Marker peptides for measuring ALT1 and ALT2

Abstract

Biomarkers for human ALT1 and ALT2 and mouse ALT1 and ALT2, the biomarkers including a peptide for measuring human ALT1, a peptide for measuring human ALT2, a peptide for measuring mouse ALT1 and a peptide for measuring mouse ALT2; and a method for detecting human ALT1 and ALT2 and mouse ALT1 and ALT2 using the biomarkers.

Description

[Technical field]

[0001] The present invention relates to peptides capable of simultaneously quantifying ALT1 and ALT2 derived from mouse and human. [Background technology]

[0002] Alanine aminotransferase (ALT) is an enzyme contained within cells, and ALT leaked out of cells due to cell damage is used as a marker of damage. Several types of ALT (ALT1, ALT2, etc.) have been reported, and ALT1 is particularly known to be hepatotoxic, while ALT2 is specific to skeletal muscle. Conventionally, ALT testing was mainly based on enzyme activity testing, but in recent years, awareness of ALT1 and ALT2 has increased, and the ELISA method, which specifically detects ALT1 and ALT2 using an antigen-antibody reaction, has come into use.

[0003] However, it is difficult to distinguish between ALT1 and ALT2 using the ALT enzyme activity method, and it is also difficult to distinguish between human-derived ALT and mouse-derived ALT in human hepatocyte chimeric mice. Furthermore, in ELISA, it is possible to specifically measure human ALT1, human ALT2, mouse ALT1, and mouse ALT2, but a total of four measurements are required. It is also known that antigen-antibody binding disappears in ELISA depending on the individual. This is presumed to be due to genetic changes in the three-dimensional structure or sugar chain of the ALT protein. Thus, there is no method that can simultaneously quantify human-derived ALT1, human-derived ALT2, mouse-derived ALT1, and mouse-derived ALT2 in a single measurement. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7037789 [Non-patent literature]

[0005] [Non-Patent Document 1] Bioanalysis. 2022 Mar;14(5):267-278. doi: 10.4155 / bio-2021-0250. Epub 2022 Feb 23. Summary of the Invention [Problem to be solved by the invention]

[0006] Under the above circumstances, there was a need to develop a technology that would enable the simultaneous measurement of human ALT1, human ALT2, mouse ALT1, and mouse ALT2 contained in a sample in the same measurement system. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the inventors determined peptide sequences specific to each ALT from peptide sequences obtained by enzymatic digestion of human-derived ALT1, human-derived ALT2, mouse-derived ALT1, and mouse-derived ALT2 contained in a sample, and then confirmed that it is possible to simultaneously quantify four types of ALT using these peptide sequences, thereby completing the present invention.

[0008] That is, the present invention is as follows. [1] A biomarker for human ALT1 and human ALT2, and mouse ALT1 and mouse ALT2, comprising a peptide for measuring human ALT1 which contains at least one peptide selected from peptides (a1) to (a4) shown in Group A below, a peptide for measuring human ALT2 which contains at least one peptide selected from peptides (b1) to (b3) shown in Group B below, a peptide for measuring mouse ALT1 which contains at least one peptide selected from peptides (c1) to (c2) shown in Group C below, or a peptide for measuring mouse ALT2 which contains at least one peptide selected from peptides (d1) to (d7) shown in Group D below, or a combination thereof. Group A: Peptides for measuring human ALT1 (a1)LLVAGEGHTR (a2) QAVLAELAAK (a3)AWALDVAELHR (a4) MTILPPLEK Group B: Peptides for measuring human ALT2 (b1)TPSSWGR (b2)IFIPAK (b3)AGEIELELQR Group C: Peptides for measuring mouse ALT1 (c1)LLVAGEGR (c2)LTEQVFNEAPGIR Group D: Peptides for measuring mouse ALT2 (d1) SHSSAAAEASAALK (d2)ASGPWGR (d3)LLVSGGGK (d4) AAVLVR (d5)ILIPAK (d6)EDVAAFITR (d7)MTILPPVDK [2] A method for detecting at least one ALT selected from human ALT1, human ALT2, mouse ALT1 and mouse ALT2, comprising subjecting a test sample to mass spectrometry to detect the biomarker described in [1]. [3] The method according to [2], for simultaneously detecting human ALT1, human ALT2, mouse ALT1 and mouse ALT2. [4] A toxicity evaluation method comprising contacting a test chemical with a biological material, subjecting the biological material to mass spectrometry to detect the biomarker described in [1], and using the detection result as an indicator to evaluate the toxicity of the test chemical. [5] The method according to [4], which simultaneously detects at least two ALTs selected from the group consisting of human ALT1, human ALT2, mouse ALT1 and mouse ALT2. [6] The method according to [4], wherein when the ALT value in the detection results shows no difference after contact with the test chemical compared to before contact, or when the ALT value in the detection results shows no difference compared to a control group, the test chemical is judged to be non-toxic or to have low toxicity. [7] The method according to [4], wherein the test chemical is judged to be toxic when the ALT value in the detection results shows a difference after contact with the test chemical compared to before contact, or when the ALT value in the detection results shows a difference compared to a control group. Effect of the Invention

