of quality control of the analytical data obtained when measuring peptides in multiple samples.

[0011] Therefore, an object of the present invention is to provide a quality control standard for peptide measurement (including protein measurement) and quality control for peptide measurement (including protein measurement). [Means for solving the problem]

[0012] In this specification, the quality control standard solution (Quality Control Standard for Peptide Measurement) may be referred to as a "QC standard sample," "QC standard solution," "QC sample," or simply as a "standard sample" or "standard solution."

[0013] In peptide measurements of multiple samples, it is necessary to obtain accurate analytical data based on certain standards for each sample, regardless of the timing of the sample measurements. To achieve this, quality control standards are measured prior to and / or after the measurement of the actual samples (samples) to confirm that there is substantially no or little variation in the measurement results of the quality control standards. If there is substantially no or little variation in the measurement results of the quality control standards, the measurement results of the actual samples (samples) performed between the measurements of the quality control standards are considered to be adequate for quality control.

[0014] It is desirable that quality control standard solutions have the same or similar composition as the actual samples (analytes). From this perspective, in the analysis of multiple samples, it is desirable to pool small portions of each sample to create quality control standard solutions. However, if not all samples have been collected before the start of a multi-sample analysis project, or if the amount of each sample is small, it can be difficult to prepare quality control standard solutions by pooling portions of each sample.

[0015] For these reasons, commercially available (human) blood samples (serum samples, plasma samples) are used as standard solutions for quality control.

[0016] However, when commercially available (human) blood samples (serum samples, plasma samples) are used as quality control standard solutions, the content of the target peptides contained in the commercially available (human) blood samples is unknown and may differ significantly from the content of the target peptides in the actual sample (specimen).

[0017] For example, when the biomarker peptide to be measured is an amyloid beta (Aβ)-related peptide, it is unknown how much amyloid beta (Aβ)-related peptide is contained in a commercially available (human) blood sample (serum sample, plasma sample), and this may be significantly different from the content of amyloid beta (Aβ)-related peptide in the actual sample (specimen). Therefore, even if the biomarker peptide to be measured is spiked (added) to a commercially available (human) blood sample, it is not possible to determine the appropriate spike amount of the biomarker peptide.

[0018] Originally, commercially available (human) blood samples were not intended for the measurement of specific biomarker peptides.

[0019] The present inventors have conducted extensive research and found that by spiking a stable isotope-labeled peptide into a blood sample as a quality control standard solution, the amount of the stable isotope-labeled peptide (or, in the case of multiple stable isotope-labeled peptides, the ratio of their amounts) can be used as a standard to control the quality of analytical data. The stable isotope-labeled peptide is not originally present in either a blood sample derived from a living body or a pseudo-blood sample, but if it has the same or similar basic structure as the peptide to be measured, it can be analyzed with the same physicochemical behavior.

[0020] The present invention includes the following inventions.

[0021] A QC standard sample for measuring a target peptide, A QC standard sample comprising a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample.

[0022] 1. A method for measuring peptides of interest in a multi-analyte blood sample, comprising: an initial QC step of measuring a QC standard sample containing a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample; a sample measurement step of measuring one or more blood samples; an intermediate QC step of measuring the QC standard sample; a step of repeating the sample measurement step and the intermediate QC step a predetermined number of times; a sample measurement step of measuring one or more blood samples; a final QC step in which the QC standard sample is measured; A peptide measurement method comprising:

[0023] The peptide measurement method as described above, wherein in each of the sample measurement steps, one to nine blood samples are continuously measured.

[0024] moreover, a determining step of determining that the measurement of the QC standard sample is acceptable if the level of the stable isotope-labeled peptide in each QC standard sample measured in the initial QC step, the intermediate QC step, and the final QC step is within a normal range of -20% to +20% of a predetermined reference value, and that the measurement of the QC standard sample is unacceptable if the level is outside the normal range; an acceptance / rejection decision step for deciding to adopt the measurement results of the one or more blood samples in a sample measurement step in which both the measurement of the QC standard sample performed in the QC step immediately before the sample measurement step and the measurement of the QC standard sample performed in the QC step immediately after the sample measurement step are passed, and not to adopt the measurement results of the one or more blood samples in a sample measurement step in which both or either the measurement of the QC standard sample performed in the QC step immediately before the sample measurement step and the measurement of the QC standard sample performed in the QC step immediately after the sample measurement step are failed; The above peptide measurement method, comprising:

[0025] A QC standard sample kit for measuring a target peptide, comprising: a blood sample containing the peptide; a stable isotope-labeled peptide to be spiked into the blood sample; QC standard sample kit including:

[0026] In the present invention, the term "peptide" also includes proteins. The level of a peptide basically means its concentration, but may also be expressed in other units that those skilled in the art use to represent concentration, such as the detected ion intensity in mass spectrometry.

[0027] The specimen to be measured may be a biological sample (e.g., body fluids such as blood, cerebrospinal fluid (CSF), urine, bodily secretions, saliva, sputum, and feces) from humans and non-human mammals (e.g., rats, dogs, cats, etc.). The biological sample is discarded without being returned to the subject (e.g., the test subject) from which it was originally obtained. [Effects of the Invention]

[0028] According to the present invention, a QC standard sample for measuring a target peptide comprises a blood sample containing the peptide and a stable isotope-labeled peptide spiked into the blood sample. The stable isotope-labeled peptide is not originally present in either a blood sample derived from a living body or a pseudo-blood sample. Therefore, by spiking the stable isotope-labeled peptide into the blood sample as a QC standard sample, quality control of analytical data can be performed based on the amount of the stable isotope-labeled peptide, regardless of various variables.

[0029] According to the present invention, a QC standard sample for measuring a target peptide comprises a blood sample containing the peptide and a stable isotope-labeled peptide spiked into the blood sample. The stable isotope-labeled peptide is not originally present in either a blood sample derived from a living body or a simulated blood sample, but if it has the same or similar chemical structure as the target peptide, it can be analyzed with the same physicochemical behavior. Therefore, by spiking a blood sample with a stable isotope-labeled peptide that has the same or similar chemical structure as the target peptide as the QC standard sample, quality control of analytical data can be performed based on the amount of the stable isotope-labeled peptide, regardless of various variables.

[0030] When there are multiple types of stable isotope-labeled peptides, more reliable quality control of analytical data can be performed based on the ratio of their amounts.

[0031] According to the present invention, the quality of analytical data can be controlled using a QC standard sample for measuring a target peptide when measuring a target peptide in multiple blood samples.

[0032] Furthermore, the present invention provides a kit for preparing a QC standard sample for measuring the target peptide. [Brief explanation of the drawings]

[0033] [Figure 1]FIG. 1 is a schematic flow diagram for measuring multiple samples. [Figure 2] FIG. 1 is a flow chart for determining the reference value of the SIL-amyloid peptide ratio for QC samples (standard plasma) from N=27 data. [Figure 3] FIG. 1 shows an example of sample arrangement on a 96-well plate when multiple samples (number of samples: 42) are subjected to mass spectrometry. DETAILED DESCRIPTION OF THE INVENTION

[0034] In one embodiment of the present invention, the QC standard sample for measuring the target peptide comprises: The method includes a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample.

[0035] [Substances to be analyzed] First, the substance to be analyzed will be described in detail.

