LC-MS / MS method for quantification of gamma-H2AX in complex samples

Abstract

The application discloses a kind of LC-MS / MS quantitative analysis methods of γ-H2AX in complex sample, it includes the following steps: (1) preparation standard curve working solution;(2) preparation internal standard working solution;(3) preparation standard curve sample;(4) preparation standard curve sample containing internal standard;(5) to be measured sample processing;(6) to be measured sample solution, internal standard containing standard curve sample solution carries out LC-MS / MS analysis;(7) according to internal standard-standard curve method, the content of H2AX, γ-H2AX in to be measured sample is quantitatively analyzed;(8) assesses the DNA damage value of phosphorylation marker in tissue sample.The application solves the technical problem that there is no accurate quantitative analysis method of γ-H2AX in complex sample.

Description

Technical Field

[0001] This invention relates to an LC-MS / MS quantitative analysis method for γ-H2AX in complex samples, belonging to the field of pharmaceutical analysis technology. Background Technology

[0002] With the proliferation of toxic chemicals, air, water, and food pollution in the environment, genotoxic substances are ubiquitous. They cause DNA damage or chromosomal aberrations through direct or indirect interaction with DNA, posing a potential threat of inducing serious diseases such as cancer and birth defects. When a double-strand break occurs in DNA, the DNA damage response mechanism is activated. The serine residue at position 139 of H2AX is rapidly phosphorylated to form γ-H2AX, which can serve as a biomarker of DNA damage. [1] .

[0003] Our research group previously established a liquid chromatography-tandem mass spectrometry method for γ-H2AX based on cell samples, achieving accurate quantitative detection of this compound. This method was then applied to a set of 69 model compounds recommended by the European Centre for Validation of Alternative Methods (ECVAM) to dynamically monitor the effects of genotoxic compounds on the entire process of DNA damage / repair and transcription, quantitatively revealing the genotoxicity and carcinogenic risk levels. [2] .

[0004] However, due to the low levels of phosphorylated peptides in complex biological samples and the signal inhibition by high-abundance unmodified peptides, trace amounts of γ-H2AX in vivo cannot be detected using extraction methods suitable for cell samples, posing a challenge to the detection of γ-H2AX in complex biomedical samples. [3] .

[0005] γ-H2AX is a phosphorylated peptide. Currently, many methods for detecting phosphorylated peptides, including high content analysis (HCA), flow cytometry, Western blotting, and whole-cell enzyme-linked immunosorbent assay (ELISA), suffer from false positives and inability to quantify the peptides. [4-7] .

[0006] There are already relatively accurate methods for quantifying γ-H2AX in cells, such as the methods established by Qu, Xu, and Shun based on human HeLa and HepG2 cells, respectively. [8-9] However, only one Japanese paper, "Quantitative analysis of γ-H2AX reveals distinct responses in multiple mouse organs after administration of mitomycin C or ethyl methanesulfonate," has been published on the quantitative detection of γ-H2AX in complex samples such as tissues.

[10] According to the experimental conditions of the literature, we did not detect γ-H2AX from the liver tissue of γ-H2AX model mice, indicating that the method of the literature is not suitable for the quantitative detection of γ-H2AX in complex samples.

[0007] There are also some quantitative analysis methods of phosphorylated peptides in complex samples, such as "An estimate assay for low-level exposure to ionizing radiation based on mass spectrometry quantification ofγ-H2AX in human peripheral blood lymphocytes" [2] According to the experimental conditions of the literature, we did not detect γ-H2AX. The methods of reference [2] and reference

[10] cannot accurately quantify γ-H2AX, which may be due to the low content of γ-H2AX in complex biological samples and the influence of high-abundance unmodified peptides. It is not suitable for the quantitative detection of γ-H2AX in complex samples.

[0008] In summary, it is an urgent problem to establish an accurate quantitative method for γ-H2AX in complex tissue samples.

[0009] Reference:

[0010] [1]Qu MM. Mass spectrometry analysis of histone phosphorylation reveals the genotoxicity of compounds and their mechanisms of action[D]. Academy of Military Science, 2020.

[0011] [2]Qu M, Xu H, Chen J, Zhang Y, Xu B, Guo L, Xie J. Distinct orchestration and dynamic processes onγ-H2AX and p-H3 for two major types of genotoxic chemicals revealed by mass spectrometry analysis. Chem Res Toxicol. 2020 Aug 17;33(8):2108-2119.

[0012] [3] Hongling Z, Minmin Q, Yuchen L, et al. An estimate assay for low-level exposure to ionizing radiation based on mass spectrometry quantification of γ-H2AX in human peripheral blood lymphocytes [J]. Frontiers in Public Health, 2022, 101031743-1031743.

[0013] [4] Lenz KD, Klosterman KE, Mukundan H, Kubicek-Sutherland JZ. Lipoprotein capture ELISA method for the sensitive detection of amphiphilic biomarkers. Anal Biochem. 2022 Sep 1;652:114747. doi: 10.1016 / j.ab.2022.114747. Epub 2022 May 27. PMID: 35636461.

[0014] [5] Mishra M, Tiwari S, Gomes AV. Protein purification and analysis: next generation Western blotting techniques. Expert Rev Proteomics. 2017 Nov;14(11):1037-1053. doi: 10.1080 / 14789450.2017.1388167. Epub 2017 Oct 13. PMID: 28974114; PMCID: PMC6810642.

[0015] [6] Manohar SM, Shah P, Nair A. Flow cytometry: principles, applications and recent advances. Bioanalysis. 2021 Feb;13(3):181-198. doi: 10.4155 / bio-2020-0267. Epub 2021 Feb 5. PMID: 33543666.

[0016] [7] Gaopengxia, Gong Mengqiang, Li Zhi, et al. Application of high content screening technology in toxic substance screening and toxicity evaluation research [J / OL]. Chinese Journal of Pharmacology and Toxicology, 1-11 [2024-09-19].

[0017] [8] Shun M, Tsuyoshi I, Tomonari M. Absolute quantification of γH2AX using liquid chromatography-triple quadrupole tandem mass spectrometry. [J]. Analytical and bioanalytical chemistry, 2015, 407(18): 5521-7.

[0018] [9] Qu M, Xu H, Chen J, Xu B, Li Z, Ma B, Guo L, Ye Q, Xie J. Differential comparison of genotoxic effects of aristolochic acid I and II in human cells by the mass spectroscopic quantification of γ-H2AX. Toxicol In Vitro. 2022 Jun;81:105349.

[0019]

[10] Shun M, Sayaka W, Toshihiko K. Quantitative analysis of γH2AX reveals distinct responses in multiple mouse organs after administration of mitomycin C or ethyl methanesulfonate. [J]. Mutagenesis, 2018, 33(5-6): 371-378. SUMMARY

[0020] Problems to be solved by the invention:

[0021] In view of the fact that there is no accurate quantitative analysis method for gamma-H2AX in complex tissue samples at present, the application establishes a quantitative analysis method for gamma-H2AX in complex tissue samples by studying how to extract, remove impurities and enrich trace gamma-H2AX from complex tissue samples, and verifies the method, and verifies that the specificity, linear range and sensitivity, precision and accuracy, recovery rate of the method meet the requirements, in order to achieve the above purpose, the specific technical scheme of the application is as follows:

[0022] The first aspect is to verify whether the method of reference [2] and reference

[10] can detect gamma-H2AX in complex samples respectively

[0023] According to the method of reference [2], no gamma-H2AX is detected in human blood samples, and according to the method of reference

[10] , only ion pair m / z 482 / 182 is detected in the spleen and lung tissue of ICR mice, which shows that the method cannot sensitively and accurately detect gamma-H2AX in complex samples.

