LC-MS / MS quantitative analysis method for gamma-H2AX in complex sample

Abstract

The invention discloses an LC-MS / MS quantitative analysis method for gamma-H2AX in a complex sample. The LC-MS / MS quantitative analysis method comprises the following steps: (1) preparing a standard curve working solution; (2) preparing an internal standard working solution; (3) preparing a standard curve sample; (4) preparing a standard curve sample containing an internal standard; (5) treating a sample to be detected; (6) performing LC-MS / MS analysis on the sample solution to be detected and the standard curve sample solution containing the internal standard; (7) quantitatively analyzing the content of H2AX and gamma-H2AX in the sample to be detected according to an internal standard-standard curve method; and (8) evaluating the DNA damage value of the phosphorylation marker in the tissue sample. The technical problem that an accurate quantitative analysis method for gamma-H2AX in a complex sample does not exist is solved.

Description

Technical Field

[0001] The invention relates to an LC-MS / MS quantitative analysis method for gamma-H2AX in a complex sample, belonging to the technical field of drug analysis. Background Art

[0002] With the presence of toxic chemicals in the environment, air, water and food pollution, genotoxic substances are everywhere. They cause DNA damage or chromosomal aberrations by directly or indirectly interacting with DNA, and have the potential to induce major diseases such as cancer and deformity. When double-strand breaks occur in DNA, the DNA damage response mechanism is activated, and the serine residue at position 139 of H2AX is rapidly phosphorylated to form γ-H2AX, which can be used as a biomarker for DNA damage. [1] .

[0003] In the early stage, our research group established a liquid chromatography-tandem mass spectrometry method for γ-H2AX based on cell samples to achieve accurate quantitative detection of it, and applied it to a group of 69 model compounds recommended by the European Center for Validation of Alternative Methods (ECVAM) to dynamically monitor the whole process of genotoxic compounds affecting DNA damage / repair and transcription, and quantitatively reveal the level of genotoxicity and carcinogenicity risk. [2] .

[0004] However, due to the low content of phosphorylated peptides in complex biological samples and the signal suppression of high-abundance non-modified peptides, the trace amount of γ-H2AX in vivo cannot be detected using extraction methods suitable for cell samples. The detection of γ-H2AX in complex biomedical samples faces challenges. [3] .

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

[0006] There are relatively accurate quantitative methods for γ-H2AX in cells, such as the quantitative methods based on human Hela and HepG2 cells established by Qu, Xu and Shun, respectively. [8-9] However, there is only one Japanese paper on the quantitative detection of γ-H2AX in complex samples such as tissues: "Quantitative analysis of γH2AX reveals distinct responses in multiple mouse organs after administration of mitomycin C or ethyl methanesulfonate."

[10] It is recorded that we did not detect γ-H2AX in the liver tissue of γ-H2AX model mice when we followed the experimental conditions of the literature, 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 for 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] We did not detect γ-H2AX according to the experimental conditions of the reference. The methods of references [2] and

[10] cannot accurately and quantitatively detect γ-H2AX. It is speculated that this may be due to the low content of γ-H2AX in complex biological samples and the influence of the signal of high-abundance non-modified peptides, which is not suitable for the quantitative detection of γ-H2AX in complex samples.

[0008] In summary, establishing an accurate quantitative method for γ-H2AX in complex tissue samples is an urgent clinical problem to be solved.

[0009] References:

[0010] [1] Qu Minmin. Mass spectrometry analysis of histone phosphorylation modification reveals compound genotoxicity and its mechanism of action[D]. Academy of Military Sciences, 2020.

[0011] [2]Qu M, Xu H, Chen J, ZhangY, Xu B, Guo L, Xie J. Distinct Orchestration and Dynamic Processes on γ-H2AX and p-H3 for Two Major Types of GenotoxicChemicals Revealed by Mass Spectrometry Analysis. Chem Res Toxicol. 2020Aug17; 33(8): 2108-2119.

[0012] [3]Hongling Z,Minmin Q,Yuchen L,et al.An estimate assay for low-levelexposure to ionizing radiation based on mass spectrometry quantification ofγ-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.Lipoproteincapture ELISA method for the sensitive detection of amphiphilicbiomarkers.Anal Biochem.2022Sep 1;652:114747.doi:10.1016 / j.ab.2022.114747.Epub 2022May 27.PMID:35636461.

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

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

[0016] [7] Gao Pengxia, 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. 2022Jun; 81:105349.

[0019]

[10] Shun M,Sayaka W,Toshihiko K.Quantitative analysis ofγH2AXreveals distinct responses in multiple mouse organs after administration ofmitomycin C or ethyl methanesulfonate.[J].Mutagenesis,2018,33(5-6):371-378. Summary of the invention

[0020] Problems to be solved by the invention:

[0021] In view of the fact that there is currently no accurate quantitative analysis method for γ-H2AX in complex tissue samples, this application studies how to extract, remove impurities and enrich trace amounts of γ-H2AX from complex tissue samples, establishes a quantitative analysis method for γ-H2AX in complex tissue samples, and conducts methodological verification of the method, verifying that the specificity, linear range and sensitivity, precision and accuracy, and recovery rate of the method all meet the requirements. To achieve the above objectives, the specific technical solutions of the present invention are as follows:

[0022] First, we verified whether the methods in references [2] and

[10] were feasible for detecting γ-H2AX in complex samples.

[0023] When human blood samples were tested according to the method in reference [2], no γ-H2AX was detected. When spleen and lung tissues of ICR mice were tested according to the method in reference

[10] , only the ion pair of m / z 482 / 182 was detected in the spleen and lung tissue samples, indicating that this method cannot sensitively and accurately detect γ-H2AX in complex samples.

[0024] Second aspect: Research on extraction, removal of impurities and enrichment of trace amounts of γ-H2AX from complex tissue samples

[0025] (I) Research on extraction methods

[0026] Four extraction methods were studied:

[0027] Option 1: Cell nucleus extraction kit; Option 2: hypotonic buffer and hypertonic buffer; Option 3: high-purity cell nucleus separation and extraction kit; Option 4: histone extraction kit.

[0028] Results: All four extraction schemes detected γ-H2AX. The results were compared based on the response intensity of H2AX. Figure 5 As shown, although the H2AX response intensity of scheme 4 was the largest, in order not to affect the detection of phosphorylation modification, the hypotonic buffer and hypertonic buffer of scheme 2 were finally selected.

[0029] (II) Research on desalination methods

[0030] Three desalination methods were studied. The three desalination methods are:

[0031] Scheme 1: GL-Tip-GC; Scheme 2: GL-Tip-SDB; Scheme 3: homemade C18 membrane desalting column.