[0036] The analyte is not particularly limited, but may include, for example, peptides, glycopeptides, sugar chains, proteins, lipids, glycolipids, etc. Various peptides, glycopeptides, sugar chains, proteins, lipids, and glycolipids may be included. More specifically, it may be Aβ and Aβ-related peptides. "Aβ and Aβ-related peptides" may also be collectively referred to simply as "Aβ-related peptides." "Aβ and Aβ-related peptides" include peptides containing even a portion of the Aβ sequence that are generated by cleavage of amyloid precursor protein (APP).

[0037] For example, Aβ-related peptides include the following:

[0038] APP677-709 (Aβ6-38) (SEQ ID NO: 1): HDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG APP672-704 (Aβ1-33) (SEQ ID NO: 2): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIG APP677-711 (Aβ6-40) (SEQ ID NO: 3): HDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672-706 (Aβ1-35) (SEQ ID NO: 4): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM APP672-708 (Aβ1-37) (SEQ ID NO: 5): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG APP674-711 (Aβ3-40) (SEQ ID NO: 6): EFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672-711 (Aβ1-40) (SEQ ID NO: 7): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV OxAPP672-711 (OxAβ1-40) (SEQ ID NO: 8): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL M VGGVV (Met 706 is oxidized) APP672-713 (Aβ1-42) (SEQ ID NO: 9): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA APP669-711 (SEQ ID NO: 10): VKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV APP672-709 (Aβ1-38) (SEQ ID NO: 11): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG APP672-710 (Aβ1-39) (SEQ ID NO: 12): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV

[0039] The amyloid precursor protein (APP) is a single-pass transmembrane protein consisting of 770 amino acid residues. APP undergoes proteolysis by β-secretase and γ-secretase, producing amyloid beta peptide (Aβ). APP672-713 and Aβ1-42 represent the same peptide (SEQ ID NO: 9). APP672-711 and Aβ1-40 represent the same peptide (SEQ ID NO: 7).

[0040] Among the Aβ-related peptides described above, Aβ1-42 (SEQ ID NO: 9), Aβ1-40 (SEQ ID NO: 7), APP669-711 (SEQ ID NO: 10), and Aβ1-39 (SEQ ID NO: 12) are effective biomarkers for Alzheimer's disease. Ratio of APP669-711 levels to APP672-713 (Aβ1-42) levels: APP669-711 / APP672-713(Aβ1-42), Ratio of APP672-711 (Aβ1-40) levels to APP672-713 (Aβ1-42) levels: APP672-711(Aβ1-40) / APP672-713(Aβ1-42), Ratio of APP674-711 (Aβ3-40) levels to APP672-713 (Aβ1-42) levels: APP674-711 (Aβ3-40) / APP672-713 (Aβ1-42), and Ratio of APP672-710 (Aβ1-39) levels to APP672-713 (Aβ1-42) levels: APP672-710(Aβ1-39) / APP672-713(Aβ1-42) are also effective biomarkers for Alzheimer's disease.

[0041] The peptide may also be a peptide obtained by immunoprecipitation (IP), a peptide generated by digesting a protein with an enzyme such as a peptidase, or a peptide fractionated by chromatography.

[0042] The analyte may contain an internal standard. The internal standard can be appropriately selected by those skilled in the art. For example, a stable isotope-labeled substance may be used. One of the analyte substances may be labeled with a stable isotope. In the examples, stable isotope-labeled Aβ1-38 (SIL-Aβ1-38) is used as the internal standard. In this specification, "SIL-" means stable isotope labeled.

[0043] A sample containing an analyte is subjected to mass spectrometry. The sample to be subjected to mass spectrometry is not particularly limited and may be, for example, a biological sample. Examples of biological samples include body fluids such as blood, cerebrospinal fluid (CSF), urine, bodily secretions, saliva, and sputum; and feces. Examples of 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 a blood sample from collected whole blood is not particularly limited, and any clinically acceptable processing may be performed. For example, centrifugation may be performed. Furthermore, the blood sample to be subjected to mass spectrometry may be stored at a low temperature, such as by freezing, during or after the preparation process. In the present invention, when a biological sample such as a blood sample is subjected to mass spectrometry, the biological sample is discarded without being returned to the subject from whom it was obtained.

[0044] The sample to be subjected to mass spectrometry may be a sample that has undergone various pretreatments. For example, it may be a sample that has undergone immunoprecipitation (IP). It may be a sample that has undergone protein digestion with an enzyme such as a peptidase. It may be a sample that has undergone chromatography. The sample to be subjected to mass spectrometry may be a sample to which a certain amount of an internal standard substance has been added.

[0045] The sample to be subjected to mass spectrometry may be subjected to immunoprecipitation in advance, and the eluate obtained by immunoprecipitation may then be subjected to mass spectrometry (immunoprecipitation-mass spectrometry; IP-MS). Immunoprecipitation may be performed using an antibody-immobilized support prepared using an immunoglobulin having an antigen-binding site capable of recognizing the analyte, or an immunoglobulin fragment containing an antigen-binding site capable of recognizing the analyte.

[0046] Alternatively, the sample to be subjected to mass spectrometry may be subjected to consecutive immunoprecipitation (cIP) followed by detection of peptides in the sample using a mass spectrometer (cIP-MS). By performing two consecutive affinity purifications, contaminants that were not completely removed by the first affinity purification can be further reduced by the second affinity purification. This prevents the suppression of polypeptide ionization by contaminants, enabling highly sensitive measurement of even trace amounts of polypeptides in biological samples by mass spectrometry.

[0047] [QC standard sample] The QC standard for the target peptide measurement is: The method includes a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample.

[0048] When the blood sample used as the QC standard sample is a biological sample, it usually contains various peptides. The peptides contained in the blood sample may include peptides to be measured, but may also include peptides that are not to be measured. The blood sample may be selected from the group consisting of whole blood, plasma, and serum. Conventional pretreatment may be performed.

[0049] When the blood sample of the QC standard sample is not a biological sample, it may be selected from the group consisting of pseudo-blood, pseudo-plasma, and pseudo-serum. In the case of these pseudo-samples, it is recommended to spike (add) a desired peptide depending on the target to be measured.

[0050] The stable isotope-labeled peptide spiked into the blood sample of the QC standard sample preferably contains a stable isotope-labeled peptide that has the same or similar chemical structure as the peptide to be measured. By containing a stable isotope-labeled peptide that has the same or similar chemical structure as the peptide to be measured, the composition of the QC standard sample becomes closer to the compositional conditions of the actual sample (analyte) to be measured, which is preferable.

[0051] Here, with respect to peptides having a chemically "similar" structure to the target peptide, for example, if the target peptide is a peptide selected from amyloid beta (Aβ)-related peptides, peptides contained in the amyloid beta (Aβ)-related peptides would be peptides having a chemically "similar" structure to the target peptide. In such cases, it would be preferable for the QC standard sample to contain a stable isotope-labeled peptide with a chemically "similar" structure corresponding to the target peptide, so that the composition of the QC standard sample is closer to the compositional conditions of the actual target sample (analyte).

[0052] A stable isotope-labeled peptide that has the same or similar chemical structure as the target peptide is not originally present in a blood sample derived from a living body or in a simulated blood sample, but if it has the same or similar chemical structure as the target peptide, it can be analyzed by mass spectrometry with the same physicochemical behavior. Therefore, by spiking a stable isotope-labeled peptide that has the same or similar chemical structure as the target peptide into a blood sample as the QC standard sample, quality control of analytical data can be performed based on the amount of stable isotope-labeled peptide, regardless of various variables.

[0053] It would be preferable if the QC standard sample contained a stable isotope-labeled peptide of the same chemical structure as the peptide to be measured, as this would result in a composition that is closer to the compositional conditions of the actual sample (analyte) to be measured than if the QC standard sample contained a stable isotope-labeled peptide of a similar structure.