[0009] The present invention makes it possible to simultaneously measure four types of ALT in one measurement and simultaneously detect cytotoxicity in humans and mice. In addition, the present invention avoids the phenomenon of loss of antigen-antibody binding in ELISA depending on the individual, ensuring universality of the simultaneous detection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention relates to biomarkers for human ALT1 and ALT2, and mouse ALT1 and ALT2, and to a method for detecting human ALT1 and human ALT2, and mouse ALT1 and mouse ALT2 by measuring the biomarkers in a test sample.

[0011] The present inventors searched for amino acid sequences that can distinguish human ALT1 and ALT2 from human samples, and for amino acid sequences that can distinguish mouse ALT1 and ALT2 from mouse samples. The amino acid sequences of each ALT were cut into several peptide fragments, and the fragments were detected using a high-resolution mass spectrometer. As a result, the amino acid sequences of the candidate fragments were identified, and detection work was performed using actual test samples, targeting the candidate peptides, making it possible to distinguish between each ALT.

[0012] The biomarkers to be detected in the present invention are peptides for measuring human ALT1 which contain at least one peptide selected from peptides (a1) to (a4) shown in Group A below, peptides for measuring human ALT2 which contain at least one peptide selected from peptides (b1) to (b3) shown in Group B below, peptides for measuring mouse ALT1 which contain at least one peptide selected from peptides (c1) to (c2) shown in Group C below, or peptides for measuring mouse ALT2 which contain at least one peptide selected from peptides (d1) to (d7) shown in Group D below, and include peptides that are combinations of each of the peptides belonging to Groups A to D.

[0013] Group A: Peptides for measuring human ALT1 (a1) LLVAGEGHTR (sequence number 1) (a2) QAVLAELAAK (sequence number 2) (a3) AWALDVAELHR (sequence number 3) (a4) MTILPPLEK (sequence number 4)

[0014] Group B: Peptides for measuring human ALT2 (b1) TPSSWGR (SEQ ID NO: 5) (b2) IFIPAK (sequence number 6) (b3) AGEIELELQR (sequence number 7)

[0015] Group C: Peptides for measuring mouse ALT1 (c1) LLVAGEGR (sequence number 8) (c2) LTEQVFNEAPGIR (sequence number 9)

[0016] Group D: Peptides for measuring mouse ALT2 (d1) SHSSAAAEASAALK (sequence number 10) (d2) ASGPWGR (SEQ ID NO: 11) (d3) LLVSGGGK (sequence number 12) (d4) AAVLVR (SEQ ID NO: 13) (d5) ILIPAK (SEQ ID NO: 14) (d6) EDVAAFITR (sequence number 15) (d7) MTILPPVDK (SEQ ID NO: 16)

[0017] In another aspect of the present invention, there is provided a method for detecting at least one ALT selected from human ALT1, human ALT2, mouse ALT1 and mouse ALT2, characterized by subjecting a test sample to mass spectrometry (MS) to detect the biomarker. In the present invention, by using these biomarkers, it becomes possible to simultaneously detect human ALT1, human ALT2, mouse ALT1 and mouse ALT2.

[0018] Here, "simultaneous detection" means that, as long as each peptide belonging to the A, B, C, and D groups can be detected in the same test system, peptides belonging to all groups may be measured at the same time, but it is not necessary to measure peptides belonging to all groups at the same time. For example, the above human ALT1, human ALT2, mouse ALT1, and mouse ALT2 may be detected simultaneously for one test sample, or one test sample may be divided into four, and human ALT1, human ALT2, mouse ALT1, and mouse ALT2 may be detected for each sample. Alternatively, human ALT1, human ALT2, mouse ALT1, and mouse ALT2 may be detected in order for one test sample (the order in this case is arbitrary).