[0024] The second aspect is to study the extraction, removal of impurities and enrichment of trace gamma-H2AX from complex tissue samples

[0025] (I) Extraction method research

[0026] Four extraction methods are studied, which are:

[0027] Scheme one, cell nucleus extraction kit; scheme two, low-osmotic buffer and high-osmotic buffer; scheme three, high-purity cell nucleus separation extraction kit; scheme four, histone extraction kit.

[0028] Results: Four extraction schemes can detect gamma-H2AX, and the response intensity based on H2AX is compared, as shown in Figure 5 Although the H2AX response intensity of scheme four is the largest, it does not affect the detection of phosphorylation modification, and finally scheme two low-osmotic buffer and high-osmotic buffer is selected.

[0029] (II) Desalination method research

[0030] Three desalination methods are studied, which are:

[0031] Scheme one, GL-Tip-GC; scheme two, GL-Tip-SDB; scheme three, self-made C18 membrane desalination column.

[0032] Results: GL-Tip-GC has the best recovery rate, and is suitable for LC-MS / MS quantitative analysis of gamma-H2AX and H2AX in complex tissue samples.

[0033] (III) Enrichment method research

[0034] Two enrichment methods were studied, which were:

[0035] Scheme one, Titansphere phos-TiO; Scheme two, polyMAC phosphopeptide enrichment.

[0036] Results: The response of γ-H2AX, the polyMAC phosphopeptide enrichment method of scheme two was better than the Titansphere phos-TiO enrichment method of scheme one.

[0037] (Four) Comparison of chromatographic columns

[0038] The results of γ-H2AX and H2AX detection of three chromatographic columns were compared, and the results were: ACQUITY UPLC BEH C18, 100 mm x 2.1 mm, 1.7 μm was more suitable for the "a LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of the application.

[0039] Third aspect, method for screening mouse tissues rich in γ-H2AX

[0040] Three methods for constructing mouse tissues rich in γ-H2AX were compared, and the results showed that the method of taking tissue samples 4 hours after intraperitoneal injection of nitrogen mustard (NH2, 10 mg / Kg) had the strongest γ-H2AX response intensity (genotoxic effect).

[0041] Fourth aspect, verification of whether it is feasible to detect γ-H2AX in complex samples by using the method not confirmed after the research of the second aspect for extraction and / or desalination

[0042] (I) Neither extraction nor desalination uses the method confirmed after the research

[0043] Extraction: Use scheme one, cell nucleus extraction kit; Desalination: Use scheme three, self-made C18 membrane desalination column.

[0044] Results: No γ-H2AX was detected

[0045] Conclusion: It is proved that the above-mentioned extraction and desalination methods are not suitable for the "a LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of the application.

[0046] (II) Extraction does not use the method confirmed after the research, desalination uses the method confirmed after the research, and does not perform enrichment

[0047] Extraction: Use scheme one, cell nucleus extraction kit; Desalination: Use the method confirmed after the research, that is, scheme one, GL-Tip-GC; Do not perform enrichment.

[0048] Results: Only ion pair of m / z 482 / 182 was detected; it was indicated that γ-H2AX and H2AX in complex samples could not be detected completely, and the detection results were lower than the true values.

[0049] Conclusion: It was indicated that the above extraction and desalination methods were not suitable for the "LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of the application.

[0050] (Three) Extraction and enrichment do not use the method of post-study confirmation, and desalination uses the method of post-study confirmation

[0051] Extraction: use scheme one, cell nucleus extraction kit; desalination: use the method of post-study confirmation, that is, scheme one, GL-Tip-GC; enrichment uses the method of post-study confirmation, that is, use scheme two, polyMAC phosphopeptide enrichment.

[0052] Results: γ-H2AX can be detected, but stable and repeated detection cannot be achieved.

[0053] Conclusion: It was indicated that the above extraction and desalination methods were not suitable for the "LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of the application.

[0054] Fifth aspect, establishing a LC-MS / MS quantitative analysis method of γ-H2AX in complex samples

[0055] The steps include: 1, preparing standard curve working solution; 2, preparing internal standard working solution; 3, preparing standard curve sample; 4, preparing standard curve sample containing internal standard; 5, processing the sample to be tested; 6, LC-MS / MS analysis of the sample to be tested solution and the standard curve sample containing internal standard; 7, quantitative analysis of H2AX and γ-H2AX content in the sample to be tested according to the internal standard-standard curve method; 8, evaluation of DNA damage value of phosphorylation marker in tissue sample.

[0056] Sixth aspect, methodological verification of the method established in the fifth aspect

[0057] The specificity, linear range, accuracy and precision and recovery rate of the method established in the fifth aspect for detecting γ-H2AX and H2AX were verified.

[0058] Results: The verification results meet the requirements, and it is indicated that the method established in the fifth aspect is suitable for accurate quantification of γ-H2AX and H2AX in complex tissue samples.

[0059] Compared with the prior art, the application has the beneficial effects of:

[0060] The application provides a method for establishing LC-MS / MS quantitative analysis of gamma-H2AX in complex tissue samples, and solves the technical problem that there is no accurate quantitative analysis method for gamma-H2AX in complex tissue samples at present. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 , mass spectrum response of blank matrix + H2AX + H2AX internal standard;

[0062] Figure 2 , mass spectrum response of blank matrix + gamma-H2AX + gamma-H2AX internal standard;

[0063] Figure 3 , linear range of H2AX and gamma-H2AX, wherein,

[0064] A is the linear range of H2AX,

[0065] B is the linear range of gamma-H2AX;

[0066] Figure 4 , Four Comparison results of four histone extraction schemes, wherein 1 is scheme one (nuclear extraction kit), 2 is scheme two (hypotonic buffer and hypertonic buffer), 3 is scheme three (high-purity nuclear separation extraction), and 4 is scheme four (histone extraction kit)

[0067] Figure 5 , Two Comparison results of enrichment schemes;

[0068] Figure 6 , Three Comparison results of three chromatographic columns, wherein,

[0069] A is the peak area of H2AX / internal standard of the three chromatographic columns,

[0070] B is the peak area of gamma-H2AX / internal standard of the three chromatographic columns;

[0071] Figure 7 , screening results of methods for constructing mouse tissues rich in gamma-H2AX;

[0072] Figure 8 , screening of nitrogen mustard species for constructing mouse tissues rich in gamma-H2AX;

[0073] Figure 9 , mass spectrum response of gamma-H2AX detected according to the method of reference [2];

[0074] Figure 10 , mass spectrum response of gamma-H2AX detected according to the method of reference

[10] , wherein,

[0075] A is the mass spectrum response of γ-H2AX in spleen tissue,

[0076] B is the mass spectrum response of γ-H2AX in lung tissue;

[0077] Figure 11 , using a nuclear extraction kit extraction + self-made C18 membrane desalination column desalination, mass spectrum response of γ-H2AX;

[0078] Figure 12 , using a nuclear extraction kit extraction + GL-Tip-GC desalination, mass spectrum response of γ-H2AX detection;

[0079] Figure 13 , using a nuclear extraction kit extraction + GL-Tip-GC desalination + Titansphere phos-TiO phosphopeptide enrichment, mass spectrum response of γ-H2AX.

[0080] Figures 1-13 ,

[0081] *, **, *** and **** represent: P<0.05, P<0.01, P<0.001 and P<0.0001, respectively, indicating that the difference between the two groups has statistical significance. DETAILED DESCRIPTION

[0082] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0083] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0084] All materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0085] Experimental materials

[0086] 1. Control, internal standard

[0087]

[0088] 2. Animals

[0089] ICR male mice (6-7 weeks), BALB / c mice (6-7 weeks) were purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd.