[0054] Alternatively, the peptides to be measured may be a plurality of peptides. In this case, the stable isotope-labeled peptides spiked into the blood sample of the QC standard sample may contain a plurality of stable isotope-labeled peptides that have chemically the same or similar structures as the plurality of peptides to be measured. By including stable isotope-labeled peptides corresponding to the peptides to be measured, the composition of the QC standard sample will be closer to the compositional conditions of the actual sample (analyte) to be measured, which is preferable.

[0055] The stable isotope-labeled peptide spiked into the blood sample of the QC standard sample should be contained in an amount within a predetermined standard range, taking into account values ​​close to the compositional conditions of the actual sample (analyte) to be measured. The actual amount contained can be the "standard value."

[0056] Specifically, when the peptide to be measured is an amyloid beta (Aβ)-related peptide, the stable isotope-labeled peptide spiked into the blood sample is preferably an amyloid beta (Aβ)-related peptide, which is preferable because the composition of the QC standard sample is closer to the compositional conditions of the actual sample (analyte) to be measured.

[0057] When the peptide to be measured includes at least one selected from the group consisting of Aβ1-42 (SEQ ID NO: 9), Aβ1-38 (SEQ ID NO: 11), Aβ1-40 (SEQ ID NO: 7), and APP669-711 (SEQ ID NO: 10), The stable isotope-labeled peptide spiked into the blood sample may include at least one selected from the group consisting of SIL-Aβ1-42, SIL-Aβ1-38, SIL-Aβ1-40, and SIL-APP669-711 (wherein "SIL-" means stable isotope labeled).

[0058] For example, stable isotope-labeled SIL-Aβ1-38 has carbon atoms of Phe and Ile. 13 C, or all nitrogen atoms 14 N 15 It may be substituted with N. Stable isotope-labeled SIL-Aβ1-42 has carbon atoms of Phe and Ile. 13 C, or all nitrogen atoms 14 N 15 It may be substituted with N. Stable isotope-labeled SIL-Aβ1-40 has carbon atoms of Phe and Ile. 13 C, or all nitrogen atoms 14 N 15 It may be substituted with N. Stable isotope-labeled SIL-APP669-711 has carbon atoms of Phe and Ile. 13C, or all nitrogen atoms 14 N 15 It may be substituted with N.

[0059] In this way, the carbon atoms of Phe and Ile in the stable isotope-labeled peptide are 13 C, or all nitrogen atoms 14 N 15 They may be substituted with N. These may be obtained by chemical synthesis or may be commercially available.

[0060] Specifically, the peptide to be measured is Aβ1-42 (SEQ ID NO: 9), At least one selected from the group consisting of Aβ1-40 (SEQ ID NO: 7) and APP669-711 (SEQ ID NO: 10); Including, Ratio of measured peptide levels: Aβ1-40 / Aβ1-42, and / or APP669-711 / Aβ1-42 When used as a biomarker, The stable isotope-labeled peptide spiked into the blood sample comprises: SIL-Aβ1-42 and At least one selected from the group consisting of SIL-Aβ1-40 and SIL-APP669-711; Including, Ratio of abundance of the stable isotope-labeled peptides: SIL-Aβ1-40 / SIL-Aβ1-42, and / or SIL-APP669-711 / SIL-Aβ1-42 It is preferable that the composition of the QC standard sample is close to the composition conditions of the actual sample (specimen) to be measured.

[0061] More specifically, the ratio of mass spectrometry peak intensities as the abundance of the stable isotope-labeled peptide: SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and / or SIL-APP669-711 / SIL-Aβ1-42: 0.7-1.3 It is preferable that the composition of the QC standard sample is close to the composition conditions of the actual sample (specimen) to be measured.

[0062] The amount of the stable isotope-labeled peptide added may be adjusted so that the peak intensity ratio falls within the above range.

[0063] The peak intensity ratio can be calculated by, for example, normalizing each peak intensity with an internal standard peptide (SIL-Aβ1-38) and then calculating the ratio. Furthermore, corrected values ​​can be obtained for each mass spectrometer used and then calculating the ratio.

[0064] In this way, the carbon atoms of Phe and Ile in the stable isotope-labeled peptide are 13 C, or all nitrogen atoms 14 N 15 They may be substituted with N. These may be obtained by chemical synthesis or may be commercially available.

[0065] Regarding the ratio of mass spectrometry peak intensities representing the abundance of the stable isotope-labeled peptide spiked into the blood sample of the QC standard sample, the amount actually contained in the QC standard sample can be the "reference value."

[0066] The amounts of the stable isotope-labeled peptides spiked into the blood sample of the QC standard sample can be expressed as concentrations in the QC standard sample, respectively, in consideration of making the composition conditions closer to those of the actual sample (specimen) to be measured. SIL-Aβ1-40 concentration: 45-121 pM SIL-Aβ1-42 concentration: 4-13 pM SIL-APP669-711 concentration: 4-11 pM However, the range is not limited to this. In addition, since the ratio of actual abundance (pM) and the ratio of mass spectrometry peak intensities do not necessarily coincide, it is useful to use the above-mentioned mass spectrometry peak intensity ratio as a standard. Since immunoprecipitation (IP) yields and MS ionization efficiencies vary depending on the peptide, the actual peptide concentration ratio and the ratio of mass spectrometry peak intensities may differ.

[0067] [Peptide measurement method] In one embodiment of the present invention, referring to FIG. 1, a method for measuring target peptides in a multi-analyte blood sample includes: an initial QC step of measuring a QC standard sample containing a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample; a sample measurement step of measuring one or more blood samples; an intermediate QC step of measuring the QC standard sample; a step of repeating the sample measurement step and the intermediate QC step a predetermined number of times; a sample measurement step of measuring one or more blood samples; a final QC step in which the QC standard sample is measured; Includes.

[0068] In each of the sample measurement steps, consecutive blood samples are measured. For example, consecutive measurements of 1 to 9 blood samples may be performed.

[0069] In one embodiment of the present invention, the method for measuring peptides of interest in a multi-analyte blood sample further comprises: a determining step of determining that the measurement of the QC standard sample is acceptable if the level of the stable isotope-labeled peptide in each QC standard sample measured in the initial QC step, the intermediate QC step, and the final QC step is within a normal range of -20% to +20% of a predetermined reference value, and that the measurement of the QC standard sample is unacceptable if the level is outside the normal range; an acceptance / rejection decision step for deciding to adopt the measurement results of the one or more blood samples in a sample measurement step in which both the measurement of the QC standard sample performed in the QC step immediately before the sample measurement step and the measurement of the QC standard sample performed in the QC step immediately after the sample measurement step are passed, and not to adopt the measurement results of the one or more blood samples in a sample measurement step in which both or either the measurement of the QC standard sample performed in the QC step immediately before the sample measurement step and the measurement of the QC standard sample performed in the QC step immediately after the sample measurement step are failed; may also include:

[0070] The peptide to be measured may be an amyloid beta (Aβ) related peptide.

[0071] The stable isotope-labeled peptide contained in the QC standard sample may be a stable isotope-labeled amyloid beta (Aβ)-related peptide.