[0019] The test sample is a biological material isolated from a subject, and examples thereof include blood, plasma, serum, urine, and culture supernatant of liver tissue collected from a subject. The method for measuring the test sample is mass spectrometry as described above. The method involves subjecting a sample to an ionization source under conditions suitable to produce one or more ions detectable by mass spectrometry from each of one or more analytes. The analytes may be extracted from the sample and separated by chromatography prior to detection by mass spectrometry.

[0020] Mass spectrometry is an analytical technique that involves ionizing, or ionizing and fragmenting, target molecules and analyzing the ions based on their mass / charge ratio to produce a mass spectrum. The mass / charge ratio of an object is determined by determining the wavelengths at which electromagnetic energy is absorbed by the object. There are several commonly used methods for determining the mass-to-charge ratio of an ion, including measuring the interaction of the ion trajectory with an electromagnetic wave, measuring the time it takes for the ion to travel a given distance, or a combination of both.

[0021] The mass spectrometer itself is composed of a sample introduction section, an ionization section (ion source), a mass separation section (analyzer), a detection section (detector), a vacuum exhaust section (vacuum pump), an equipment control section, a data processing section (data system), etc. There are various types of analyzers. One of them is the quadrupole type, and a mass spectrometer with a quadrupole analyzer is called a quadrupole mass spectrometer. In the present invention, it is preferable to use a tandem quadrupole mass spectrometer.

[0022] It should be noted that an internal standard can be added to the calibration standards and / or test samples. To obtain a more accurate value of the measured analyte in the sample, the internal standard may be used to account for analyte losses during sample processing. The ratio of the analyte peak area at the level of the calibration standard to the peak area of ​​the internal standard can also be used to generate a calibration curve and quantify the sample.

[0023] In yet another aspect of the present invention, there is provided a toxicity evaluation method, characterized in that a test chemical is contacted with a biological material, the biological material is subjected to mass spectrometry to detect the biomarker, and the toxicity of the test chemical is evaluated using the detection result as an indicator. In this case, it is preferable to simultaneously detect at least two ALTs selected from the group consisting of human ALT1, human ALT2, mouse ALT1 and mouse ALT2. If at least two ALTs are detected, it is possible to identify the organ in which toxicity is expressed (utilizing the background that ALT1 is hepatotoxic and ALT2 is specific to skeletal muscle) and the animal species in which toxicity is expressed (comparing the measurement results of human ALT and mouse ALT) using the same sample.

[0024] "Contact" means placing an organism or biological material and a candidate substance to be evaluated (test chemical) in the same environment, reaction system, or culture system, and includes, for example, administering the test chemical to an organism, adding the test chemical to a cell culture container or a blood sample container, mixing the test chemical with biological material, culturing cells in the presence of the test chemical, and adding the test chemical to a cell culture medium. The term "organism" includes animals, plants, fungi, protozoa, archaea, bacteria, etc., but in the present invention, the type of organism is not limited as long as ALT can be detected, and animals are preferred. Examples of animals include humans, mice, rats, rabbits, goats, cows, sheep, horses, dogs, cats, monkeys, etc.

[0025] The term "biological material" has the same meaning as the test sample used in the method for detecting ALT, and examples thereof include blood, plasma, serum, urine, and culture supernatant of liver tissue collected from a subject.

[0026] A "test chemical substance" is a candidate substance to be evaluated for toxicity, and examples of the candidate substance include peptides, proteins, non-peptidic compounds, polymeric compounds, or low molecular weight compounds (which may be organic or inorganic compounds, and include both chemically synthesized and natural products), and these chemical substances may be new or known substances. When the test chemical substance is a polymeric or low molecular weight compound, it may form a salt, and examples of such salts include salts with physiologically acceptable acids (e.g., inorganic acids, organic acids, etc.) and bases (e.g., metallic acids, etc.).

[0027] When the ALT value in the detection results shows no difference after contact with the test chemical compared to before contact, or when the ALT value in the detection results shows no difference compared to a control group, it can be determined that the test chemical is non-toxic or has low toxicity. For example, (i) when no statistically significant difference is observed in the ALT level in the detection results after contact with the test chemical when comparing the levels before and after contact between two groups, (ii) when the value after contact with the test chemical is less than 200% of the value before contact, or (iii) when no statistically significant difference is observed in a two-group comparison between a control group and a group that has come into contact with the test chemical, or between a control group and multiple groups that have come into contact with the test chemical, it can be determined that the test chemical is non-toxic or has low toxicity.