[0090] 3. Reagents

[0091]

[0092]

[0093] 4. Instruments

[0094]

[0095]

[0096] 5. Commonly used buffer solutions and reagent preparation

[0097] (1) 1x PBS buffer solution

[0098] 10x PBS buffer solution dry powder is supplemented with ultrapure water to 2000 mL, and after high-pressure sterilization, it is stored at 4°C for use;

[0099] (2) 0.2M H2SO4

[0100] Concentrated sulfuric acid 5.43 mL is supplemented with ultrapure water to 500 mL, and stored at 4°C for use;

[0101] (3) 100% trichloroacetic acid

[0102] Trichloroacetic acid 50 g is supplemented with ultrapure water to 22.7 mL, and stored at 4°C for use;

[0103] (4) 50mM NH4HCO3

[0104] 0.395 g of NH4HCO3 is supplemented with ultrapure water to 100 mL, and stored at 4°C for use;

[0105] (5) 20% acetic acid

[0106] 20 mL of acetic acid is supplemented with ultrapure water to 100 mL, and stored at 4°C for use;

[0107] Example 1, LC-MS / MS quantitative analysis method of γ-H2AX in complex samples

[0108] 1. Preparation of standard curve working solution

[0109] H2AX standard curve working solution: Take the peptide segment ASQASQEY (SEQ ID No. 1), i.e., H2AX control, and prepare 1 mg / mL of H2AX standard curve working solution with sterilized ultrapure water;

[0110] γ-H2AX standard curve working solution: Take the peptide segment ASQApSQEY (SEQ ID No. 2), i.e., γ-H2AX control, and prepare 1 mg / mL of γ-H2AX standard curve working solution with sterilized ultrapure water;

[0111] 2. Preparation of internal standard working solution

[0112] H2AX internal standard working solution: Take the peptide segment [13 C3, 15 [N]ASQASQEY (SEQ ID No. 3) is the H2AX internal standard, prepared as a 1 mg / mL H2AX internal standard working solution using sterile ultrapure water;

[0113] γ-H2AX internal standard working solution: Take peptide fragments [ 13 C3, 15 [N]ASQApSQEY (SEQ ID No. 4) is the γ-H2AX internal standard, which is prepared into a 1 mg / mL γ-H2AX internal standard working solution using sterile ultrapure water;

[0114] 3. Prepare standard curve samples

[0115] H2AX standard curve samples: Ultrapure water and H2AX standard curve working solution are mixed in an appropriate ratio to prepare H2AX standard curve samples. The concentrations of H2AX in the H2AX standard curve samples are 0.5, 5, 50, 100, 150, 200, 100, and 250 ng / mL, respectively.

[0116] γ-H2AX standard curve samples: Ultrapure water and γ-H2AX standard curve working solution were mixed in an appropriate ratio to prepare γ-H2AX standard curve samples. The concentrations of γ-H2AX in the γ-H2AX standard curve samples were 0.5, 5, 50, 100, 150, 200, 100, and 250 ng / mL, respectively.

[0117] 4. Prepare standard curve samples containing internal standards.

[0118] H2AX standard curve samples containing internal standard: H2AX standard curve samples were added to H2AX internal standard working solution to prepare a solution with an H2AX internal standard concentration of 50 ng / mL;

[0119] γ-H2AX standard curve samples containing internal standard: The γ-H2AX standard curve samples were added to the γ-H2AX internal standard working solution to prepare a solution with an internal standard concentration of 50 ng / mL.

[0120] 5. Sample preparation

[0121] 5.1 Extraction

[0122] (1) Add 100mg of tissue to 1mL of 1×PBS homogenate for 60s, centrifuge at 300g for 5min at 4℃ to precipitate;

[0123] (2) Add 1 mL of hypotonic buffer and shake at 4°C for 10 min;

[0124] (3) 1500g centrifuge at 4°C for 5 min, resuspend the pellet with 1 mL high salt buffer, 4°C shake for 10 min;

[0125] (4) 1500g centrifuge at 4°C for 5 min to precipitate the chromatin, resuspend in 400 μL 0.4M H2SO4, 4°C shake for 30 min;

[0126] (5) 16000g centrifuge at 4°C for 10 min to precipitate the sample, take the supernatant and add 132 μL trichloroacetic acid, ice bath for 30 min to precipitate histone;

[0127] (6) 16000g centrifuge at 4°C for 10 min to precipitate the histone, wash twice with 500 μL pre-cooled acetone, and dry at room temperature;

[0128] 5.2 Enzymolysis

[0129] (1) Add sequencing grade trypsin at a ratio of 1:50 overnight;

[0130] (2) The next day, add 2 μL of 20% acetic acid to terminate;

[0131] 5.3 Desalination

[0132] Use GL-Tip-GC desalination column to desalt, the steps are as follows:

[0133] (1) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile 3000g centrifuge for 2 min to rinse;

[0134] (2) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile 3000g centrifuge for 2 min to balance;

[0135] (3) Discard the liquid, add the sample to the GL-Tip-GC desalination column;

[0136] (4) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile 3000g centrifuge for 2 min to wash;

[0137] (5) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile 3000g centrifuge for 2 min to elute;

[0138] (6) Vacuum centrifuge the obtained peptide sample to dryness

[0139] 5.4 Enrichment

[0140] Use polyMAC phosphopeptide enrichment kit for enrichment, the steps are as follows:

[0141] (1) Dry the sample and resuspend in 200 μL of buffer;

[0142] (2) PolyMAC peptide enrichment magnetic beads were fully vortexed (10-20 s), and 50 μL was taken in a 1.5 mL centrifuge tube, and the upper storage solution was removed after centrifugation for 2-3 s;

[0143] (3) The resuspended sample was added to the centrifuge tube containing the polyMAC peptide enrichment magnetic beads, and then placed in a vortexer, 26°C, 1200 rpm, and shaken vigorously for 25 min;

[0144] (4) The sample was added to the PolyMAC Tips, centrifuged at 20g for 2 min, and if there was residual liquid, centrifuged at 100g for 1 min to ensure that the liquid flowed out of the Tips tip into the centrifuge tube;

[0145] (5) A new centrifuge tube was used, 200 μL of buffer I was added to the PolyMAC Tips, centrifuged at 20g for 2 min, and if there was residual liquid, centrifuged at 100g for 1 min to ensure that the liquid flowed out of the Tips tip into the centrifuge tube;

[0146] (6) 200 μL of wash solution I was added, centrifuged at 20g for 2 min, and if there was residual liquid, centrifuged at 100g for 1 min;

[0147] (7) Another 200 μL of wash solution II was added, centrifuged at 20g for 2 min, and if there was residual liquid, centrifuged at 100g for 1 min, and the H2AX in (5), (6), and (7) was collected;

[0148] (8) A new centrifuge tube was used, 50 μL of eluent was added, centrifuged at 20g for 2 min, and repeated once at 100g for 1 min, and the γ-H2AX was collected;

[0149] (9) The collected γ-H2AX was lyophilized and stored at -80°C or reconstituted with 0.1% formic acid water for LC-MS / MS detection;

[0150] 6. The sample solution to be tested and the standard curve sample solution containing the internal standard were analyzed by LC-MS / MS

[0151] 6.1 Chromatographic conditions:

[0152] Chromatographic column: ACQUITY UPLC BEH C18, 100 mm x 2.1 mm, 1.7 μm;

[0153] Mobile phase

[0154] Mobile phase A: 0.1% formic acid solution by volume;

[0155] Mobile phase B: acetonitrile;

[0156] Flow rate: 0.3 mL / min;

[0157] Column temperature: 40 °C;

[0158] Injection volume: 10 μL;

[0159] Gradient elution, elution program as follows:

[0160]

[0161] The percentage of mobile phase A and mobile phase B is volume percentage

[0162] 6.2 Mass spectrometry conditions

[0163] Multiple reaction monitoring (MRM) mode, electrospray ionization (ESI), positive ion mode, mass spectrometry conditions as follows:

[0164]

[0165] 6.3 The parameters of the detector are as follows:

[0166]

[0167] 7. Quantitative analysis of H2AX and γ-H2AX in the sample to be tested according to the internal standard-standard curve method

[0168] The ratio of the concentration of the analyte to the internal standard is taken as the abscissa, and the peak area ratio is taken as the ordinate. Linear regression is performed to obtain the internal standard-H2AX-standard curve. The peak area ratio of the chromatographic peak in the sample to be tested is substituted into the internal standard-H2AX-standard curve to calculate the content of H2AX in the sample to be tested.