[0072] When the peptide to be measured includes at least one selected from the group consisting of Aβ1-42 (SEQ ID NO: 9), Aβ1-38 (SEQ ID NO: 11), Aβ1-40 (SEQ ID NO: 7), and APP669-711 (SEQ ID NO: 10), The stable isotope-labeled peptide contained in the QC standard sample may include at least one selected from the group consisting of SIL-Aβ1-42, SIL-Aβ1-38, SIL-Aβ1-40, and SIL-APP669-711 (here, "SIL-" means stable isotope labeled).

[0073] Specifically, the peptide to be measured is Aβ1-42 (SEQ ID NO: 9), At least one selected from the group consisting of Aβ1-40 (SEQ ID NO: 7) and APP669-711 (SEQ ID NO: 10); Including, Ratio of measured peptide levels: Aβ1-40 / Aβ1-42, and / or APP669-711 / Aβ1-42 is used as a biomarker, The stable isotope-labeled peptide contained in the QC standard sample is SIL-Aβ1-42 and At least one selected from the group consisting of SIL-Aβ1-40 and SIL-APP669-711; Including, The ratio of abundance of the stable isotope-labeled peptides in the QC standard sample: SIL-Aβ1-40 / SIL-Aβ1-42, and / or SIL-APP669-711 / SIL-Aβ1-42 It is preferable that the composition of the QC standard sample is close to the composition conditions of the actual sample (specimen) to be measured.

[0074] More specifically, in the QC standard sample, the ratio of mass spectrometry peak intensities representing the abundance of the stable isotope-labeled peptide: SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and / or SIL-APP669-711 / SIL-Aβ1-42: 0.7-1.3 It is preferable that the composition of the QC standard sample is close to the composition conditions of the actual sample (specimen) to be measured.

[0075] The ratios of the mass spectrometry peak intensities of the stable isotope-labeled SIL-peptides are not limited to the above ranges. The values ​​of these ratios may differ from the actual ratios of peptide concentration ratios and mass spectrometry peak intensities because the immunoprecipitation (IP) yield and MS ionization efficiency vary depending on the peptide. Therefore, the optimal range values ​​may vary depending on the mass spectrometer technique (MALDI, ESI, etc.) and the type of mass spectrometer (manufacturer, model, laser type, etc.). The above ranges of the ratios are determined using an AXIMA-Performance mass spectrometer manufactured by Shimadzu. TM This is an example of measurement.

[0076] Regarding the ratio of mass spectrometry peak intensities representing the abundance of the stable isotope-labeled peptides spiked into the blood sample of the QC standard sample, the ratio of peptide peak intensities obtained by actually measuring the QC standard plasma by mass spectrometry can be the "reference value."

[0077] Peptides can be measured by immunoprecipitation (IP)-mass spectrometry (IP-MS), liquid chromatography-mass spectrometry (LC-MS), or gas chromatography-mass spectrometry (GC-MS).

[0078] [Mass spectrometry] Mass spectrometry is not particularly limited, but includes mass spectrometry by matrix-assisted laser desorption / ionization (MALDI) mass spectrometry, electrospray ionization (ESI) mass spectrometry, etc. 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, an ESI-FTICR (electrospray ionization Fourier transform ion cyclotron resonance) mass spectrometer, etc. can be used.

[0079] The matrix and matrix solvent can be appropriately determined by those skilled in the art depending on the substance to be analyzed.

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

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

[0082] 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, and more preferably 0.5 to 10 mg / mL.

[0083] When using a detection system based on MALDI mass spectrometry, it is preferable to use a matrix additive (comatrix). The matrix additive can be appropriately selected by those skilled in the art depending on the analyte (polypeptide) and / or matrix. For example, a phosphonic acid group-containing compound can be used as the matrix additive. Specific examples of compounds containing one phosphonic acid group include phosphonic acid, methylphosphonic acid, phenylphosphonic acid, and 1-naphthylmethylphosphonic acid. 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 phosphonic acid group-containing compounds, compounds having two or more, preferably 2 to 4, phosphonic acid groups in one molecule are preferred.

[0084] The use of a phosphonic acid group-containing compound is useful, for example, when metal ions remaining on the surface of the antibody-immobilized carrier in the washing solution are contaminated in the eluate after the dissociation step. These metal ions adversely affect the background in mass spectrometry. The use of a phosphonic acid group-containing compound has the effect of suppressing such adverse effects.

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

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

[0087] [QC Standard Sample Kit] In one embodiment of the present invention, a QC standard sample kit for measuring a target peptide comprises: a blood sample containing the peptide; a stable isotope-labeled peptide to be spiked into the blood sample; Includes.

[0088] In the QC standard sample kit, the stable isotope-labeled peptide is SIL-Aβ1-42 and At least one selected from the group consisting of SIL-Aβ1-40 and SIL-APP669-711 (wherein "SIL-" means stable isotope labeled), The QC standard sample kit may also contain other necessary components, such as SIL-Aβ1-38 used as an internal standard, various buffer solutions, and the like. [Example]

[0089] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following, the amount of a substance shown in % is shown on a volume basis if it is a liquid, unless otherwise specified.

[0090] [Experimental Example 1: Preparation of standard plasma] An example protocol for preparing a QC sample (standard plasma) is shown below.

[0091] SIL-Aβ1-38 was prepared using AnaSpec (San Jose, CA, USA). SIL-Aβ1-38 was prepared by cleaving the carbon atoms of Phe and Ile in Aβ1-38. 13 It is substituted with C. SIL-Aβ1-40 was prepared using rPeptide (Watkinsville, GA, USA). SIL-Aβ1-40 was prepared by converting all nitrogen atoms of Aβ1-40 to rPeptide. 15 It is substituted with N. SIL-Aβ1-42 was prepared using rPeptide (Watkinsville, GA, USA). SIL-Aβ1-42 was prepared by cleaving all nitrogen atoms of Aβ1-42. 15 It is substituted with N. SIL-APP669-711 was prepared using rPeptide (Watkinsville, GA, USA). SIL-APP669-711 was prepared by converting all nitrogen atoms of APP669-711 to rPeptide. 15 It is substituted with N.

[0092] [Experimental Example 1-1: Spiking of SIL-peptide into plasma] [1] 100 μL of each of 50 nM SIL-Aβ1-40, 5 nM SIL-Aβ1-42, and 5 nM SIL-APP669-711 diluted in 1 mg / mL BSA, 0.1% (w / v) DDM, and 50 mM Tris-HCl buffer (pH 9.0) was added to 100 mL of human plasma. This mixture is designated "SIL-spiked plasma-1." [2] SIL spiked plasma-1 was mixed thoroughly by inversion.

[0093] [3] 900 μL of SIL-spiked plasma-1 was dispensed into two 1.5 mL microtubes, and the two aliquots and the remaining SIL-spiked plasma-1 were frozen at -60°C or below. [4] One of the 900 μL aliquots was thawed and subjected to sequential immunoprecipitation (cIP)-mass spectrometry (cIP-MS) (IP was repeated three times). The details of the IP-MS procedure were as follows:

[0094] [Continuous Immunoprecipitation (cIP)]

[0095] (Preparation of antibody-immobilized beads) An anti-Aβ antibody (IgG) clone 6E10 (Covance) was prepared, which has an epitope consisting of residues 3-8 of amyloid β protein (Aβ).

[0096] Approximately 3.3 × 10 magnetic beads (Dynabeads (registered trademark) M-270 Epoxy) were used for 100 μg of anti-Aβ antibody (IgG). 8 The beads were reacted in an immobilization buffer (0.1 M phosphate buffer (pH 7.4) containing 1.3 M ammonium sulfate) at 37° C. for 16 to 24 hours to prepare anti-Aβ IgG-immobilized beads.