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

[0033] (III) Enrichment method research

[0034] Two enrichment methods were studied:

[0035] Scheme 1: Titansphere phos-TiO; Scheme 2: polyMAC phosphopeptide enrichment.

[0036] Results: For the response of γ-H2AX, the polyMAC phosphopeptide enrichment method of scheme 2 was superior to the Titansphere phos-TiO enrichment method of scheme 1.

[0037] (IV) Comparison of chromatographic columns

[0038] The γ-H2AX and H2AX detection results of the three chromatographic columns were compared, and the result showed that ACQUITY UPLC BEHC18, 100 mm × 2.1 mm, 1.7 μm was more suitable for the "LC-MS / MS quantitative analysis method for γ-H2AX in complex samples" of the present application.

[0039] Third aspect: screening of methods for constructing mouse tissues rich in γ-H2AX

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

[0041] Fourth, verify whether extraction and / or desalting is feasible to detect γ-H2AX in complex samples without using the method confirmed after the second aspect of the study

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

[0043] Extraction: using scheme 1, cell nucleus extraction kit; desalting: using scheme 3, homemade C18 membrane desalting column.

[0044] Results: γ-H2AX was not detected

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

[0046] (ii) The extraction method was not used and the desalting method was used and no enrichment was performed.

[0047] Extraction: using protocol 1, nuclear extraction kit; Desalting: using the method confirmed after the study, i.e. protocol 1, GL-Tip-GC; no enrichment was performed.

[0048] Results: Only the ion pair of m / z 482 / 182 was detected, indicating that it was not possible to detect all γ-H2AX and H2AX in the complex sample, and the detection result was lower than the true value.

[0049] Conclusion: It is shown that the above extraction and desalting methods are not suitable for the "LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of this application.

[0050] (III) Extraction and enrichment do not use methods confirmed after research, and desalination uses methods confirmed after research

[0051] Extraction: using protocol 1, cell nucleus extraction kit; desalting: using a method confirmed after the study, i.e. protocol 1, GL-Tip-GC; enrichment: using a method confirmed after the study, i.e. protocol 2, polyMAC phosphopeptide enrichment.

[0052] Results: γ-H2AX could be detected, but could not be stably and repeatedly detected.

[0053] Conclusion: It is shown that the above extraction and desalting methods are not suitable for the "LC-MS / MS quantitative analysis method of γ-H2AX in complex samples" of this application.

[0054] Fifth aspect: Establishment of a LC-MS / MS quantitative analysis method for γ-H2AX in complex samples

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

[0056] Aspect 6: Methodological verification of the method established in Aspect 5

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

[0058] Results: The validation results all met the requirements, indicating that the method established in the fifth aspect is suitable for the accurate quantification of γ-H2AX and H2AX in complex tissue samples.

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

[0060] The present invention provides a LC-MS / MS quantitative analysis method for γ-H2AX in complex tissue samples, which solves the technical problem that there is currently no accurate quantitative analysis method for γ-H2AX in complex tissue samples. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0063] Figure 3 , H2AX and γ-H2AX linear range, where

[0064] A is the linear range of H2AX,

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

[0066] Figure 4 , Four Comparison results of the histone extraction schemes, among which 1 is scheme 1 (nuclear extraction kit), 2 is scheme 2 (hypotonic buffer and hypertonic buffer), 3 is scheme 3 (high-purity nucleus separation and extraction), and 4 is scheme 4 (histone extraction kit)

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

[0068] Figure 6 , three The results of the comparison of the chromatographic columns are shown in Figure 2.

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

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

[0071] Figure 7 , screening results of methods for constructing mouse tissues enriched with γ-H2AX;

[0072] Figure 8 , construct a nitrogen mustard species screening for mouse tissues rich in γ-H2AX;

[0073] Fig. 9 , the mass spectrometry response of γ-H2AX was detected according to the method of reference [2];

[0074] Fig.10 , according to the method of reference

[10] , the mass spectrometry response of γ-H2AX is,

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

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

[0077] Fig.11 , extraction with a cell nucleus extraction kit + desalting with a homemade C18 membrane desalting column, mass spectrometry response of γ-H2AX;

[0078] Fig.12 , extraction with a cell nucleus extraction kit + GL-Tip-GC desalting, mass spectrometry response of γ-H2AX detection;

[0079] Fig.13 , extraction using a cell nuclear extraction kit + GL-Tip-GC desalting + Titansphere phos-TiO phosphopeptide enrichment, and mass spectrometry response of γ-H2AX.

[0080] Figure 1-13 middle,

[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 was statistically significant. DETAILED DESCRIPTION

[0082] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

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

[0085] Experimental Materials

[0086] 1. Reference substances and internal standards

[0087]

[0088] 2. Animals

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

[0090] 3. Reagents

[0091]

[0092]

[0093] 4. Instruments

[0094]

[0095]

[0096] 5. Commonly used buffer solutions and reagents configuration

[0097] (1) 1× PBS buffer

[0098] 10×PBS buffer powder was made up to 2000mL with ultrapure water, sterilized by high pressure, and stored at 4℃ for later use;

[0099] (2)0.2MH 2 SO 4

[0100] Add 5.43 mL of concentrated sulfuric acid to 500 mL with ultrapure water and store at 4°C until use;

[0101] (3) 100% trichloroacetic acid

[0102] 50 g of trichloroacetic acid was made up to 22.7 mL with ultrapure water and stored at 4 °C until use;

[0103] (4) 50 mM NH 4 HCO 3

[0104] 0.395 g of NH 4 HCO 3 Make up to 100 mL with ultrapure water and store at 4°C until use;

[0105] (5) 20% acetic acid

[0106] Add ultrapure water to make up 20 mL of acetic acid to 100 mL and store at 4°C until use;

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

[0108] 1. Prepare standard curve working solution

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

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

[0111] 2. Prepare internal standard working solution

[0112] H2AX internal standard working solution: Take peptide [ 13 C3, 15 N]ASQASQEY (SEQ ID No. 3), i.e., H2AX internal standard, was prepared into 1 mg / mL H2AX internal standard working solution with sterile ultrapure water;

[0113] γ-H2AX internal standard working solution: Take peptide [ 13 C3, 15 N]ASQApSQEY (SEQ ID No. 4), i.e., the γ-H2AX internal standard, was 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 were mixed in an appropriate ratio to prepare H2AX standard curve samples. The concentrations of H2AX in the H2AX standard curve samples were 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: γ-H2AX standard curve samples were added to γ-H2AX internal standard working solution to prepare a solution with a γ-H2AX internal standard concentration of 50 ng / mL;

[0120] 5. Sample processing

[0121] 5.1 Extraction

[0122] (1) 100 mg of tissue was added to 1 mL of 1× PBS and homogenized for 60 s. The pellet was then centrifuged at 300 g for 5 min at 4°C.