[0169] The ratio of the concentration of the analyte to the internal standard is taken as the abscissa, and the peak area ratio is taken as the ordinate. Linear regression is performed to obtain the internal standard-H2AX-standard curve. The peak area ratio of the chromatographic peak in the sample to be tested is substituted into the internal standard-H2AX-standard curve to calculate the content of H2AX in the sample to be tested.

[0170] 8. Evaluation of DNA damage values of phosphorylation markers in tissue samples

[0171] Rγ / T Value = γ-H2AX / H2AX x 100%

[0172] Example 2, Methodology verification

[0173] 1. Specificity

[0174] Preparation of blank matrix: 100 mg of spleen tissue was ground and then processed according to the steps “5.1 extraction”, “5.2 purification” and “5.3 enrichment” in Example 1 to obtain blank matrix;

[0175] Sample preparation

[0176] 32 μL blank matrix + 4 μL H2AX at a final concentration of 2.5 ng / mL + 4 μL H2AX internal standard at a final concentration of 50 ng / mL

[0177] 32 μL blank matrix + 4 μL γ-H2AX at a final concentration of 2.5 nL / mL + 4 μL γ-H2AX internal standard at a final concentration of 50 ng / mL

[0178] LC-MS / MS analysis was performed according to "6. Analyze the test sample solution and the standard curve sample solution containing internal standard" in Example 1.

[0179] The results are as follows Figure 1 As shown, the method of this application does not interfere with the determination of H2AX and internal standard;

[0180] The results are as follows Figure 2 As shown, the method of this application does not interfere with the determination of γ-H2AX and internal standard.

[0181] 2. Linear range and sensitivity

[0182] Add γ-H2AX and H2AX reference standards to the blank solution to prepare mixed solutions of γ-H2AX and H2AX with concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, and 250 ng / mL, respectively. Add internal standards H2AX(IS) and γ-H2AX(IS) to make the concentration of H2AX and γ-H2AX(IS) 50 ng / mL. Finally, perform LC-MS / MS analysis according to "6. LC-MS / MS analysis of the sample solution to be tested and the standard curve sample solution containing internal standards" in Example 1.

[0183] Using the peak area ratio of the above-mentioned reference standard and internal standard as the ordinate (y), and the mass concentration of the reference standard as the abscissa (x, mg / mL), a working curve is constructed: y = ax + b; the lowest concentration with a signal-to-noise ratio S / N > 3 in the working curve is taken as the limit of detection, and the lowest concentration with a signal-to-noise ratio S / N > 10 is taken as its lowest limit of quantitation.

[0184] See results Figure 3 According to Table 1, the linear equation of γ-H2AX is: y = 0.022x + 0.034, R0 2 =0.994, the linear range of γ-H2AX is 1-250 ng / mL, and the limit of quantitation is 1 ng / mL; the linear equation of H2AX is: y = 0.023x + 0.132, R2 = 0.992, H2AX linear range is 1-250 ng / mL, the lowest quantification limit is 1 ng / mL.

[0185] Table 1, γ-H2AX and H2AX detection limit, quantification limit and quality control sample concentration

[0186]

[0187] 3. Accuracy and precision

[0188] The γ-H2AX control and H2AX control were added to the blank solution, respectively, and the γ-H2AX quality control samples with final concentrations of 1, 2.5, 125, 180 ng / mL and the H2AX quality control samples with final concentrations of 1, 2.5, 125, 180 ng / mL were prepared according to the experimental results of the linear range above, and the internal standard H2AX (IS) and γ-H2AX (IS) were added, respectively, so that the internal standard concentration was 50 ng / mL. Each concentration of quality control sample was prepared in 6 replicates, and was analyzed and detected according to “5, preparation of the sample solution to be tested”, “6, LC-MS / MS analysis of the sample solution to be tested, the standard curve sample solution containing the internal standard” in Example 1. The accuracy was calculated by measuring the content and the percentage of the true content. The precision was calculated by calculating the relative standard deviation of the measured concentrations of the 6 quality control samples at each concentration. The results are shown in Tables 2 and 3.

[0189] Table 2, accuracy and precision of γ-H2AX

[0190]

[0191] From Table 2, it can be seen that:

[0192] The within-run accuracy and coefficient of variation of γ-H2AX of the method of the present application were 94.4%-99.8% and 5.8%-14.7%, respectively, indicating that the within-run accuracy and precision of the method of the present application for detecting γ-H2AX in solution samples met the Chinese Pharmacopoeia 2024 edition Biological Sample Quantitative Analysis Method Validation Guideline;

[0193] The between-run accuracy and coefficient of variation of γ-H2AX of the method of the present application were 104.8%-121.1% and 10.3%-16.6%, respectively, indicating that the between-run accuracy and precision of the method of the present application for detecting γ-H2AX in solution samples met the Chinese Pharmacopoeia 2024 edition Biological Sample Quantitative Analysis Method Validation Guideline.

[0194] Table 3, accuracy and precision of H2AX

[0195]

[0196] From Table 3, it can be seen that:

[0197] The intra-batch accuracy and coefficient of variation of the H2AX method of the application were 97.8% to 101.4% and 5.2% to 17.7%, respectively, indicating that the intra-batch accuracy and precision of the H2AX method of the application for detecting solution samples met the Chinese Pharmacopoeia 2024 edition Biological Sample Quantitative Analysis Method Validation Guidelines;

[0198] The inter-batch accuracy and coefficient of variation of the H2AX method of the application were 95.2% to 102.7% and 8.5% to 17.5%, respectively, indicating that the inter-batch accuracy and precision of the H2AX method of the application for detecting solution samples met the Chinese Pharmacopoeia 2024 edition Biological Sample Quantitative Analysis Method Validation Guidelines.

[0199] 4. Recovery rate

[0200] The γ-H2AX control and H2AX control were added to the blank solution to prepare γ-H2AX quality control samples and H2AX quality control samples at concentrations of 2.5, 5, 125, and 180 ng / mL, and the internal standard was added to a final concentration of 50 ng / mL. Six samples were taken for each concentration of the quality control sample, which was analyzed and detected according to “5, Preparation of the test sample” in Example 1, “6, LC-MS / MS analysis of the test sample solution, standard curve sample solution containing the internal standard” and “7, Drawing a standard curve, and quantitatively analyzing the content of γ-H2AX and H2AX in the test sample according to the standard curve method” in Example 1. The results are shown in Tables 4 and 5.