[0097] (First reaction step) 250 μL of the SIL-spiked plasma-1 was mixed with 250 μL of the first IP reaction buffer (0.2% (w / v) DDM, 0.2% (w / v) NTM, 800 mM GlcNAc, 100 mM Tris-HCl (pH 7.4), 300 mM NaCl) containing 11 pM stable isotope-labeled Aβ1-38 (SIL-Aβ1-38), and then incubated on ice for 5 to 60 minutes. SIL-Aβ1-38 has carbon atoms of Phe and Ile. 13 This was substituted with C and used as an internal standard to normalize the signal intensity of the mass spectrum. The plasma was mixed with anti-Aβ IgG-immobilized beads and shaken and mixed by pipetting in a Peltier at 4°C for 1 hour.

[0098] (First washing step, first elution step) The antibody beads were then washed once with 100 μL of first IP wash buffer (0.1% DDM, 0.1% NTM, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl) and once with 50 μL of 50 mM ammonium acetate buffer, and then Aβ and Aβ-like peptides (i.e., APP-derived peptides) bound to the antibody beads were eluted with a first IP elution solution (50 mM glycine buffer (pH 2.8) containing 0.1% DDM).

[0099] (Neutralization process) The resulting eluate was mixed with a second IP reaction buffer (0.2% (w / v) DDM, 800 mM GlcNAc, 300 mM Tris-HCl (pH 7.4), 300 mM NaCl) to obtain a first purified solution.

[0100] (Second reaction step) The obtained first purified solution was mixed with separate anti-Aβ antibody-immobilized beads, and the mixture was shaken and mixed by pipetting in a Peltier at 4°C for 1 hour.

[0101] (Second washing step, second elution step) The anti-Aβ antibody-immobilized beads were then washed twice with 50 μL of second wash buffer (0.1% DDM, 150 mM Tris-HCl (pH 7.4), 150 mM NaCl), once with 50 μL of 50 mM ammonium acetate buffer, and once with 30 μL of HO. The Aβ and Aβ-like peptides (APP-derived peptides) bound to the antibody beads were then eluted with 8 μL (or 9 μL, depending on the humidity in the laboratory) of second IP elution solution (70% (v / v) acetonitrile containing 5 mM hydrochloric acid). This yielded a second purified solution, which was then subjected to mass spectrometry.

[0102] n-Dodecyl-β-D-maltoside (DDM) [Critical micelle concentration cmc:0.009%] n-Nonyl-β-D-thiomaltoside (NTM) [cmc:0.116%]

[0103] [Detection by MALDI-TOF MS] α-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix for Linear TOF. The matrix solution was prepared by dissolving 1 mg of CHCA in 1 mL of 70% (v / v) acetonitrile. 0.4% (w / v) methanediphosphonic acid (MDPNA) was used as the matrix additive. Equal volumes of 1 mg / mL CHCA solution and 0.4% (w / v) MDPNA were mixed, and 0.5 μL of the mixture was applied to a μFocus MALDI plate. TM The solution was dropped onto a 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ) and allowed to dry.

[0104] 1 μL of the second purified solution obtained by the above immunoprecipitation was taken and placed on a μFocus MALDI plate. TM The drop was dropped onto the matrix 900 μm above.

[0105] Mass spectral data were acquired using an AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) in positive ion mode with Linear TOF. 400 spots per well were accumulated for 16,000 shots. The peak detection limit was a signal-to-noise ratio of 3 or higher. Linear TOF m / z values ​​were expressed as the average mass of the peak. The m / z values ​​were calibrated using external standards: human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, and bovine insulin.

[0106] [5] The SIL amyloid peptide ratios (SIL-Aβ1-40 / SIL-Aβ1-42 and SIL-APP669-711 / SIL-Aβ1-42, mean values ​​of N=3) were calculated. If data cannot be obtained due to poor instrument performance or unexpected trouble, cIP-MS can be performed again using another 900 μL aliquot frozen.

[0107] [6] If the calculated value is within the reference range: the ratio of SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and The ratio of SIL-APP669-711 / SIL-Aβ1-42 was 0.7 to 1.3 If the result was outside the reference range, the procedure went to the next section [Experimental Example 1-2: Adjustment of the SIL amyloid peptide ratio].

[0108] [Experimental Example 1-2: Adjusting the SIL-Amyloid Peptide Ratio] [1] The remaining SIL-spiked plasma-1 that had been frozen and stored in [3] of Experimental Example 1-1 above was left to stand at room temperature and thawed. [2] If another tube of the 900 μL aliquots taken and frozen in [3] of Experimental Example 1-1 remained, thaw it and mix it with the thawing solution in [1] above.

[0109] [3] The target value for the SIL amyloid peptide ratio is: The ratio of SIL-Aβ1-40 / SIL-Aβ1-42 was 28.0, and The ratio of SIL-APP669-711 / SIL-Aβ1-42 was 1.0 Aiming for the midpoint (intermediate target value) between the measured value in [5] of Experimental Example 1-1 above and the target value, the required amounts of 50 nM SIL-Aβ1-40, 5 nM SIL-Aβ1-42, and 5 nM SIL-APP669-711 were added to the thawing solution in [1] above. (Adding them aiming for the final target value often results in adding too much, so we deliberately added them aiming for the intermediate target value.) [4] The liquid [3] above was thoroughly mixed by inversion. This is called "SIL spiked plasma-2."

[0110] [5] 900 μL of SIL-spiked plasma-2 was dispensed into two 1.5 mL microtubes, and the two aliquots and the remaining SIL-spiked plasma-2 were frozen at -60°C or below. [6] One of the 900 μL aliquots was thawed and subjected to sequential immunoprecipitation (cIP)-mass spectrometry (cIP-MS) (IP was repeated three times). The details of the IP-MS procedure were as described in the previous section, Example 1-1.

[0111] [7] The SIL-amyloid peptide ratios (SIL-Aβ1-40 / SIL-Aβ1-42 and SIL-APP669-711 / SIL-Aβ1-42, mean values ​​of N=3) were calculated. If data cannot be obtained due to poor instrument performance or unexpected trouble, cIP-MS can be performed again using another 900 μL aliquot frozen.

[0112] [8] If the calculated value is within the reference range: the ratio of SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and The ratio of SIL-APP669-711 / SIL-Aβ1-42 was 0.7 to 1.3 If the SIL amyloid peptide ratio was outside the reference range, proceed to the next section [9] and perform a third spike.

[0113] [9] The remaining SIL-spiked plasma-2 that had been frozen and stored in [5] above was left to thaw at room temperature.

[10] If another tube of the 900 μL aliquot frozen in [5] above remained, thaw it and mix it with the thawing solution in [9] above.

[0114]

[11] For the third spike, the target value for the SIL-amyloid peptide ratio was: The ratio of SIL-Aβ1-40 / SIL-Aβ1-42 was 28.0, and The ratio of SIL-APP669-711 / SIL-Aβ1-42 was 1.0 To achieve this, the required amounts of 50 nM SIL-Aβ1-40, 5 nM SIL-Aβ1-42, and 5 nM SIL-APP669-711 were added to the thawing solution described above [9].

[12] The liquid

[11] above was thoroughly mixed by inversion. This is called "SIL spiked plasma-3."

[0115]

[13] 900 μL of SIL-spiked plasma-3 was dispensed into two 1.5 mL microtubes, and the two aliquots and the remaining SIL-spiked plasma-3 were frozen at -60°C or below.