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

[0124] (3) Centrifuge at 1500 g for 5 min at 4°C, resuspend the pellet in 1 mL of hypertonic buffer, and shake at 4°C for 10 min;

[0125] (4) Centrifuge at 1500 g for 5 min at 4°C to precipitate the chromatin, and resuspend in 400 μL of 0.4 M HO. 2 SO 4 , shake at 4°C for 30 min;

[0126] (5) Centrifuge at 16000 g for 10 min at 4°C to precipitate the sample, add 132 μL of trichloroacetic acid to the supernatant, and incubate on ice for 30 min to precipitate histones;

[0127] (6) Centrifuge at 16,000 g for 10 min at 4°C to precipitate histones, wash twice with 500 μL of pre-cooled acetone, and air-dry at room temperature;

[0128] 5.2 Enzymatic hydrolysis

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

[0130] (2) The next day, 2 μL of 20% acetic acid was added to terminate the reaction;

[0131] 5.3 Desalination

[0132] Desalting is done using a GL-Tip-GC desalting column. The steps are as follows:

[0133] (1) Rinse with 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile by centrifugation at 3000 g for 2 min;

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

[0135] (3) discarding the waste liquid and adding the sample to the GL-Tip-GC desalting column;

[0136] (4) Wash with 20 μL of 0.1% trifluoroacetic acid and 20% acetonitrile by centrifugation at 3000 g for 2 min;

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

[0138] (6) The eluted peptide samples were concentrated to dryness by vacuum centrifugation.

[0139] 5.4 Enrichment

[0140] The polyMAC phosphopeptide enrichment kit was used for enrichment, and the steps were as follows:

[0141] (1) Add 200 μL of buffer to the dried sample and resuspend it;

[0142] (2) After fully vortexing the PolyMAC peptide enrichment magnetic beads (10-20 seconds), take 50 μL into a 1.5 mL centrifuge tube, centrifuge for 2-3 seconds, and then remove the upper storage solution;

[0143] (3) Add the resuspended sample to a centrifuge tube containing polyMAC peptide-enriched magnetic beads, and then place it in a mixer at 26°C and 1200 rpm for 25 min of vigorous shaking;

[0144] (4) Add the sample to the PolyMAC Tips and centrifuge at 20g for 2 min. If there is residual liquid, centrifuge at 100g for 1 min to ensure that the liquid flows out of the Tips into the centrifuge tube.

[0145] (5) Use a new centrifuge tube and add 200 μL of buffer I to the PolyMAC Tips. Centrifuge at 20 g for 2 min. If there is residual liquid, centrifuge at 100 g for 1 min to ensure that the liquid flows out of the Tips into the centrifuge tube.

[0146] (6) Add 200 μL of cleaning solution I and centrifuge at 20 g for 2 min. If there is residual liquid, centrifuge at 100 g for 1 min.

[0147] (7) Add 200 μL of cleaning solution II and centrifuge at 20 g for 2 min. If there is residual liquid, centrifuge at 100 g for 1 min to collect H2AX in (5), (6), and (7) for detection;

[0148] (8) Add 50 μL of the eluent to a new centrifuge tube and centrifuge at 20 g for 2 min. Repeat the centrifugation at 100 g for 1 min to collect γ-H2AX.

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

[0150] 6. LC-MS / MS analysis of the sample solution to be tested and the standard curve sample solution containing the internal standard

[0151] 6.1 Chromatographic conditions:

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

[0153] Mobile phase

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

[0155] Mobile phase B: acetonitrile;

[0156] Flow rate: 0.3 mL / min;

[0157] Column temperature: 40°C;

[0158] The injection volume was 10 μL;

[0159] Gradient elution, the elution program is as follows:

[0160]

[0161] ★The percentages of mobile phase A and mobile phase B are by volume

[0162] 6.2 Mass spectrometry conditions

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

[0164]

[0165] 6.3 The test object parameters are as follows:

[0166]

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

[0168] The ratio of the analyte to the internal standard concentration is used as the horizontal coordinate, and the peak area ratio is used as the vertical coordinate to perform linear regression 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 H2AX content in the sample to be tested;

[0169] Linear regression was performed with the ratio of the analyte to the internal standard concentration as the horizontal coordinate and the peak area ratio as the vertical coordinate to obtain the internal standard γ-H2AX-standard curve; the peak area ratio of the chromatographic peaks in the sample to be tested was substituted into the internal standard γ-H2AX-standard curve for calculation to obtain the γ-H2AX content in the sample to be tested.

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

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

[0172] Example 2: Methodological Verification

[0173] 1. Exclusivity

[0174] Preparation of blank matrix: 100 mg of spleen tissue was ground and then prepared according to "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 with a final concentration of 2.5 ng / mL + 4 μL H2AX internal standard with a final concentration of 50 ng / mL

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

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

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

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

[0181] 2. Linear range and sensitivity

[0182] Add γ-H2AX and H2AX reference substances to the blank solution, prepare γ-H2AX and H2AX mixed solutions with concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, and 250 ng / mL, respectively, and add internal standards H2AX(IS) and γ-H2AX(IS) to make H2AX and γ-H2AX(IS) 50 ng / mL, and 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 the internal standard" in Example 1;

[0183] The working curve was prepared with the peak area ratio of the reference substance and the internal standard as the ordinate (y) and the mass concentration of the reference substance as the abscissa (x, mg / mL): y=ax+b; the lowest concentration with a spectrum signal-to-noise ratio S / N>3 in the working curve was taken as the detection limit, and the lowest concentration with a signal-to-noise ratio S / N>10 was taken as its minimum quantification limit;

[0184] Results Figure 3 and Table 1, γ-H2AX linear equation: y = 0.022x + 0.034, R 2=0.994, the linear range of γ-H2AX is 1-250ng / mL, and the minimum limit of quantification is 1ng / mL; H2AX linear equation: y=0.023x+0.132, R 2 =0.992, the linear range of H2AX was 1-250 ng / mL, and the minimum limit of quantification was 1 ng / mL.