[0201] Table 4, γ-H2AX recovery rate (n = 6)

[0202]

[0203]

[0204] a: CV is the relative standard deviation, relative standard deviation (%) = standard deviation / mean x 100 (retained to one decimal place)

[0205] The results of the γ-H2AX recovery rate showed that the determination of γ-H2AX was not affected using the test sample processing method of the application.

[0206] Table 5, H2AX recovery rate (n = 6)

[0207]

[0208] a: CV is the relative standard deviation, relative standard deviation (%) = standard deviation / mean x 100 (retained to one decimal place)

[0209] The H2AX recovery results show that the determination of H2AX is not affected by using the sample processing method of the present application.

[0210] Example 3, extraction method research

[0211] 1. Scheme

[0212] Phosphatase and protease inhibitors were added in advance, and the treatment process was maintained at 4°C. Four extraction methods were studied.

[0213] Scheme 1, nuclear extraction kit

[0214] (1) Wash the cells with 1x PBS buffer for 1-2 times, collect into a 15 mL centrifuge tube, and centrifuge at 300g for 5 min at 4°C;

[0215] (2) Discard the supernatant, leave about 1 mL of supernatant and the precipitate, and transfer into a 1.5 mL EP tube, centrifuge at 300g for 5 min at 4°C;

[0216] (3) Discard the supernatant, and the order of the number of cells used is 10 6 In the EP tube with the precipitate, add 500 μL of ice-precooled lysis buffer to resuspend the cells, and then add 20 μL of reagent A to shock the cells for 40 min;

[0217] (4) Directly centrifuge at 300g for 5 min at 4°C, the cells and precipitate are at the bottom of the collection tube, discard the supernatant, and add 0.25 mL of ice-precooled lysis buffer to resuspend the precipitate;

[0218] (5) Take another new centrifuge tube and add 0.25 mL of medium salt buffer to the resuspended liquid of the previous step, carefully add it into the centrifuge tube along the wall of the tube, place it on top of the medium salt buffer, and centrifuge at 700g for 5 min at 4°C, the cell nucleus precipitate is precipitated at the bottom;

[0219] (6) Discard the supernatant, add 0.25 mL of lysis buffer to the cell nucleus precipitate, and centrifuge at 1000g for 10 min to discard the supernatant, and obtain a relatively pure cell nucleus precipitate;

[0220] (7) Completely aspirate the supernatant and resuspend the nucleus in 400 μL of 0.2M H2SO4, and shake at 4°C for 60 min

[0221] (8) Centrifuge the sample at 4°C to remove nuclear debris at 16000g for 10 min, and transfer the supernatant containing histones to a clean 1.5 mL Ep tube (400 μL is aspirated);

[0222] (9) Add 132 μL of 100% trichloroacetic acid to the solution containing histone, drop by drop, and invert the solution up and down to mix the solution, and the histone will precipitate in 100% trichloroacetic acid;

[0223] (10) Incubate the solution on ice for 30 min;

[0224] (11) 16000g 4℃ centrifuge for 10 min, remove the supernatant with a pipette and wash the histone with pre-cooled acetone;

[0225] (12) 16000g 4℃ centrifuge for 5 min, repeat the above two steps, remove the supernatant and dry at room temperature for 10 min;

[0226] (13) Evaporate the acetone to obtain relatively pure histone;

[0227] When the protein is attached to the tube wall in solid form, wash with 40 μL of 50 mM NH4HCO3, then 4℃ 16000g centrifuge for 10 min.

[0228] Scheme II, low-osmotic buffer and high-osmotic buffer

[0229] (1) 1 mL of low-osmotic buffer homogenized for 60 s, 4℃ 300g centrifuged for 5 min to precipitate;

[0230] (2) Homogenize the sample with 1 mL of low-osmotic buffer, 4℃ shake for 10 min;

[0231] (3) 4℃ 1500g centrifuge for 5 min, resuspend the precipitate with 1 mL of high-osmotic buffer, 4℃ shake for 10 min;

[0232] (4) 4℃ 1500g centrifuge for 5 min to precipitate the chromatin, and resuspend in 400 μL of 0.4M H2SO4, 4℃ shake for 30 min;

[0233] (5) 4℃ 16000g centrifuge for 10 min to precipitate the sample, add 132 μL of trichloroacetic acid to the supernatant, and incubate on ice for 30 min to precipitate the histone;

[0234] (6) 4℃ 16000g centrifuge for 10 min to precipitate the histone, wash twice with 500 μL of pre-cooled acetone, and air dry at room temperature;

[0235] Scheme III, high-purity nuclear separation extraction kit

[0236] (1) Resuspend the tissue cells with 1 mL of pre-cooled lysis buffer;

[0237] (2) 1 mL of 1.8M sucrose buffer is operated on ice: add ice-cold to each lysis solution sample; mix by gently pipetting and inverting the test tube;

[0238] (3) 1 mL of 1.8M sucrose buffer For each sample preparation, take a clean ultracentrifuge tube and place it on ice, add ice-cold ice box to the bottom;

[0239] (4) 1 mL of 1.8M sucrose buffer Carefully and slowly place the lysate solution of step (1) on top, avoiding disturbing the sucrose cushion;

[0240] (5) Carefully place the ultracentrifuge tube into the pre-cooled bucket of the ultracentrifuge, 30000g at 4°C for 45 min;

[0241] (6) Carefully and completely aspirate the supernatant (cytoplasm and cell debris) and the transparent sucrose cushion without disturbing the underlying purified nuclei pellet;

[0242] (7) Resuspend in 400 μL of 0.4M H2SO4, shake at 4°C for 30 min;

[0243] (8) 4°C 16000g centrifuge for 10 min to precipitate the sample, add 132 μL of trichloroacetic acid to the supernatant, and ice bath for 30 min to precipitate histone;

[0244] (9) 4°C 16000g centrifuge for 10 min to precipitate histone, wash twice with 500 μL of pre-cooled acetone, and air dry at room temperature;

[0245] Protocol Four, Histone Extraction Kit

[0246] (1) Preparation of extraction solution: add 2 μL of reagent G to each 1 mL of reagent A, mix after adding 10 μL of reagent C, and store on ice;

[0247] (2) Take 30 mg of sample tissue and cut it into small pieces, add 1 mL of reagent A, homogenize, and aspirate into a clean centrifuge tube on ice to lyse for 30 min;

[0248] (3) 4°C 800g centrifuge for 10 min, discard the supernatant;

[0249] (4) Add 500 μL of reagent B, 4°C 800g centrifuge for 10 min, discard the supernatant;

[0250] (5) Resuspend the precipitate with 500 μL of reagent C, and react on ice for 30 min;

[0251] (6) 10000 rpm 4°C centrifuge for 5 min, collect the supernatant;

[0252] (7) Add 130 μL of reagent D to the supernatant and mix well, react on ice for 60 min;

[0253] (8) 14000 rpm, 4°C centrifuge for 10 min, discard the supernatant (protein);

[0254] (9) 500 μL reagent E precipitated twice, 14000 rpm, 4°C centrifugation 5 min;

[0255] 1, sample

[0256] The sample is ICR mouse (male, 6 weeks) NH2 10 mg / kg 0.5 h liver, spleen, lung and kidney tissue.

[0257] 2, detection

[0258] Except for extraction according to the "1, scheme" of the present embodiment, the rest are carried out according to the steps of Example 1.

[0259] 3, results, conclusions

[0260] Since the four extraction schemes are all detected for γ-H2AX, based on the response intensity results of H2AX, as shown in Figure 4 , although the H2AX response intensity of scheme four is the largest, the sample amount is small, and the cost is high, while the sample amount of scheme two is large, and the cost is low, scheme two is temporarily selected for subsequent research of low osmotic buffer and high osmotic buffer.