[14] One of the 900 μL aliquots was thawed and subjected to sequential immunoprecipitation (cIP)-mass spectrometry (cIP-MS) (IP was repeated three times). The details of the IP-MS procedure were as described in the previous section, Example 1-1.

[0116]

[15] The SIL-amyloid peptide ratios (SIL-Aβ1-40 / SIL-Aβ1-42 and SIL-APP669-711 / SIL-Aβ1-42, mean values ​​of N=3) were calculated. If data cannot be obtained due to poor instrument performance or unexpected trouble, cIP-MS can be performed again using another 900 μL aliquot frozen.

[0117]

[16] If the calculated value is within the reference range: the ratio of SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and The ratio of SIL-APP669-711 / SIL-Aβ1-42 was 0.7 to 1.3 If the value was outside the reference range, we returned to [9] above, and repeated the steps [9] to

[16] above, repeating the spike and IP-MS procedure until the calculated value was within the reference range.

[0118] [Experimental Example 2: Determination of the reference value of the SIL-amyloid peptide ratio in standard plasma] [1] The SIL-amyloid peptide ratio is within the reference range: the ratio of SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and The ratio of SIL-APP669-711 / SIL-Aβ1-42 was 0.7 to 1.3 The SIL-spiked plasma in the 100 ml sample was used as the standard plasma. [2] The frozen SIL-spiked plasma was left to thaw at room temperature. [3] If another 900 μL aliquot was left, thaw it and mix it with the thawing solution from [2] above.

[0119] [4] The total volume of the thawing solution was dispensed into 1.5 mL microtubes in 900 μL portions. [5] All aliquots were frozen at -60°C or below.

[0120] [6] Three of the frozen samples from [5] above were thawed, and IP-MS was performed using three lots of antibody beads, each with N = 3 (total N = 9). The details of the IP-MS procedure were as described in the previous section, Example 1-1.

[0121] [7] The IP-MS procedure was repeated three batches (total N = 27).

[0122] [8] Amyloid peptide ratios were calculated from IP-MS results of N=27. Both calculated amyloid peptide ratios were within the reference range: the ratio of SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and The ratio of SIL-APP669-711 / SIL-Aβ1-42 was 0.7 to 1.3 If either or both of the calculated amyloid peptide ratios were outside the above-mentioned reference ranges, the procedure was repeated starting from the preparation of standard plasma.

[0123] A QC sample (standard plasma) was prepared according to the above example protocol.

[0124] The flow of determining the reference value of the SIL-amyloid peptide ratio for QC samples (standard plasma) from N=27 data is shown in FIG.

[0125] In Figure 2, <1> Regarding the S / N threshold, S / N threshold for SIL-Aβ1-38: 15 S / N threshold for SIL-Aβ1-40: 3 S / N threshold for SIL-Aβ1-42: 3 SIL-APP669-711 S / N threshold: 3

[0126] <2> Regarding the standard values, Reference value 1: the ratio of SIL-Aβ1-40 / SIL-Aβ1-42, and Reference value 2: SIL-APP669-711 / SIL-Aβ1-42 ratio

[0127] <3> How to remove it, - Data with a relative error of more than 20% from the reference value will be removed, starting with the largest data, and recalculated. Each standard value is independent. In other words, even if standard value 1 exceeds 20% and is removed, standard value 2 will be adopted if it is 20% or less.

[0128] More specifically, it is as follows. When determining the reference value, outliers are removed, taking into account measurement error. Finally, if 22 or more data points (80% or more) remain out of the 27 data points (N = 27), the average value of the remaining data is calculated and set as the reference value. For example, if a contaminant peak overlaps the SIL-Aβ1-40 peak position in a certain spectrum, causing the SIL-Aβ1-40 peak intensity to be abnormal, the data for reference value 1 (SIL-Aβ1-40 / SIL-Aβ1-42 ratio) is excluded, but the peaks of SIL-APP669-711 and SIL-Aβ1-42 are correctly detected, so reference value 2 (SIL-APP669-711 / SIL-Aβ1-42 ratio) is adopted.

[0129] The reference values ​​1 and 2 can be determined from N=27 measurement data by automatic calculation using software.

[0130] <4> Regarding the acceptable standard value range, Reference value 1: 25.0~32.5 Reference value 2: 0.7 to 1.3

[0131] [Experimental Example 3: Measurement of amyloid peptide samples using QC samples (standard plasma)] Here, the blood amyloid peptide samples (number of samples: 42) to be measured were subjected to IP-MS measurement using QC samples (standard plasma) prepared according to the above protocol example.

[0132] (Basic operation of IP-MS measurement) Each sample to be measured was added with SIL-Aβ1-38 (AnaSpec, San Jose, CA, USA) as an internal standard, and subjected to IP operation according to the sequential immunoprecipitation method described in the previous Experimental Example 1-1.

[0133] MS measurements were performed by MALDI-TOF MS analysis.

[0134] More specifically, mass spectral data were acquired using an AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) with Linear TOF in positive ion mode. α-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix for Linear TOF. The matrix solution was prepared by dissolving 1 mg of CHCA in 1 mL of 70% (v / v) acetonitrile. 0.4% (w / v) methanediphosphonic acid (MDPNA) was used as the matrix additive. Equal volumes of 1 mg / mL CHCA solution and 0.4% (w / v) MDPNA were mixed, and 0.5 μL of the mixture was applied to a μFocus MALDI plate. TM The solution was dropped onto a 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ) and allowed to dry.

[0135] 1 μL of the purified solution from the IP sample was taken and placed on a μFocus MALDI plate. TM The drop was dropped onto the matrix 900 μm above.

[0136] Each well contained 400 spots, with 16,000 shots accumulated. The peak detection limit was a signal-to-noise ratio of 3 or higher. The m / z values ​​of the linear TOF were expressed as the average mass of the peaks. The m / z values ​​were calibrated using external standards: human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, and bovine insulin.

[0137] Referring to Figure 1, the following is an example of processing the maximum number of samples (42 samples): First, the QC standard sample was subjected to MS measurement (initial QC step). Next, MS measurement was carried out on each of the samples (number of samples: 9) to be measured after IP (sample measurement step). Subsequently, the QC standard sample was subjected to MS measurement (intermediate QC step). Next, the sample measurement step and the intermediate QC step were repeated a predetermined number of times (three times) (repeated step). Next, MS measurement was carried out on each of the samples (number of samples: 6) to be measured after IP (sample measurement step). Subsequently, the QC standard sample was subjected to MS measurement (final QC step).

[0138] Figure 3 shows an example of sample arrangement on a 96-well plate when performing mass spectrometry on multiple samples (number of samples: 42). The measurement order is indicated by arrows (→).

[0139] According to FIG. 3, first, QC standard 1 is placed on plate well 1A (initial QC step), and then: Samples 1 to 9 are placed on wells 1B to 2G (sample measurement process), and QC standard sample 2 is placed on well 2F (intermediate QC process). specimen 10~18 are placed on wells 2E to 3D (sample measurement process), and QC standard sample 3 is placed on well 3E (intermediate QC process), specimen 19 ~27 is well 3F ~ is placed on well 4C (sample measurement process), and QC standard sample 4 is placed on well 4B (intermediate QC process), Sample 28~ 36 Gawell 4A~ 5H (sample measurement process), and QC standard sample 5 is placed on well 6H (intermediate QC process). Samples 37 to 42 are placed on wells 6G to 6B (sample measurement step), and finally, QC standard 6 is placed on plate well 6A (final QC step).