[0028] Furthermore, when the ALT value in the detection results shows a difference after contact with the test chemical compared to before contact, or when the ALT value in the detection results shows a difference compared to a control group, the test chemical can be determined to be toxic. For example, (i) when a statistically significant difference is observed in the ALT of the detection results after contact with the test chemical substance in a comparison between two groups before and after contact, (ii) when the value after contact with the test chemical substance is 200% or more compared to before contact, or (iii) when a statistically significant difference is observed in a comparison between two groups, a control group and a group exposed to the test chemical substance, or a multi-group comparison between a control group and multiple groups exposed to the test chemical substance, the test chemical substance can be determined to be toxic.

[0029] However, the above criteria are not limited to these, and the threshold value can be set appropriately depending on the type and properties of the test chemical substance. Chemical substances that are determined to be non-toxic or have low toxicity as a result of the above assessment are selected as non-toxic substances and used for various purposes (for example, as a basis for deciding whether to continue development as reagents, agricultural chemicals, or pharmaceuticals).

[0030] On the other hand, chemical substances that are determined to be toxic as a result of the above assessment are also selected as toxic substances and used for various purposes (for example, as a basis for deciding whether to continue development as reagents, agricultural chemicals, or pharmaceuticals with limited uses or methods). EXAMPLES

[0031] The present invention will be described in more detail below with reference to examples, although the scope of the present invention is not limited to these examples. [Example 1] Identification of candidate peptide sequences to serve as markers

[0032] 1.1. Standard material The following recombinant proteins were used as standards: (1) Recombinant mouse ALT1 / GPT1 protein (mouse ALT1, hereafter referred to as m-ALT1) Manufacturer: NKMAX Co., Ltd. Product number: ATGP3530 Amino acid region: 1-496aa UniProt No.: Q8QZR5 Storage conditions: -20℃ or below

[0033] (2) Recombinant mouse ALT2 / GPT2 protein (mouse ALT2, hereafter referred to as m-ALT2) Manufacturer: NKMAX Co., Ltd. Product number: ATGP3881 Amino acid region: 1-522aa UniProt No.: Q8BGT5 Storage conditions: below -20°C

[0034] (3) Recombinant human ALT1 / GPT1 protein (human ALT1, hereafter referred to as h-ALT1) Manufacturer: NKMAX Co., Ltd. Product number: ATGP1283 Amino acid region: 1-496aa UniProt No.: P24298 Storage conditions: below -20°C

[0035] (4) Recombinant human ALT2 / GPT2 protein (human ALT2, hereinafter referred to as h-ALT2) Manufacturer: NKMAX Co., Ltd. Product number: ATGP1914 Amino acid region: 1-523aa UniProt No.: Q8TD30 Storage conditions: below -20°C

[0036] 1.2.Blank Matrix The blank matrix is ​​a biological sample that does not contain or contains a small amount of the target protein, and is used for the purpose of diluting the standard substance. (1) Mouse plasma Strain: Crl:CD1(ICR) Anticoagulant: Heparin Li (2) Pooled mouse plasma Strain: Crl:CD1(ICR) Anticoagulant: Sodium heparin (3) Pooled rat plasma Lineage: Crl:CD(SD) Anticoagulant: Sodium heparin

[0037] 1.3. Reagents used (Table 1) [Table 1]

[0038] 1.4.Solid Plate (1) SOLAμ HRP 2 mg / 1 mL 96 well plate Model number: 60209-001 Manufacturer: Thermo Fisher Scientific (2) Oasis MCX 96-well μElution Plate, 30 μm Model number: 186001830BA Manufacturer: Waters

[0039] 1.5. Digestive enzymes Product name: SMART Digest Trypsin Kit, Bulk Resin option Model Number: 60109-101-B Manufacturer: Thermo Fisher Scientific

[0040] 1.6. Main Equipment (Table 2) [Table 2]

[0041] 1.7. Treatment Method 1. Trypsin digestion and sample processing of various matrices and standards 160.0 μL of Smart buffer was added to 40.0 μL of h-ALT1 (500 μg / mL), 40.0 μL of m-ALT1 (500 μg / mL), and 40.0 μL of h-ALT2 (500 μg / mL), respectively, and 180.0 μL of Smart buffer was added to 20.0 μL of m-ALT2 (1000 μg / mL) to prepare 200 μL samples. In addition, 200 μL of samples were prepared by adding 180.0 μL of Smart buffer to 20.0 μL of human plasma or 20.0 μL of mouse plasma.