[0261] Example 4, desalination method research

[0262] 1, scheme

[0263] Three desalination methods are studied

[0264] Scheme one, GL-Tip-GC (suitable for hydrophilic peptides, referred to as GC)

[0265] (1) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile 3000g centrifugation 2 min rinse;

[0266] (2) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile 3000g centrifugation 2 min balance;

[0267] (3) Discard the liquid, load the sample;

[0268] (4) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile 3000g centrifugation 2 min washing;

[0269] (5) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile 3000g centrifugation 2 min elution.

[0270] Scheme two, GL-Tip-SDB (suitable for hydrophobic peptides, referred to as SDB)

[0271] (1) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile 3000g centrifugation 2 min rinse;

[0272] (2) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile 3000g centrifugation 2 min balance;

[0273] (3) Discard the liquid, sample;

[0274] (4) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile 3000g centrifugation 2 min wash;

[0275] (5) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile 3000g centrifugation 2 min elution;

[0276] Scheme three, self-made C18 membrane desalination column (referred to as C18)

[0277] (1) 20 μL of 0.1% formic acid 80% acetonitrile 3000g centrifugation 2 min rinse;

[0278] (2) 20 μL of 0.1% formic acid 20% acetonitrile 3000g centrifugation 2 min balance;

[0279] (3) Discard the liquid, sample;

[0280] (4) 20 μL of 0.1% formic acid 20% acetonitrile 3000g centrifugation 2 min wash;

[0281] (5) 20 μL of 0.1% formic acid 80% acetonitrile 3000g centrifugation 2 min elution;

[0282] 2, sample

[0283] ICR mice (male, 6 weeks) NH2 10mg / kg administration 30min after the decapitation method, take the liver, spleen, lung, kidney tissue.

[0284] 3, detection

[0285] In addition to desalination according to the "1, scheme" in this example, the remaining steps are carried out according to example 1.

[0286] 4, results, conclusions

[0287] The recovery rates of γ-H2AX and H2AX of the three desalination methods are shown in Table 4. The GL-Tip-GC has the best recovery rate, which is suitable for LC-MS / MS quantitative analysis of γ-H2AX and H2AX in complex tissue samples.

[0288] Table 4, recovery rate of three desalination columns

[0289]

[0290] Example 5, enrichment method research

[0291] 1, scheme

[0292] Two enrichment protocols were investigated

[0293] Protocol 1, Titansphere phos-TiO (TIO)

[0294] (1) Activation: 20 μL Buffer A (0.5% trifluoroacetic acid in 80% acetonitrile) was used, 3000g centrifugation for 2 min;

[0295] (2) Equilibration: 20 μL Buffer B (0.5% trifluoroacetic acid in 80% acetonitrile + lactic acid 300 mg / mL) was used, 3000g centrifugation for 2 min. Discard 40 μL effluent;

[0296] (3) Adsorption: 30 μL sample was mixed with 100 μL Buffer B (mix well) and eluted at 1000g for 10 min (repeat once);

[0297] (4) Elution: 20 μL Buffer B was added, 3000g centrifugation for 2 min, 20 μL Buffer A was added, and 3000g centrifugation for 2 min, and the effluent was discarded (twice);

[0298] (5) Elution: 50 μL Elution C (5% ammonium hydroxide + 5% pyrrolidine) was used, 1000g centrifugation for 5 min (each 1 time);

[0299] (6) The obtained phosphopeptide was dried in a vacuum centrifuge and stored at -80°C

[0300] Protocol 2, polyMAC phosphopeptide enrichment kit (polyMAC)

[0301] (1) Dry sample was resuspended with 200 μL of buffer;

[0302] (2) 50 μL of polyMAC peptide segment enrichment magnetic beads was taken in a 1.5 mL centrifuge tube after vortexing (10-20 s), and the upper storage solution was removed after instantaneous centrifugation for 2-3 s;

[0303] (3) The resuspended sample was added to the centrifuge tube containing the peptide segment enrichment magnetic beads, and then placed in a vortex instrument, 26°C, 1200 rpm, and shaken vigorously for 25 min;

[0304] (4) The sample was added to the polyMAC enrichment column, 20g centrifugation for 2 min, and then 100g centrifugation for 1 min to ensure that the liquid flowed out of the tip end of the centrifuge tube;

[0305] (5) 200 μL of buffer was added to the polyMAC Tips, 20g centrifugation for 2 min, and 100g centrifugation for 1 min to ensure that the liquid flowed out of the tip end of the centrifuge tube (collecting waste liquid);

[0306] (6) Use a new centrifuge tube, add 200 μL of wash solution I, 20g centrifuge for 2 min, 100g centrifuge for 1 min;

[0307] (7) Add 200 μL of wash solution II again, 20g centrifuge for 2 min, 100g centrifuge for 1 min (collect waste liquid);

[0308] (8) New centrifuge tube, add 50 μL of elution solution, 20g centrifuge for 2 min, repeat once, and 100g centrifuge for 1 min; collect γ-H2AX;

[0309] (9) The collected γ-H2AX and H2AX are freeze-dried and stored at -80°C or reconstituted with 0.1% formic acid water, and detected by LC-MS / MS.

[0310] 2、Sample

[0311] Prepare a mixed standard solution of 2.5 ng / mL γ-H2AX and 50 ng / mL H2AX (n = 3), and add 50 ng / mL γ-H2AX internal standard and 50 ng / mL H2AX internal standard solution after treatment, respectively.

[0312] 3、Detection

[0313] Without sample extraction, enzymolysis, desalting, enrichment according to the "1, scheme" of this embodiment, and the remaining steps are carried out according to Example 1.

[0314] 4、Results, Conclusion

[0315] Results: As shown in Figure 5 , the response of γ-H2AX, polyMAC is stronger than TIO.

[0316] Conclusion: The polyMAC enrichment method of scheme two is better than the TIO enrichment method of scheme one.

[0317] Example 6, column comparison

[0318] 1、Scheme

[0319] Comparison of three kinds of chromatographic columns

[0320] Scheme one, BioBasic 8HPLC4, 250mm×4.6mm, 5μm, abbreviated as Basic;

[0321] Scheme two, AdvanceBio HIC, 100mm×4.6mm, 3.5μm, abbreviated as Advance;

[0322] Scheme three, ACQUITY UPLC BEH C18, 100mm x 2.1mm, 1.7μm, abbreviated as BEH;

[0323] 2. Preparation of the sample

[0324] 1mg / mL of γ-H2AX and H2AX control solution was diluted with ultrapure water to a solution of 125ng / mL.

[0325] 3. Detection

[0326] The chromatographic column was prepared according to the method of the present embodiment "1, scheme", the sample was prepared according to the method of the present embodiment "2, sample preparation", and the remaining steps were performed according to the method of Example 1.

[0327] 4. Results, Conclusion

[0328] Results: The sample was injected into the three chromatographic columns of schemes one, two and three, respectively,

[0329] The statistical diagram of the peak area ratio of H2AX to internal standard of the three chromatographic columns is shown in Figure 6 A, BEH and Advance have no significant difference, and Advance is significantly greater than Basic (P<0.05);

[0330] The statistical diagram of the peak area ratio of γ-H2AX to internal standard of the three chromatographic columns is shown in Figure 6 B, BEH is significantly greater than Advance (P<0.05), and BEH is significantly greater than Basic (P<0.01).

[0331] 5. Conclusion

[0332] Conclusion: BEH chromatographic column is more suitable for the method of "a method for quantitatively analyzing γ-H2AX in complex samples by LC-MS / MS".