[0185] Table 1. Detection limits, quantification limits and quality control sample concentrations of γ-H2AX and H2AX

[0186]

[0187] 3. Precision and Accuracy

[0188] γ-H2AX reference substance and H2AX reference substance were added to the blank solution, and γ-H2AX quality control samples with final concentrations of 1, 2.5, 125, and 180 ng / mL and H2AX quality control samples with final concentrations of 1, 2.5, 125, and 180 ng / mL were prepared according to the above linear range experimental results, and internal standards H2AX (IS) and γ-H2AX (IS) were added to make the internal standard concentration 50 ng / mL. Each concentration quality control sample was prepared in 6 copies, and analyzed and detected according to "5. Preparation of sample solution to be tested" and "6. LC-MS / MS analysis of sample solution to be tested and standard curve sample solution containing internal standard" in Example 1, and 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 concentration of 6 quality control samples of each concentration, and the results are shown in Tables 2 and 3.

[0189] Table 2. Accuracy and precision of γ-H2AX

[0190]

[0191] From Table 2, we can see that:

[0192] The intra-batch accuracy and coefficient of variation of γ-H2AX of the method of this application are between 94.4% and 99.8% and between 5.8% and 14.7%, respectively, indicating that the intra-batch accuracy and precision of the method of this application for detecting γ-H2AX in solution samples are in line with the guidelines for validation of quantitative analysis methods for biological samples in the 2024 edition of the Chinese Pharmacopoeia;

[0193] The inter-batch accuracy and coefficient of variation of γ-H2AX in the method of the present application were 104.8% to 121.1% and 10.3% to 16.6%, respectively, indicating that the inter-batch accuracy and precision of the method of the present application for detecting γ-H2AX in solution samples are in compliance with the guidelines for validation of quantitative analysis methods for biological samples in the 2024 edition of the Chinese Pharmacopoeia.

[0194] Table 3. Accuracy and precision of H2AX

[0195]

[0196] From Table 3, we can see that:

[0197] The intra-batch accuracy and coefficient of variation of H2AX in the method of this application are between 97.8% and 101.4% and between 5.2% and 17.7%, respectively, indicating that the intra-batch accuracy and precision of the method of this application for detecting H2AX in solution samples are in line with the guidelines for validation of quantitative analysis methods for biological samples in the 2024 edition of the Chinese Pharmacopoeia;

[0198] The inter-batch accuracy and coefficient of variation of H2AX in the method of the present application were 95.2% to 102.7% and 8.5% to 17.5%, respectively, indicating that the inter-batch accuracy and precision of the method of the present application for detecting H2AX in solution samples are in compliance with the guidelines for validation of quantitative analysis methods for biological samples in the 2024 edition of the Chinese Pharmacopoeia.

[0199] 4. Recovery rate

[0200] γ-H2AX reference substance and H2AX reference substance were added to the blank solution, respectively, and γ-H2AX quality control samples and H2AX quality control samples with concentrations of 2.5, 5, 125, and 180 ng / mL were prepared, and the internal standard was added to a final concentration of 50 ng / mL, respectively. Six samples were taken for each concentration of quality control sample, and they were analyzed and detected according to "5. Preparation of samples to be tested", "6. LC-MS / MS analysis of sample solutions to be tested and standard curve sample solutions containing internal standards" and "7. Drawing a standard curve, and quantitatively analyzing the contents of γ-H2AX and H2AX in the samples to be tested 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 × 100 (retain to one decimal place)

[0205] The γ-H2AX recovery rate results show that the sample processing method of the present application has no effect on the determination of γ-H2AX.

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

[0207]

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

[0209] The H2AX recovery rate results show that the use of the test sample processing method of the present application has no effect on the determination of H2AX.

[0210] Example 3: Study on extraction method

[0211] 1. Solution

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

[0213] Solution 1: Cell Nuclear Extraction Kit

[0214] (1) Wash the cells once or twice with 1× PBS buffer, collect them into a 15 mL centrifuge tube, and centrifuge at 300 g for 5 min at 4°C;

[0215] (2) Discard the supernatant and mix the supernatant with the precipitate in about 1 mL. Transfer the mixture into a 1.5 mL EP tube and centrifuge at 300 g for 5 min at 4°C.

[0216] (3) The supernatant was discarded and the number of cells used was 10 6 , add 500 μL of ice-cold lysis buffer to the EP tube with the pellet to resuspend the cells, then add 20 μL of reagent A and shake the cells on a cell shaker for 40 min;

[0217] (4) Centrifuge directly at 4°C, 300 g for 5 min. The cells and precipitate are at the bottom of the collection tube. Discard the supernatant and add 0.25 mL of ice-cold lysis buffer to resuspend the precipitate.

[0218] (5) Take another new centrifuge tube and add 0.25 mL of medium salt buffer to absorb the resuspension in the previous step. Carefully add it to the centrifuge tube along the tube wall and place it on top of the medium salt buffer. Centrifuge at 700 g for 5 min at 4°C. The cell nuclei precipitate will settle to the bottom.

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

[0220] (7) Remove the supernatant completely and add 400 μL 0.2 MH 2 SO 4 Resuspend nuclei in medium and shake at 4°C for 60 min

[0221] (8) Centrifuge the sample at 16,000 g for 10 min at 4°C to remove nuclear debris, and transfer the supernatant containing histones to a clean 1.5 mL Eppendorf tube (aspirate 400 μL);

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

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

[0224] (11) Centrifuge at 16,000 g for 10 min at 4 °C, remove the supernatant with a pipette, and wash the histones with pre-cooled acetone;

[0225] (12) Centrifuge at 16000 g for 5 min at 4 °C. Repeat the above two steps, remove the supernatant and dry at room temperature for 10 min.

[0226] (13) Volatilize the acetone to obtain purer histones;

[0227] When the protein adheres to the tube wall in a solid form, add 40 μL 50 mM NH 4 HCO 3 After washing, centrifugation was performed at 16000 g for 10 min at 4°C.

[0228] Solution 2: Hypotonic and hypertonic buffers

[0229] (1) Homogenize with 1 mL of hypotonic buffer for 60 s and centrifuge at 300 g for 5 min at 4°C to precipitate.