[0042] 15 μL of trypsin was added to each of the above samples, and the mixture was heated and then centrifuged at 1400 rpm at 70° C. for 30 minutes, 60 minutes, or 120 minutes. After centrifugation, the entire amount was added to SOLAμ HRP (conditioned with 200 μL of methanol and water), and washed with 300 μL of 1% formic acid. Next, it was eluted twice with 30 μL of 1% formic acid / 80% methanol, and 440 μL of water was added to prepare a sample for mass spectrometry.

[0043] 2. Detection of digestion fragments by high-resolution mass spectrometer (HRMS) The digestion fragments were analyzed using an Orbitrap QExactive plus. Liquid chromatography (LC) was performed using a Vanquish TM Horizon UHPLC system (Thermo Scientific TM ).

[0044] The column was YMC-Triart C18, with a pore size of 12 nM, a particle size of 1.9 μm, an inner diameter of 2.0 mm, and a length of 100 mm (model number: TA12SP9-1002PT). The column temperature was set at 60°C. Mobile phase A was water / acetonitrile / formic acid (980:20:1 v / v / v), mobile phase B was acetonitrile. The analysis time was 70 minutes and the injection volume was 25 μL.

[0045] 3. Identification and analysis of peptide fragments by protein analysis software Regarding the data obtained by HRMS analysis, protein analysis software (BioPharmaFinder) was used for analysis to search for peptide fragments derived from m_ALT.

[0046] 4. Method creation (transition creation) for tandem quadrupole mass spectrometer (MS / MS) measurement Candidate peptides and their transitions (combinations of m / z of Q1 and m / z of Q3) for each ALT were created as primary candidates using BioPharmaFinder. For these transitions, the DP (pull-in voltage to the mass spectrometer) and CE (collision energy) were optimized using the solution of each digested peptide prepared in item 1 above. The LC conditions for the optimization are shown below. MS was performed using a TripleQuad6500+ manufactured by SCIEX.

[0047] <LC conditions> LC used: NANOSPACE NASCA2 Column: YMC Triart C18, pore size 12 nM, particle size 1.9 μm, inner diameter 2.0 mm, length 100 mm (model number TA12SP9 1002PT) Column temperature: 60℃ Mobile phase A: Water / acetonitrile / formic acid (980:20:1 v / v / v) Mobile phase B: Acetonitrile Analysis time: 10 minutes Injection volume: 5μL

[0048] 5. Selection of candidate peptides and confirmation of sensitivity As a result of measurements using a tandem quadrupole mass spectrometer (MS / MS), 8 digested peptide fragments and 73 transitions were selected for mouse ALT1, 10 digested peptide fragments and 130 transitions for human ALT1, 10 digested peptide fragments and 112 transitions for mouse ALT2, and 12 digested peptide fragments and 109 transitions for human ALT2.

[0049] From these, a total of 144 transitions for four substances were selected as second-order candidates. For these selected transitions, the parameters of the ion source of the tandem mass spectrometer were set as follows: IS (Ion spray voltage (V)): 5000 CUR(Curtain Gas(psi)):32 TEM (heater temperature (℃)): 500 CAD(Collision Gas(psi)):10 GS1 (Nebulizer Gas (psi)): 65 GS2 (Heater Gas (psi)): 60

[0050] Subsequently, the solutions containing h-ALT1 (500,000 ng / mL), m-ALT1 (500,000 ng / mL), h-ALT2 (500,000 ng / mL), and m-ALT2 (1,000,000 ng / mL) were serially diluted to 1,000, 500, 200, 100, 50, 20, 10, 5, 2, and 1 ng / mL. A solution containing 10 μL of each sample, 190 μL of Buffer (Smart Digest Buffer), and 15 μL of enzyme (SMART Digest Trypsin) was centrifuged at 70°C and 1400 rpm for 60 minutes, dissolved in 300 μL of 1% formic acid, and loaded onto the solid phase (500×g 10 sec) (conditioned with 500 μL of methanol and 500 μL of water). After washing with 500 μL of 1% formic acid (1000×g 10 sec) and elution with 1% formic acid and 50% methanol (25 μL twice, 2000×g 10 sec), the sample was dissolved in 50 μL of 1% formic acid and injected into the LC-MS / MS.