[0140] (Quality control of amyloid peptide sample measurements using QC samples) The ratio of the levels of the stable isotope-labeled peptide in each QC standard sample used in the initial QC step, the intermediate QC step, and the final QC step is Reference value 1: the ratio of SIL-Aβ1-40 / SIL-Aβ1-42 is within the range of 25.0 to 32.5, and Reference value 2: The ratio of SIL-APP669-711 / SIL-Aβ1-42 is within the range of 0.7 to 1.3 It meets the following criteria.

[0141] the ratio of the levels of the stable isotope-labeled peptide in each QC standard sample actually measured in the initial QC step, the intermediate QC step, and the final QC step is If the actual standard value 1 and standard value 2 were within the normal range of -20% or more and +20% or less, the QC standard sample was judged to have passed, whereas if the actual standard value 1 and standard value 2 were outside the normal range, the QC standard sample was judged to have failed (judgment process).

[0142] The measurement results obtained in the sample measurement process in which both the QC standard sample used in the QC process immediately before the sample measurement process and the QC standard sample used in the QC process immediately after the sample measurement process were determined to be acceptable were adopted.On the other hand, the measurement results obtained in the sample measurement process in which either or both of the QC standard sample used in the QC process immediately before the sample measurement process and the QC standard sample used in the QC process immediately after the sample measurement process were determined to be unacceptable were not adopted (acceptance / rejection decision process).

[0143] Quality control was automatically assessed by software, demonstrating that all samples were measured properly.

[0144] In this example, the maximum number of samples to be measured in one batch is 48, and an example of measurement and quality control for 42 specimens is shown. Although the present invention is typically applied to cases with multiple specimens, it can also be implemented when there is only one specimen by performing the following sequence: initial QC step → specimen measurement step → final QC step. The number of specimens measured in each specimen measurement step can be adjusted depending on the total number of specimens, and measurement and quality control can be performed using the QC standard of the present invention.

[0145] As described above, in the present invention, a QC sample is prepared by spiking commercially available human plasma or pseudo-plasma with stable isotope-labeled amyloid β peptide (SIL-Aβ peptide) that is not present in the plasma. The spiked amount of SIL-Aβ peptide is adjusted to approximate the amount of the corresponding amyloid β peptide contained in the actual sample (analyte), and the ratio of SIL-Aβ peptide is adjusted so that the above ratio falls within a certain range.

[0146] The prepared QC sample is measured in multiple batches using multiple lots of antibody beads, and the average is calculated to set a "reference value" for the SIL-amyloid β peptide ratio in the QC sample of that lot.

[0147] For each QC sample measured within the same batch as the actual sample, a determination is made as to whether the ratio of the two SIL-amyloid β peptides is within ±20% of the "reference value." Only the measured values ​​of the actual samples sandwiched between QC samples that meet the acceptance criteria are used.

[0148] By controlling the amount of SIL-Aβ peptide in the QC sample, it is possible to prepare a QC sample that is close to the actual sample. By presetting a reference value for the SIL-Aβ peptide ratio of the QC sample, the risk of overlooking gradual problems in the analytical system (instrumental problems) can be reduced. Furthermore, for QC samples measured at regular intervals, only the measurements of actual samples sandwiched between QC samples that meet the reference can be used to determine whether or not to use the data without overlooking localized sensitivity declines within a batch.

[0149] The present invention includes, for example, the following embodiments.

[0150] (1) A QC standard sample for measuring a target peptide, A QC standard sample comprising a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample.

[0151] (2) The QC standard sample according to (1) above, wherein the blood sample is selected from the group consisting of whole blood, plasma, and serum.

[0152] (3) The QC standard sample according to (1) above, wherein the blood sample is selected from the group consisting of peptide-spiked pseudo-blood, pseudo-plasma, and pseudo-serum.

[0153] (4) The QC standard sample according to any one of (1) to (3) above, wherein the stable isotope-labeled peptide spiked into the blood sample has a chemically identical or similar structure to the peptide to be measured and contains a stable isotope-labeled peptide.

[0154] (5) The peptides to be measured include a plurality of types of peptides, The QC standard sample according to any one of (1) to (4) above, wherein the stable isotope-labeled peptides spiked into the blood sample contain multiple peptides that are chemically identical or similar in structure to the multiple peptides to be measured and are stable isotope-labeled.

[0155] (6) The QC standard sample according to any one of (1) to (5) above, wherein the stable isotope-labeled peptide spiked into the blood sample is contained in an amount within a predetermined standard range.

[0156] (7) The QC standard sample according to any one of (1) to (6) above, wherein the peptide to be measured is an amyloid beta (Aβ)-related peptide.

[0157] (8) A QC standard sample according to any one of (1) to (7) above, wherein the stable isotope-labeled peptide spiked into the blood sample is a stable isotope-labeled amyloid beta (Aβ)-related peptide.

[0158] (9) The QC standard sample according to any one of (1) to (8) above, wherein the peptide to be measured comprises at least one selected from the group consisting of Aβ1-42 (SEQ ID NO: 9), Aβ1-38 (SEQ ID NO: 11), Aβ1-40 (SEQ ID NO: 7), and APP669-711 (SEQ ID NO: 10).

[0159] (10) The QC standard sample according to any one of (1) to (9) above, wherein the stable isotope-labeled peptide spiked into the blood sample comprises at least one selected from the group consisting of SIL-Aβ1-42, SIL-Aβ1-38, SIL-Aβ1-40, and SIL-APP669-711 (wherein "SIL-" means stable isotope labeled).

[0160] (11) The peptide to be measured is Aβ1-42 (SEQ ID NO: 9), At least one selected from the group consisting of Aβ1-40 (SEQ ID NO: 7) and APP669-711 (SEQ ID NO: 10); Including, Ratio of measured peptide levels: Aβ1-40 / Aβ1-42, and / or APP669-711 / Aβ1-42 is used as a biomarker, The stable isotope-labeled peptide spiked into the blood sample comprises: SIL-Aβ1-42 and At least one selected from the group consisting of SIL-Aβ1-40 and SIL-APP669-711; Including, Ratio of abundance of the stable isotope-labeled peptides: SIL-Aβ1-40 / SIL-Aβ1-42, and / or SIL-APP669-711 / SIL-Aβ1-42 The QC standard sample according to any one of (1) to (10) above, which is within a predetermined standard range.

[0161] (12) Ratio of abundance of the stable isotope-labeled peptides: SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and / or SIL-APP669-711 / SIL-Aβ1-42: 0.7-1.3 The QC standard sample described in (11) above is within the reference range.

[0162] (13) 1. A method for measuring peptides of interest in a multi-analyte blood sample, comprising: an initial QC step of measuring a QC standard sample containing a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample; a sample measurement step of measuring one or more blood samples; an intermediate QC step of measuring the QC standard sample; a step of repeating the sample measurement step and the intermediate QC step a predetermined number of times; a sample measurement step of measuring one or more blood samples; a final QC step in which the QC standard sample is measured; A peptide measurement method comprising:

[0163] (14) The peptide measurement method according to (13) above, wherein one to nine blood samples are continuously measured in each of the sample measurement steps.