[0333] Example 7, screening of methods for constructing γ-H2AX-rich mouse tissues

[0334] 1. Scheme

[0335] Three methods for constructing γ-H2AX-rich mouse tissues were compared, and the mice were ICR mice (male, 6 weeks).

[0336] Scheme one, tissue samples were taken 4h after the mice were exposed to ionizing radiation IR (4GY);

[0337] Scheme two, tissue samples were taken 4h after intraperitoneal injection of nitrogen mustard (NH2, 10mg / Kg)

[0338] Option 3: Tissue samples were taken 30 days after intraperitoneal injection of nitrogen mustard (NH2, 1 mg / kg / week);

[0339] 2. Testing

[0340] Mice were euthanized by cervical dislocation, and spleen tissue was extracted, frozen, and tested according to the steps in Example 1.

[0341] 3. Results

[0342] like Figure 7 As shown, LC-MS / MS detection revealed that all monitored target ion pairs exhibited good signals, and γ-H2AX was successfully detected without interference. A comparison of the γ-H2AX response intensity (genotoxic effect) results is also presented.

[0343] Nitrogen mustard (10 mg / kg, 4 hours) > Ionizing radiation (4 Gy, 4 hours) > Nitrogen mustard (1 mg / kg / week, 4 injections)

[0344] 4. Conclusion

[0345] Mouse tissues rich in γ-H2AX were constructed by intraperitoneal injection of nitrogen mustard (10 mg / kg, 4 hours).

[0346] Example 8: Screening for nitrogen mustard species in mouse tissues rich in γ-H2AX

[0347] 1. Plan

[0348] Nitrogen mustard species were screened for constructing mouse tissues rich in γ-H2AX. All mice were male ICR mice (6 weeks old).

[0349] Option 1: Mice were intraperitoneally injected with nitrogen mustard (NH1, 2 mg / Kg) and exposed for 10 min;

[0350] Option 2: Mice were intraperitoneally injected with nitrogen mustard (NH2, 2 mg / Kg) and exposed for 10 min;

[0351] Option 3: Intraperitoneal injection of nitrogen mustard (NH3, 2 mg / Kg) for 10 min exposure.

[0352] 2. Testing

[0353] Mice were euthanized by cervical dislocation, and spleen tissue was extracted, frozen, and tested according to the steps in Example 1.

[0354] 3. Results

[0355] like Figure 8 As shown, γ-H2AX can be successfully and stably detected by LC-MS / MS. A comparison of the γ-H2AX response intensity (genotoxic effect) results is presented.

[0356] NH2>NH1>NH34

[0357] 4. Conclusion

[0358] The intensity of γ-H2AX (genotoxic effect) in the spleen tissue of mice injected with nitrogen mustard NH2 was greater.

[0359] Example 9, Reference [2] Detection of γ-H2AX in complex samples (Comparative Example 1)

[0360] 1. Sample collection

[0361] Clinical coronary heart disease patient, 61-year-old female, no smoking history, blood sample

[0362] 2. Sample processing

[0363] Blood processing: 5 mL of fresh blood was diluted with PBS at 1:1, 3 mL of lymphocyte separation medium was added to the PBMC centrifuge tube, the diluted blood was laid on the separation medium, the interface between the two liquids was clear, and centrifugation was performed at 2200 rpm for 25 min, and the PBMC layer was carefully aspirated.

[0364] Extraction: according to "Scheme One (nuclei extraction kit)" in Example 3;

[0365] Desalination: according to "Scheme Three, self-made C18" in Example 4;

[0366] 3. Detection

[0367] Chromatographic conditions

[0368] The HPLC chromatographic column was ACQUITY UPLC BEH C18 (100 mm x 2.1 mm, 1.7 μm); the column temperature was 40°C; the mobile phase A was 0.1% formic acid solution, and the mobile phase B was acetonitrile; the injection volume was 10 μL; the flow rate was 0.25 mL / min; the gradient elution program was 0-8.0 min, 1%-30% B; 8.0-8.5 min, 30%-80% B; 8.5-10.1 min, 80%-1% B; 10.1-12.0 min, 1% B.

[0369] Mass spectrometry conditions

[0370] Monitoring mode: multiple reaction monitoring (MRM) mode; Ion source: electro spray ionization (ESI), positive ion mode; Ion source temperature: 500℃; Gas 1 pressure: 276 kPa; Gas 2 pressure: 414 kPa; Collision cell exit potential (CEX): 15 eV, Entrance potential (EP): 10 eV, MRM method parameters are shown in Table 5.

[0371] Table 5, MRM method parameters of H2AX peptide and H3 peptide standards

[0372]

[0373]

[0374] The remaining steps were performed according to Example 1.

[0375] 4. Results, Conclusion

[0376] As shown in Table 6, no γ-H2AX was detected. Figure 9

[0377] Example 10, Detection of γ-H2AX in complex samples by Japanese literature method (Reference

[10] ) (Comparative Example 2)

[0378] 1. Sample collection

[0379] ICR mice (male, 6 weeks) were exposed to mitomycin C 2 mg / kg for 12 h, and spleen and lung tissues were taken;

[0380] 2. Preparation of sample to be tested

[0381] 2.1 Extraction

[0382] (1) Homogenize 1 mL of hypotonic buffer for 60 s, and centrifuge at 300 g at 4℃ for 5 min to precipitate;

[0383] (2) Add 1 mL of hypotonic buffer to the homogenized sample, and shake at 4℃ for 10 min;

[0384] (3) Centrifuge at 1500 g at 4℃ for 5 min, resuspend the precipitate with 1 mL of hypertonic buffer, and shake at 4℃ for 10 min;

[0385] (4) Centrifuge at 1500 g at 4℃ for 5 min to precipitate the chromatin, and resuspend in 400 μL of 0.4 M H2SO4, and shake at 4℃ for 30 min; ​

[0386] (5) 4°C, 16000g centrifugation for 10 min to precipitate the sample, and 132 μL of trichloroacetic acid was added to the supernatant, and ice-bath for 30 min to precipitate histone;

[0387] (6) 4°C, 16000g centrifugation for 10 min to precipitate histone, and washed twice with 500 μL of pre-cooled acetone, and air-dried at room temperature;

[0388] 2.2 Enzymolysis

[0389] (1) 1:50 ratio of sequencing grade trypsin was added overnight;

[0390] (2) 2 μL of 20% acetic acid was added the next day to terminate;

[0391] 2.3 Desalination

[0392] (1) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile, 3000g centrifugation for 2 min, and rinsing;

[0393] (2) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile, 3000g 2 min equilibration;

[0394] (3) Discard the liquid, and load the sample;

[0395] (4) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile, 3000g 2 min washing;

[0396] (5) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile, 3000g 2 min elution;

[0397] (6) Rotary evaporation for re-dissolving for LC-MS / MS;

[0398] 3. Detection

[0399] Except for the sample processing according to the "2. Sample processing" of the present example, the remaining steps were performed according to Example 1.

[0400] 4. Results, Conclusion

[0401] As shown in Table 1, the ion pair of 482 / 182 was detected in the spleen and lung tissue samples according to the Japanese literature method, but the ion pair of 482 / 433 could not be detected, and it could not be confirmed that the detected peak was γ-H2AX. Figure 10 Example 11, Comparative Example 3

[0402] 1. Scheme

[0403] Extraction: according to the "Scheme One (nuclear extraction kit)" of Example 4;

[0404]

[0405] ​Desalting: according to "Scheme 3, desalting column with self-made C18 membrane" in Example 5;

[0406] 2. Sample

[0407] ICR mice (male, 6 weeks) were intraperitoneally injected with NH2, 1 mg / kg / week, and the liver tissue was taken 4 hours after the last exposure;

[0408] 3. Detection

[0409] In addition to extraction and desalting according to the "1, scheme" of this example, the remaining steps were performed according to Example 1.