[0230] (2) Add 1 mL of hypotonic buffer to the homogenized sample and shake at 4°C for 10 min;

[0231] (3) Centrifuge at 1500 g for 5 min at 4°C, resuspend the pellet in 1 mL of hypertonic buffer, and shake at 4°C for 10 min;

[0232] (4) Centrifuge at 1500 g for 5 min at 4°C to precipitate the chromatin and resuspend in 400 μL of 0.4 MH 2 SO 4 , shake at 4°C for 30 min;

[0233] (5) Centrifuge at 16000 g for 10 min at 4°C to precipitate the sample, add 132 μL of trichloroacetic acid to the supernatant, and incubate on ice for 30 min to precipitate histones;

[0234] (6) Centrifuge at 16,000 g for 10 min at 4°C to precipitate histones, wash twice with 500 μL of pre-cooled acetone, and air-dry at room temperature;

[0235] Solution 3: High-purity cell nucleus isolation and extraction kit

[0236] (1) Resuspend tissue cells in 1 mL of pre-chilled lysis buffer;

[0237] (2) 1 mL of 1.8 M sucrose buffer on ice: Add ice-cold to each lysate sample; mix by gently pipetting and inverting the tube;

[0238] (3) Prepare 1 mL of 1.8 M sucrose buffer for each sample. Place a clean ultracentrifuge tube on ice and add an ice-cold ice box to the bottom.

[0239] (4) Carefully and slowly add 1 mL of 1.8 M sucrose buffer onto the lysate solution from step (1), avoiding disturbing the sucrose cushion.

[0240] (5) Carefully place the ultracentrifuge tube into the precooled bucket of an ultracentrifuge and centrifuge at 30,000 g for 45 min at 4°C.

[0241] (6) Carefully and completely discard the supernatant (cytoplasm and cell debris) and the clear sucrose cushion without disturbing the purified nuclear pellet below;

[0242] (7) Resuspend in 400 μL of 0.4 MH 2 SO 4 , shake at 4°C for 30 min;

[0243] (8) Centrifuge at 16,000 g for 10 min at 4°C to precipitate the sample. Add 132 μL of trichloroacetic acid to the supernatant and incubate on ice for 30 min to precipitate histones.

[0244] (9) Centrifuge at 16,000 g for 10 min at 4°C to precipitate histones, wash twice with 500 μL of pre-cooled acetone, and air-dry at room temperature;

[0245] Solution 4: Histone Extraction Kit

[0246] (1) Preparation of extract: Add 2 μL of reagent G to every 1 mL of reagent A and 10 μL of reagent C, mix well and place on ice for later use;

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

[0248] (3) Centrifuge at 800 g for 10 min at 4°C and discard the supernatant;

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

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

[0251] (6) Centrifuge at 10,000 rpm at 4°C for 5 min and collect the supernatant;

[0252] (7) Add 130 μL of reagent D to the supernatant and mix thoroughly. Incubate on ice for 60 min.

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

[0254] (9) 500 μL of reagent E was precipitated twice, centrifuged at 14000 rpm, 4°C for 5 min;

[0255] 1. Sample

[0256] The samples were: liver, spleen, lung and kidney tissues of ICR mice (male, 6 weeks) treated with 10 mg / kg NH2 for 0.5 h.

[0257] 2. Detection

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

[0259] 3. Results and Conclusions

[0260] Since all four extraction schemes detected γ-H2AX, the results based on the response intensity of H2AX were compared, e.g. Figure 4 As shown, although Scheme 4 has the strongest H2AX response intensity, the sample volume to be processed is small and the cost is very high, while Scheme 2 processes a large sample volume and has a lower cost. Therefore, Scheme 2 hypotonic buffer and hypertonic buffer are temporarily selected for subsequent research.

[0261] Example 4: Study on desalination method

[0262] 1. Solution

[0263] Study on three desalination methods

[0264] Scheme 1: GL-Tip-GC (applicable to hydrophilic peptides, referred to as GC)

[0265] (1) Rinse with 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile by centrifugation at 3000 g for 2 min;

[0266] (2) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile was centrifuged at 3000 g for 2 min to balance;

[0267] (3) Discard the waste liquid and add the sample;

[0268] (4) Wash with 20 μL of 0.1% trifluoroacetic acid and 20% acetonitrile by centrifugation at 3000 g for 2 min;

[0269] (5) Elution was performed by centrifugation at 3000 g for 2 min with 20 μL of 0.1% trifluoroacetic acid and 80% acetonitrile.

[0270] Scheme 2: GL-Tip-SDB (applicable to hydrophobic peptides, referred to as SDB)

[0271] (1) Rinse with 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile by centrifugation at 3000 g for 2 min;

[0272] (2) 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile was centrifuged at 3000 g for 2 min to balance;

[0273] (3) Discard the waste liquid and add the sample;

[0274] (4) Wash with 20 μL of 0.1% trifluoroacetic acid and 20% acetonitrile by centrifugation at 3000 g for 2 min;

[0275] (5) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile was centrifuged at 3000 g for 2 min to elute;

[0276] Solution 3: Homemade C18 membrane desalting column (referred to as C18)

[0277] (1) Rinse with 20 μL of 0.1% formic acid 80% acetonitrile by centrifugation at 3000 g for 2 min;

[0278] (2) 20 μL of 0.1% formic acid 20% acetonitrile was centrifuged at 3000 g for 2 min to balance;

[0279] (3) Discard the waste liquid and add the sample;

[0280] (4) Wash with 20 μL of 0.1% formic acid and 20% acetonitrile by centrifugation at 3000 g for 2 min;

[0281] (5) 20 μL of 0.1% formic acid 80% acetonitrile was centrifuged at 3000 g for 2 min to elute;

[0282] 2. Sample

[0283] ICR mice (male, 6 weeks) were treated with 10 mg / kg NH2 and then sacrificed by cervical dislocation 30 minutes later, and the liver, spleen, lung and kidney tissues were obtained.

[0284] 3. Detection

[0285] Except that desalination is carried out according to "1. Scheme" in this embodiment, the remaining steps are carried out according to Example 1.

[0286] 4. Results and Conclusions

[0287] The recoveries of γ-H2AX and H2AX by the three desalting methods are shown in Table 4 . GL-Tip-GC had the best recovery rate and was suitable for LC-MS / MS quantitative analysis of γ-H2AX and H2AX in complex tissue samples.