[0051] The LC conditions were as follows. The experiment was performed at least twice to confirm reproducibility. LC used: NANOSPACE NASCA2 Column: YMC Triart C18, pore size 12 nM, particle size 1.9 μm, inner diameter 2.0 mm, length 100 mm (model number TA12SP9 1002PT) Column temperature: 60℃ Mobile phase A: Water, acetonitrile, formic acid (980:20:1 v / v / v) Mobile phase B: Acetonitrile Analysis time: 12~15 minutes Injection volume: 20~25μL

[0052] result: The selected fragment peptides, as well as their transitions and calibration curve data, are shown below (Table 3). [Table 3]

[0053] In the table, "Analyte Peak Name" is the ID of the peptide fragment tested, "Peptide Sequence" is the amino acid sequence of the peptide fragment, "Q1 / Q3" is the precursor ion / product ion, "Slope" is the slope of the calibration curve, "Y-intercept" is the Y-intercept, and "r" is the correlation coefficient. All calibration curves are weighted by 1 / x 2 It was carried out in. In Table 3, the numbers such as "+2" and "+3" on the C-terminal side of the peptide sequence represent the positive charge of the measured ion (the charge carried by the peptide), and the alphabet and number at the end represent the type of fragment.

[0054] [Example 2] Sample measurement Serum from human hepatocyte chimeric mice (PXB mice, Phoenix Bio) and culture supernatants of human hepatocytes obtained by perfusing the livers of PXB mice were used as samples, and ALT levels were measured using the following peptide fragments from the candidate peptide sequences obtained in Example 1 as markers. h-ALT1:AWALDVAELHR (SEQ ID NO: 3) h-ALT2: IFIPAK (SEQ ID NO: 6) m-ALT1: LLVAGEGR (SEQ ID NO: 8) m-ALT2: ILIPAK (SEQ ID NO: 14)

[0055] In order to create a more accurate calibration curve, rat plasma that does not contain the target protein was used as a blank matrix. In addition, stable isotope labels of each peptide were used as internal standards (IS) to correct for the sensitivity difference between rat plasma and mouse serum and measurement errors.

[0056] (1) Prepared Reagents The following reagents were mixed in the specified ratio to prepare a solution. (1) Water / acetonitrile (1:1, v / v) (2) Water / methanol (1:1, v / v) (3) Water / formic acid (100:2, v / v) (4) Water / methanol / 10% ammonia water (22:75:3, v / v / v) (5) Mobile phase A: Water / formic acid (1000:1, v / v) (6) Mobile phase B: acetonitrile / 2-propanol (1:1, v / v) (7) Cleaning solution: water / methanol (1:1, v / v)

[0057] (2) Preparation of IS solution Each IS was dissolved in water / acetonitrile (1:1, v / v) to prepare a 5 mg / mL IS stock solution. The IS stock solutions were mixed and diluted to prepare the IS solutions (Table 4). [Table 4]

[0058] (3) Preparation of high-concentration samples Pooled mouse plasma was aliquoted and then spiked with each standard (Table 5). [Table 5]

[0059] (4) Preparation of samples for calibration curve High concentration samples were serially diluted with pooled rat plasma (Table 6). [Table 6]

[0060] (5) QC sample preparation method High concentration samples were serially diluted with pooled mouse plasma (Table 7). [Table 7]

[0061] 190 μL of SMART Digest Buffer (buffer solution included in digestion enzyme kit) and 15 μL of Bulk Non-magnetic SMART Digest Resin (enzyme solution included in digestion enzyme kit) were added to the sample (10 μL) and stirred in a Thermomixer C (70°C, 1400 rpm, 60 min). 10 μL of IS solution and 200 μL of water / formic acid (100:2, v / v) were added to the mixture and stirred in a vortex mixer. The entire volume of the solution after stirring was loaded onto an Oasis MCX 96-well μElution Plate and centrifuged (4°C, 200×g, 6 min). Then, 400 μL of water / formic acid (100:2, v / v) was added and centrifuged (4°C, 200×g, 1 min).

[0062] Next, washing was performed three times under the following conditions. Add 400 μL of water / formic acid (100:2, v / v) and centrifuge (4°C, 200×g, 4 min). Add 400 μL of water / methanol (1:1, v / v) and centrifuge (4°C, 200×g, 4 min). Add 400 μL of methanol and centrifuge (4°C, 200×g, 4 min).