[0164] (15) moreover, a determining step of determining that the measurement of the QC standard sample is acceptable if the level of the stable isotope-labeled peptide in each QC standard sample measured in the initial QC step, the intermediate QC step, and the final QC step is within a normal range of -20% to +20% of a predetermined reference value, and that the measurement of the QC standard sample is unacceptable if the level is outside the normal range; an acceptance / rejection decision step for deciding to adopt the measurement results of the one or more blood samples in a sample measurement step in which both the measurement of the QC standard sample performed in the QC step immediately before the sample measurement step and the measurement of the QC standard sample performed in the QC step immediately after the sample measurement step are passed, and not to adopt the measurement results of the one or more blood samples in a sample measurement step in which both or either the measurement of the QC standard sample performed in the QC step immediately before the sample measurement step and the measurement of the QC standard sample performed in the QC step immediately after the sample measurement step are failed; The peptide measurement method according to (13) or (14) above, comprising:

[0165] (16) The peptide measurement method according to any one of (13) to (15) above, wherein the peptide to be measured is an amyloid beta (Aβ) related peptide.

[0166] (17) The peptide measurement method according to any one of (13) to (16) above, wherein the stable isotope-labeled peptide contained in the QC standard sample is a stable isotope-labeled amyloid beta (Aβ)-related peptide.

[0167] (18) The peptide measurement method according to any one of (13) to (17) above, wherein the peptide to be measured includes at least one selected from the group consisting of Aβ1-42 (SEQ ID NO: 9), Aβ1-38 (SEQ ID NO: 11), Aβ1-40 (SEQ ID NO: 7), and APP669-711 (SEQ ID NO: 10).

[0168] (19) The peptide measurement method according to any one of (13) to (18) above, wherein the stable isotope-labeled peptide contained in the QC standard sample includes at least one selected from the group consisting of SIL-Aβ1-42, SIL-Aβ1-38, SIL-Aβ1-40, and SIL-APP669-711 (here, "SIL-" means stable isotope labeled).

[0169] (20) The peptide to be measured is Aβ1-42 (SEQ ID NO: 9), At least one selected from the group consisting of Aβ1-40 (SEQ ID NO: 7) and APP669-711 (SEQ ID NO: 10); Including, Ratio of measured peptide levels: Aβ1-40 / Aβ1-42, and / or APP669-711 / Aβ1-42 is used as a biomarker, The stable isotope-labeled peptide contained in the QC standard sample is SIL-Aβ1-42 and At least one selected from the group consisting of SIL-Aβ1-40 and SIL-APP669-711; Including, The ratio of abundance of the stable isotope-labeled peptides in the QC standard sample: SIL-Aβ1-40 / SIL-Aβ1-42, and / or SIL-APP669-711 / SIL-Aβ1-42 The peptide measurement method according to any one of (13) to (19) above, wherein the value is within a predetermined reference range.

[0170] (twenty one) The ratio of abundance of the stable isotope-labeled peptides in the QC standard sample: SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and / or SIL-APP669-711 / SIL-Aβ1-42: 0.7-1.3 The peptide measurement method according to (20) above, wherein the peptide concentration is within the reference range.

[0171] (twenty two) The peptide measurement method according to any one of (13) to (21) above, wherein the measurement is performed by immunoprecipitation (IP)-mass spectrometry (IP-MS), liquid chromatography-mass spectrometry (LC-MS), or gas chromatography-mass spectrometry (GC-MS).

[0172] (twenty three) A QC standard sample kit for measuring a target peptide, comprising: a blood sample containing the peptide; a stable isotope-labeled peptide to be spiked into the blood sample; QC standard sample kit including:

[0173] (twenty four) The stable isotope-labeled peptide is SIL-Aβ1-42 and At least one selected from the group consisting of SIL-Aβ1-40 and SIL-APP669-711 (wherein "SIL-" means stable isotope labeled), The QC standard sample kit according to (23) above, comprising:

Claims

1. 1. A method for measuring peptides of interest in a multi-analyte blood sample, comprising: an initial QC step of measuring a QC standard sample containing a blood sample containing a peptide and a stable isotope-labeled peptide spiked into the blood sample; a sample measurement step of measuring one or more blood samples; an intermediate QC step of measuring the QC standard sample; a step of repeating the sample measurement step and the intermediate QC step a predetermined number of times; a sample measurement step of measuring one or more blood samples; a final QC step in which the QC standard sample is measured; Including, moreover, The level of the stable isotope-labeled peptide in each QC standard sample measured in the initial QC step, the intermediate QC step, and the final QC step is compared with a predetermined reference value. a determining step of determining that the measurement of the QC standard sample is acceptable if the result is within a predetermined normal range, and failing the measurement of the QC standard sample if the result is outside the normal range; an acceptance / rejection decision step for deciding to adopt the measurement results of the one or more blood samples in a specimen measurement step for which both the measurement of the QC standard sample performed in the QC step immediately before the specimen measurement step and the measurement of the QC standard sample performed in the QC step immediately after the specimen measurement step are passed, and not to adopt the measurement results of the one or more blood samples in a specimen measurement step for which both or either the measurement of the QC standard sample performed in the QC step immediately before the specimen measurement step and the measurement of the QC standard sample performed in the QC step immediately after the specimen measurement step are failed; A peptide measurement method comprising:

2. The peptide measurement method according to claim 1 , wherein one to nine blood samples are continuously measured in each of the sample measurement steps.

3. 2. The peptide measurement method according to claim 1, wherein the predetermined normal range in the determination step is between −20% and +20% of the predetermined reference value.

4. The peptide measurement method according to claim 1 , wherein the peptide to be measured is an amyloid beta (Aβ)-related peptide.

5. The peptide measurement method according to claim 1 , wherein the stable isotope-labeled peptide contained in the QC standard sample is a stable isotope-labeled amyloid beta (Aβ)-related peptide.

6. The peptide measurement method according to claim 1, wherein the peptide to be measured includes at least one selected from the group consisting of Aβ1-42 (SEQ ID NO: 9), Aβ1-38 (SEQ ID NO: 11), Aβ1-40 (SEQ ID NO: 7), and APP669-711 (SEQ ID NO: 10).

7. The peptide measurement method according to claim 1, wherein the stable isotope-labeled peptide contained in the QC standard sample includes at least one peptide selected from the group consisting of SIL-Aβ1-42, SIL-Aβ1-38, SIL-Aβ1-40, and SIL-APP669-711 (here, "SIL-" means stable isotope labeled).

8. The peptide to be measured is Aβ1-42 (SEQ ID NO: 9), and At least one selected from the group consisting of Aβ1-40 (SEQ ID NO: 7) and APP669-711 (SEQ ID NO: 10); Including, Ratio of measured peptide levels: Aβ1-40 / Aβ1-42, and / or APP669-711 / Aβ1-42 is used as a biomarker, The stable isotope-labeled peptide contained in the QC standard sample is SIL-Aβ1-42, At least one selected from the group consisting of SIL-Aβ1-40 and SIL-APP669-711; Including, In the QC standard sample, the ratio of abundance of the stable isotope-labeled peptides: SIL-Aβ1-40 / SIL-Aβ1-42, and / or SIL-APP669-711 / SIL-Aβ1-42 The peptide measurement method according to claim 1, wherein the amount of the peptide is within a predetermined reference range.

9. In the QC standard sample, the ratio of abundance of the stable isotope-labeled peptides: SIL-Aβ1-40 / SIL-Aβ1-42 is 25.0 to 32.5, and / or SIL-APP669-711 / SIL-Aβ1-42: 0.7 to 1.3 The peptide measurement method according to claim 8, wherein the peptide concentration is within a reference range of 0.1 to 0.

5.

10. The peptide measurement method according to claim 1, wherein the measurement is performed by immunoprecipitation (IP)-mass spectrometry (IP-MS), liquid chromatography-mass spectrometry (LC-MS), or gas chromatography-mass spectrometry (GC-MS).

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