[0288] Table 4. Recovery rates of three desalting columns

[0289]

[0290] Example 5: Enrichment Method Study

[0291] 1. Solution

[0292] Two enrichment schemes were studied

[0293] Solution 1: Titansphere phos-TiO (TIO for short)

[0294] (1) Activation: Use 20 μL buffer A (0.5% trifluoroacetic acid in 80% acetonitrile) and centrifuge at 3000 g for 2 min;

[0295] (2) Balance: Use 20 μL of buffer B (0.5% trifluoroacetic acid in 80% acetonitrile + 300 mg / mL lactic acid), centrifuge at 3000 g for 2 min. Discard 40 μL of the flow-through;

[0296] (3) Adsorption: add 30 μL sample and mix with 100 μL buffer B (mix well) and extract at 1000 g for 10 min (repeat once);

[0297] (4) Washing: add 20 μL buffer B, centrifuge at 3000 g for 2 min, add 20 μL buffer A, centrifuge at 3000 g for 2 min, and discard the flow-through (twice);

[0298] (5) Elution: centrifuge at 1000 g for 5 min using 50 μL of eluent C (5% ammonium hydroxide + 5% pyrrolidine) (once each);

[0299] (6) Dry the obtained phosphopeptide in a vacuum centrifuge and store at -80°C

[0300] Solution 2: polyMAC Phosphopeptide Enrichment Kit (polyMAC for short)

[0301] (1) Add 200 μL of buffer to the dried sample and resuspend it;

[0302] (2) After fully vortexing the PolyMAC peptide enrichment magnetic beads (10-20 seconds), take 50 μL into a 1.5 mL centrifuge tube, centrifuge for 2-3 seconds, and then remove the upper storage solution;

[0303] (3) Add the resuspended sample to a centrifuge tube containing peptide-enriched magnetic beads, and then place it in a mixer at 26°C and 1200 rpm for 25 min of vigorous shaking;

[0304] (4) Add the sample to the PolyMAC enrichment column, centrifuge at 20g for 2 min, and then centrifuge at 100g for 1 min, ensuring that the liquid flows out of the tip into the centrifuge tube;

[0305] (5) Add 200 μL buffer to the PolyMAC Tips, centrifuge at 20 g for 2 min, and then centrifuge at 100 g for 1 min, ensuring that the liquid flows out of the Tips into the centrifuge tube (to collect the waste liquid);

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

[0307] (7) Add 200 μL of cleaning solution II, centrifuge at 20 g for 2 min, and then centrifuge at 100 g for 1 min (collect the waste liquid);

[0308] (8) Add 50 μL of elution buffer to a new centrifuge tube, centrifuge at 20 g for 2 min, repeat once, and centrifuge at 100 g for 1 min to collect γ-H2AX;

[0309] (9) The collected γ-H2AX and H2AX were 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] A mixed standard solution of 2.5 ng / mL γ-H2AX and 50 ng / mL H2AX was prepared (n=3), and after treatment, 50 ng / mL γ-H2AX internal standard and 50 ng / mL H2AX internal standard solutions were added respectively.

[0312] 3. Detection

[0313] There is no need to perform sample extraction, enzymatic hydrolysis, or desalting. Enrichment is performed according to "1. Scheme" of this embodiment, and the remaining steps are performed according to Example 1.

[0314] 4. Results and Conclusions

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

[0316] Conclusion: The polyMAC enrichment method of scheme 2 is superior to the TIO enrichment method of scheme 1.

[0317] Example 6: Comparison of Chromatographic Columns

[0318] 1. Solution

[0319] Comparison of three columns

[0320] Scheme 1: BioBasic 8HPLC4, 250 mm × 4.6 mm, 5 μm, referred to as Basic;

[0321] Solution 2: AdvanceBio HIC, 100mm×4.6mm, 3.5μm, referred to as Advance;

[0322] Solution 3: ACQUITY UPLC BEH C18, 100 mm × 2.1 mm, 1.7 μm, referred to as BEH;

[0323] 2. Sample processing

[0324] The 1 mg / mL γ-H2AX and H2AX reference solutions were diluted with ultrapure water to a concentration of 125 ng / mL.

[0325] 3. Detection

[0326] The chromatographic column is prepared according to "1. Scheme" of this embodiment, the sample preparation is prepared according to "2. Treatment of the sample", and the remaining steps are carried out according to Example 1.

[0327] 4. Results and Conclusions

[0328] Results: The samples to be tested were injected into the three chromatographic columns of schemes 1, 2 and 3 respectively.

[0329] The statistical chart comparing the ratio of the peak area of ​​H2AX to the peak area of ​​the internal standard of the three chromatographic columns is as follows: Figure 6 As shown in A, there was no significant difference between BEH and Advance, and Advance was significantly greater than Basic (P < 0.05);

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

[0331] 5. Conclusion

[0332] Conclusion: BEH column is more suitable for the application of "LC-MS / MS quantitative analysis method of γ-H2AX in complex samples".

[0333] Example 7: Screening of methods for constructing mouse tissues rich in γ-H2AX

[0334] 1. Solution

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

[0336] Protocol 1: Tissue samples were obtained 4 h after mice were exposed to ionizing radiation IR (4 GY);

[0337] Scheme 2: Take tissue samples 4 hours after intraperitoneal injection of nitrogen mustard (NH2, 10 mg / Kg)

[0338] Scheme 3: Take tissue samples 30 days after intraperitoneal injection of nitrogen mustard (NH2, 1 mg / kg / week);

[0339] 2. Detection

[0340] The mice were killed by cervical dislocation, and the spleen tissues were removed and frozen, and then tested according to the steps of Example 1.

[0341] 3. Results

[0342] like Figure 7 As shown, after LC-MS / MS detection, the monitored target ion pairs all showed good signals, and γ-H2AX was successfully detected without interference. The results of γ-H2AX response intensity (genotoxic effect) were compared:

[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] The method of intraperitoneal injection of nitrogen mustard (10 mg / kg, 4 hours) was chosen to construct mouse tissues rich in γ-H2AX.

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

[0347] 1. Solution

[0348] The species of nitrogen mustard used to construct mouse tissues rich in γ-H2AX were screened, and all mice were ICR mice (male, 6 weeks old).

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

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

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

[0352] 2. Detection

[0353] The mice were killed by cervical dislocation, and the spleen tissues were removed and frozen, and then tested according to the steps of Example 1.

[0354] 3. Results

[0355] like Figure 8 As shown, γ-H2AX can be successfully and stably detected by LC-MS / MS, and the results of γ-H2AX response intensity (genotoxic effect) are compared:

[0356] NH2>NH1>NH34

[0357] 4. Conclusion

[0358] After intraperitoneal injection of nitrogen mustard NH2, the intensity of γ-H2AX (genotoxic effect) in the spleen tissue of mice was greater.

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

[0360] 1. Sample collection

[0361] Blood sample from a 61-year-old female patient with clinical coronary heart disease and no smoking history

[0362] 2. Sample processing

[0363] Blood treatment: 5 mL of fresh blood was diluted with PBS at a ratio of 1:1. 3 mL of lymphocyte separation solution was added to the PBMC centrifuge tube. The diluted blood was spread on the separation solution to make the interface between the two liquid surfaces clear. Centrifuge at 2200 rpm for 25 min and carefully aspirate the PBMC layer.