[0063] After the above washing, elution was carried out twice under the following conditions. Add 25 μL of water / methanol / 10% ammonia water (22:75:3, v / v / v) and centrifuge (4°C, 200×g, 4 min). 100 μL of water / formic acid (100:2, v / v) was added to the recovery plate in advance, and 25 μL was injected into the LC-MS / MS system.

[0064] (6)LC conditions Analytical column: CAPCELL PAK ADME-HR, particle size: 2 μm, 2.1 mm × 150 mm (Osaka Soda) Mobile phase: A: Water / formic acid (1000:1, v / v) B: Acetonitrile / 2-propanol (1:1, v / v) Gradient (Table 8): [Table 8]

[0065] Washing Solvent: Water / methanol (1:1, v / v) Column temperature: 65℃ Ambient temperature: 10℃ Injection volume: 25 μL Analysis time: 19 min

[0066] (7)MS / MS conditions Ionization mode: Electrospray ionization Polarity: Positive ion mode Scan type: MRM Curtain gas: 45 psi Collision Gas: 7 Ion spray voltage: 2000 V Heater gas temperature: 500℃ Nebulizer gas (GS1): 80 psi Turbo Gas (GS2): 85 psi Q1 Resolution: High Q3 Resolution:High Valve Positions (Table 9): [Table 9]

[0067] Monitoring ions and collision energies (Table 10): [Table 10]

[0068] result The quantitative values ​​obtained are shown in Table 11. The results of the calibration curve obtained when measuring the concentrations of the actual samples are shown in Table 12. The calibration curve met the criteria. [Table 11] [Table 12]

[0069] From Tables 11 and 12 above, it can be seen that in the calibration curves of all peptide sequences, the accuracy was 85% or more at a minimum concentration of 20 ng / mL or more. In the concentration measurements of the actual samples, the quantification of each ALT was achieved near the middle concentration of the calibration curve for most of the actual samples used for the study. Therefore, the quantitative sensitivity was considered to be good.

Claims

1. A biomarker for human ALT1 and human ALT2, and mouse ALT1 and mouse ALT2, comprising a peptide for measuring human ALT1 which contains at least one peptide selected from peptides (a2) to (a4) shown in Group A below, a peptide for measuring human ALT2 which contains at least one peptide selected from peptides (b2) to (b3) shown in Group B below, a peptide for measuring mouse ALT1 which contains peptide (c1) shown in Group C below, or a peptide for measuring mouse ALT2 which contains at least one peptide selected from peptides (d1) to (d7) shown in Group D below, or a combination thereof. Group A: Peptides for measuring human ALT1 (a2) QAVLAELAAK (a3) AWALDVAELHR (a4) MTILPPLEK Group B: Peptides for measuring human ALT2 (b2)IFIPAK (b3)AGEIELELQR Group C: Peptides for measuring mouse ALT1 (c1) LLVAGEGR Group D: Peptides for measuring mouse ALT2 (d1) SHSSAAAEAASAALK (d2) ASGPWGR (d3) LLVSGGGK (d4) AAVLVR (d5) ILIPAK (d6)EDVAAFITR (d7) MTILPPVDK

2. A method for detecting at least one ALT selected from human ALT1, human ALT2, mouse ALT1 and mouse ALT2, which comprises subjecting a test sample to mass spectrometry to detect the biomarker according to claim 1.

3. The method according to claim 2, wherein human ALT1, human ALT2, mouse ALT1 and mouse ALT2 are simultaneously detected.

4. A toxicity evaluation method comprising contacting a test chemical with an organism or biological material, subjecting the biological material to mass spectrometry to detect the biomarker described in claim 1, and using the detection result as an indicator to evaluate the toxicity of the test chemical.

5. The method according to claim 4, wherein at least two ALTs selected from the group consisting of human ALT1, human ALT2, mouse ALT1 and mouse ALT2 are simultaneously detected.

6. The method according to claim 4, wherein when there is no difference in the ALT value in the detection results after contact with the test chemical compared to before contact, or when there is no difference in the ALT value in the detection results compared to a control group, the test chemical is determined to be non-toxic or to have low toxicity.

7. The method according to claim 4, wherein the test chemical is judged to be toxic when there is a difference in the ALT value among the detection results after contact with the test chemical compared to before contact, or when there is a difference in the ALT value among the detection results compared to a control group.

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