[0410] 4. Results

[0411] As shown in Figure 11 , no γ-H2AX was detected.

[0412] 5. Conclusion

[0413] Extraction and desalting were performed according to the scheme of this example, which was not applicable to the "Method for LC-MS / MS quantitative analysis of γ-H2AX in complex samples" of the present application.

[0414] Example 12, Comparative Example 4

[0415] 1. Scheme

[0416] Extraction: according to "Scheme 1 (nuclear extraction kit)" in Example 4;

[0417] Desalting: according to "Scheme 1, GL-Tip-GC" in Example 5;

[0418] This time, no enrichment treatment

[0419] 2. Sample

[0420] ICR mice (male, 6 weeks) were intraperitoneally injected with MMC 2 mg / mkg, and the spleen tissue was taken 4 hours after exposure;

[0421] 3. Detection

[0422] In addition to extraction, desalting and enrichment according to the "1, scheme" of this example, the remaining steps were performed according to Example 1.

[0423] 4. Results

[0424] As shown in Figure 12 , only the ion pair of m / z 482 / 182 was detected; it was indicated that γ-H2AX and H2AX in complex samples could not be detected completely, and the detection results were lower than the true values.

[0425] 5. Conclusion

[0426] Extraction and desalination used the protocol of this example, which is not applicable to the "LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of the present application.

[0427] Example 13, Comparative Example 5

[0428] 1. Protocol

[0429] Extraction: according to "Protocol One (Nuclei Extraction Kit)" in Example 4;

[0430] Desalination: according to "Protocol One, GL-Tip-GC" in Example 5;

[0431] Enrichment: according to "Protocol One, Titansphere phos-TiO phosphopeptide enrichment" in Example 6.

[0432] 2. Samples

[0433] ICR mice (male, 6 weeks) were intraperitoneally injected with MMC 2 mg / kg, and the spleen tissues were collected at 4 h after exposure;

[0434] 3. Detection

[0435] In addition to extraction, desalination and enrichment according to the "1. Protocol" of this example, the remaining steps were performed according to Example 1.

[0436] 4. Results

[0437] As shown in Figure 13 , γ-H2AX could be detected, but stable and repeated detection could not be achieved.

[0438] 5. Conclusion

[0439] Extraction, desalination and enrichment used the protocol of this example, which is not applicable to the "LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of the present application.

[0440] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A method for LC-MS / MS quantitative analysis of γ-H2AX in mouse tissue samples, the method comprising the following steps: (1) Pretreatment of mouse tissue samples ① Extraction A. Homogenize 100 mg of mouse tissue in 1 mL of 1 × PBS for 60 s, and centrifuge at 300 g at 4 ℃ for 5 min to precipitate; B. Add 1 mL of hypotonic buffer and shake at 4 ℃ for 10 min; C. Centrifuge at 1500 g at 4 ℃ for 5 min, resuspend the precipitate with 1 mL of hypertonic buffer, and shake at 4 ℃ for 10 min; D. Centrifuge at 1500 g at 4 ℃ for 5 min to precipitate the chromatin, and resuspend it in 400 μL of 0.4 M H2SO4, shake at 4 ℃ for 30 min; E. Centrifuge at 16000 g at 4 ℃ for 10 min to precipitate the sample, and add 132 μL of trichloroacetic acid to the supernatant, and incubate in an ice bath for 30 min to precipitate histones; F. Centrifuge at 16000 g at 4 ℃ for 10 min to precipitate the histones, wash twice with 500 μL of pre-cooled acetone, and dry at room temperature; ② Enzymatic digestion A. Add sequencing-grade trypsin at a ratio of 1:50 overnight; B. Add 2 μL of 20% acetic acid the next day to terminate the reaction; ③ Desalting Desalt using a GL-Tip-GC desalting column, steps as follows: A. Rinse 20 μL of 0.1% trifluoroacetic acid in 80% acetonitrile at 3000 g for 2 min; B. Equilibrate 20 μL of 0.1% trifluoroacetic acid in 20% acetonitrile at 3000 g for 2 min; C. Discard the waste liquid, and add the sample to the GL-Tip-GC desalting column; D. Wash 20 μL of 0.1% trifluoroacetic acid in 20% acetonitrile at 3000 g for 2 min; E. Elute 20 μL of 0.1% trifluoroacetic acid in 80% acetonitrile at 3000 g for 2 min; F. Concentrate the obtained peptide sample to dryness by vacuum centrifugation; ④ Enrichment Enrichment is performed using a polyMAC phosphopeptide enrichment kit, steps as follows: A. Resuspend the dried sample in 200 μL of buffer; B. After vortexing the polyMAC peptide enrichment magnetic beads for 10-20 s, take 50 μL to a 1.5 mL centrifuge tube, centrifuge for 2-3 s, and then remove the upper storage liquid; C. Add the resuspended sample to the centrifuge tube containing the polyMAC peptide enrichment magnetic beads, and then place it in a vortex mixer, shake at 1200 rpm at 26 ℃ for 25 min; D. Add the sample to the polyMAC Tips, centrifuge at 20 g for 2 min, and if there is residual liquid, centrifuge at 100 g for 1 min to ensure that the liquid flows from the tip of the Tips to the centrifuge tube; E. Use a new centrifuge tube, add 200 μL of buffer I to the polyMAC Tips, centrifuge at 20 g for 2 min, and if there is residual liquid, centrifuge at 100 g for 1 min to ensure that the liquid flows from the tip of the Tips to the centrifuge tube; F. Add 200 μL of washing solution I, centrifuge at 20 g for 2 min, and if there is residual liquid, centrifuge at 100 g for 1 min; G. Add another 200 μL of washing solution II, centrifuge at 20 g for 2 min, and if there is residual liquid, centrifuge at 100 g for 1 min, collect H2AX to be tested in E, F, and G. H. Add 50 μL of eluent to a new centrifuge tube, centrifuge at 20 g for 2 min, repeat once, centrifuge at 100 g for 1 min, collect γ-H2AX; I. The collected γ-H2AX is lyophilized and stored at -80℃ or reconstituted with 0.1% formic acid water for LC-MS / MS detection; (2) LC-MS / MS analysis The liquid chromatography conditions are as follows: Chromatographic column: C18; mobile phase A: 0.1% formic acid solution; mobile phase B: acetonitrile; Flow rate: 0.2-0.4 mL / min; Column temperature: 35-45℃; Injection volume: 5-20 μL; The elution mode is gradient elution, and the gradient elution program is as follows: 0-6.0 min, A phase from 99% to 50%, B phase from 1% to 50%; 6.0-6.1 min, A phase from 50% to 45%, B phase from 50% to 55%; 6.1-8.0 min, A phase from 45% to 5%, B phase from 55% to 95%; 8.0-8.1 min, A phase is 5%, B phase is 95%; 8.1-10.0 min, A phase from 5% to 99%, B phase from 95% to 1%; Mass spectrometry conditions: multiple reaction monitoring (MRM) mode, electrospray ion source (ESI), positive ion mode, ion source temperature: 500℃; The parameters of the multiple reaction monitoring (MRM) mode are as follows:

2. The method of claim 1, wherein, The linear range of the method H2AX and γ-H2AX is 1-250 ng / mL.

3. The method of claim 1, wherein, The minimum quantification limit of the method H2AX and γ-H2AX is 1 ng / mL.

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