[0364] Extraction: according to "Scheme 1 (Cell Nuclear Extraction Kit)" in Example 3;

[0365] Desalination: according to "Scheme 3, homemade C18" in Example 4;

[0366] 3. Detection

[0367] Chromatographic conditions

[0368] The HPLC column was ACQUITY UPLC BEH C18 (100 mm × 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: electrospray ionization (ESI), positive ion mode; ion source temperature: 500°C; nebulizer gas (Gas1) pressure: 276 kPa; auxiliary heating drying gas (Gas2) pressure: 414 kPa; collision cell exit potential (CEX): 15 eV, entrance potential (EP): 10 eV. The MRM method parameters are shown in Table 5.

[0371] Table 5. MRM method parameters for H2AX peptide and H3 peptide standards

[0372]

[0373]

[0374] The remaining steps are carried out according to Example 1.

[0375] 4. Results and Conclusions

[0376] like Fig. 9 As shown, γ-H2AX was not detected.

[0377] Example 10: Detection of γ-H2AX in complex samples using the 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 obtained;

[0380] 2. Sample processing

[0381] 2.1 Extraction

[0382] (1) Homogenize with 1 mL of hypotonic buffer for 60 s and centrifuge at 300 g for 5 min at 4°C to precipitate.

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

[0384] (3) Centrifuge at 1500 g for 5 min at 4°C, resuspend the pellet in 1 mL of hypertonic buffer, and shake at 4°C for 10 min;

[0385] (4) Centrifuge at 1500 g for 5 min at 4°C to precipitate the chromatin and resuspend in 400 μL of 0.4 MH 2 SO 4, shake at 4°C for 30 min;

[0386] (5) Centrifuge at 16000 g for 10 min at 4°C to precipitate the sample, add 132 μL of trichloroacetic acid to the supernatant, and incubate on ice for 30 min to precipitate histones;

[0387] (6) Centrifuge at 16,000 g for 10 min at 4°C to precipitate histones, wash twice with 500 μL of pre-cooled acetone, and air-dry at room temperature;

[0388] 2.2 Enzymatic hydrolysis

[0389] (1) Add sequencing-grade trypsin at a ratio of 1:50 overnight;

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

[0391] 2.3 Desalination

[0392] (1) 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile, centrifuge at 3000 g for 2 min to rinse;

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

[0394] (3) Discard the waste liquid and add the sample;

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

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

[0397] (6) Reconstitute by rotary evaporation and LC-MS / MS for analysis;

[0398] 3. Detection

[0399] Except that the processing of the sample to be tested is performed according to "2. Processing of the sample to be tested" of this embodiment, the remaining steps are performed according to Embodiment 1.

[0400] 4. Results and Conclusions

[0401] like Fig.10 As shown, according to the Japanese literature method, only the ion pair of 482 / 182 was detected in the spleen and lung tissue samples, and the ion pair of 482 / 433 could not be detected, and it was impossible to confirm that the measured peak was γ-H2AX.

[0402] Example 11, Comparative Example 3

[0403] 1. Solution

[0404] Extraction: according to "Scheme 1 (Cell Nuclear Extraction Kit)" in Example 4;

[0405] Desalting: according to "Scheme 3, homemade C18 membrane desalting column" in Example 5;

[0406] 2. Sample

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

[0408] 3. Detection

[0409] Except that the extraction and desalting were carried out according to "1. Scheme" of this embodiment, the remaining steps were carried out according to Example 1.

[0410] 4. Results

[0411] like Fig.11 As shown, γ-H2AX was not detected.

[0412] 5. Conclusion

[0413] The extraction and desalting scheme adopted in this embodiment is not applicable to the "LC-MS / MS quantitative analysis method for γ-H2AX in a complex sample" of the present application.

[0414] Example 12, Comparative Example 4

[0415] 1. Solution

[0416] Extraction: according to "Scheme 1 (Cell Nuclear Extraction Kit)" in Example 4;

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

[0418] This is non-enrichment treatment

[0419] 2. Sample

[0420] ICR mice (male, 6 weeks) were intraperitoneally injected with MMC 2 mg / mkg, and spleen tissues were obtained 4 h after exposure;

[0421] 3. Detection

[0422] Except that the extraction, desalting and enrichment were carried out according to "1. Scheme" of this embodiment, the remaining steps were carried out according to Example 1.

[0423] 4. Results

[0424] like Fig.12 As shown, only the ion pair of m / z482 / 182 was detected, which means that it is not possible to detect all γ-H2AX and H2AX in the complex sample, and the detection result is lower than the true value.

[0425] 5. Conclusion

[0426] The extraction and desalting scheme adopted in this embodiment is not applicable to the "LC-MS / MS quantitative analysis method for γ-H2AX in a complex sample" of the present application.

[0427] Example 13, Comparative Example 5

[0428] 1. Solution

[0429] Extraction: according to "Scheme 1 (Cell Nuclear Extraction Kit)" in Example 4;

[0430] Desalting: according to "Scheme 1, GL-Tip-GC" in Example 5;

[0431] Enrichment: According to "Scheme 1, Titansphere phos-TiO phosphopeptide enrichment" in Example 6.

[0432] 2. Sample

[0433] Spleen tissue of ICR mice (male, 6 weeks) 4 h after intraperitoneal injection of MMC 2 mg / kg;

[0434] 3. Detection

[0435] Except that the extraction, desalting and enrichment were carried out according to "1. Scheme" of this embodiment, the remaining steps were carried out according to Example 1.

[0436] 4. Results

[0437] like Fig.13 As shown, γ-H2AX can be detected but cannot be stably and reproducibly detected.

[0438] 5. Conclusion

[0439] The extraction, desalting and enrichment schemes adopted in this embodiment are not applicable to the "LC-MS / MS quantitative analysis method for γ-H2AX in a complex sample" of the present application.

[0440] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A LC-MS / MS quantitative analysis method for γ-H2AX in complex tissue samples, the method comprising the following steps: (1) Prepare standard curve working solution H2AX standard curve working solution: Take the peptide ASQASQEY, i.e. the H2AX reference substance, and prepare a 1 mg / mL H2AX standard curve working solution with sterile ultrapure water; γ-H2AX standard curve working solution: Take the peptide ASQApSQEY, i.e., the γ-H2AX reference substance, and prepare a 1 mg / mL γ-H2AX standard curve working solution with sterile ultrapure water; (2) Preparation of internal standard working solution H2AX internal standard working solution: Take peptide [ 13 C3, 15 N]ASQASQEY is the H2AX internal standard, and a 1 mg / mL H2AX internal standard working solution is prepared with sterile ultrapure water; γ-H2AX internal standard working solution: Take peptide [ 13 C3, 15 N]ASQApSQEY is the γ-H2AX internal standard, and a 1 mg / mL γ-H2AX internal standard working solution is prepared with sterile ultrapure water; (3) Preparation of standard curve samples H2AX standard curve samples: ultrapure water and H2AX standard curve working solution were mixed in an appropriate ratio to prepare H2AX standard curve samples. The concentrations of H2AX in the H2AX standard curve samples were 0.5, 5, 50, 100, 150, 200, 100, and 250 ng / mL, respectively; 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. (4) Preparation of standard curve samples 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; γ-H2AX standard curve samples containing internal standard: γ-H2AX standard curve samples were added to γ-H2AX internal standard working solution to prepare a solution with a γ-H2AX internal standard concentration of 50 ng / mL; (5) Sample processing ① Extraction A. 100 mg of tissue was added to 1 mL of 1× PBS and homogenized for 60 s, and then centrifuged at 300 g for 5 min at 4°C to precipitate. B. Add 1 mL of hypotonic buffer and shake at 4°C for 10 min. C. Centrifuge at 1500 g for 5 min at 4°C, resuspend the pellet in 1 mL of hypertonic buffer, and shake at 4°C for 10 min; D. Centrifuge at 1500 g for 5 min at 4°C to precipitate the chromatin, resuspend in 400 μL 0.4 M H2SO4, and shake at 4°C for 30 min; E. Centrifuge at 16000 g for 10 min at 4°C to precipitate the sample, add 132 μL of trichloroacetic acid to the supernatant, and place on ice for 30 min to precipitate histones; F. Centrifuge at 16000 g for 10 min at 4°C to precipitate histones, wash twice with 500 μL pre-cooled acetone, and air-dry at room temperature; ② Enzymatic hydrolysis A. Add sequencing-grade trypsin at a ratio of 1:50 overnight; B. The next day, add 2 μL of 20% acetic acid to terminate; ③Desalination Desalting is done using a GL-Tip-GC desalting column. The steps are as follows: A, rinse with 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile and centrifuge at 3000 g for 2 min; B, 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile centrifuged at 3000 g for 2 min for equilibrium; C. Discard the waste liquid and add the sample to the GL-Tip-GC desalting column; D, wash with 20 μL of 0.1% trifluoroacetic acid 20% acetonitrile by centrifugation at 3000 g for 2 min; E, 20 μL of 0.1% trifluoroacetic acid 80% acetonitrile centrifuged at 3000 g for 2 min to elute; F. The peptide samples obtained by elution are concentrated to dryness by vacuum centrifugation; ④ Enrichment The polyMAC phosphopeptide enrichment kit was used for enrichment, and the steps were as follows: A. Add 200 μL of buffer to the dried sample and resuspend it; B. After fully vortexing the PolyMAC peptide-enriched magnetic beads for 10-20 seconds, take 50 μL into a 1.5 mL centrifuge tube, centrifuge for 2-3 seconds, and then remove the upper storage solution; C. Add the resuspended sample to the centrifuge tube containing polyMAC peptide enrichment magnetic beads, and then place it in a mixer at 26°C and 1200 rpm, shaking vigorously for 25 minutes; D. Add the sample to the PolyMAC Tips and centrifuge at 20g for 2 minutes. If there is residual liquid, centrifuge at 100g for 1 minute to ensure that the liquid flows out of the Tips into the centrifuge tube. E. Use a new centrifuge tube and add 200 μL of buffer I to the PolyMAC Tips. Centrifuge at 20 g for 2 min. If there is residual liquid, centrifuge at 100 g for 1 min to ensure that the liquid flows out of the Tips into the centrifuge tube. F. Add 200 μL of cleaning solution I and centrifuge at 20 g for 2 min. If there is residual liquid, centrifuge at 100 g for 1 min. G. Add 200 μL of cleaning solution II and centrifuge at 20 g for 2 min. If there is residual liquid, centrifuge at 100 g for 1 min and collect H2AX in E, F, and G for detection; H. Add 50 μL of elution buffer to a new centrifuge tube, centrifuge at 20 g for 2 min, repeat the centrifugation at 100 g for 1 min, and collect γ-H2AX; I. The collected γ-H2AX was freeze-dried and stored at -80°C or reconstituted with 0.1% formic acid for LC-MS / MS detection; (6) LC-MS / MS analysis The chromatographic conditions for the LC-MS / MS analysis were as follows: Column: ACQUITY UPLC BEH C18, 100 mm × 2.1 mm, 1.7 μm Mobile phase A: 0.1% formic acid solution; Mobile phase B: acetonitrile; Flow rate: 0.3 mL / min; Column temperature: 40°C; Injection volume: 10 μL; The elution method is gradient elution, and the gradient elution procedure is as follows: From 0 to 6.0 min, phase A decreased from 99% to 50%, and phase B increased from 1% to 50%; 6.0-6.1min, phase A decreased from 50% to 45%, and phase B increased from 50% to 55%; 6.1-8.0min, phase A decreased from 45% to 5%, and phase B increased from 55% to 95%; 8.0-8.1min, phase A is 5%, phase B is 95%; 8.1-10.0min, phase A increased from 5% to 99%, and phase B decreased from 95% to 1%; Mass spectrometry conditions: multiple reaction monitoring (MRM) mode, electrospray ionization (ESI), positive ion mode, Mass spectrometry parameters: Ion source temperature: 500°C; Nebulizer gas pressure: 35 kPa; Auxiliary heating drying gas pressure: 55 kPa; Collision chamber exit voltage: 21 eV; Inlet voltage: 10 eV; (7) Quantitative analysis of H2AX and γ-H2AX content in the sample to be tested based on the internal standard-standard curve method The ratio of the analyte to the internal standard concentration is used as the horizontal coordinate, and the peak area ratio is used as the vertical coordinate to perform linear regression 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 H2AX content in the sample to be tested; The ratio of the analyte to the internal standard concentration is used as the horizontal coordinate, and the peak area ratio is used as the vertical coordinate to perform linear regression to obtain the internal standard-γ-H2AX standard curve; the peak area ratio of the chromatographic peak in the sample to be tested is substituted into the internal standard-γ-H2AX standard curve to calculate the content of γ-H2AX in the sample to be tested; (8) Evaluate DNA damage values ​​of phosphorylation markers in tissue samples Rγ / T Value = γ-H2AX / H2AX × 100% 2. The method according to claim 1, characterized in that During the LC-MS / MS analysis, the parent ion m / z of the H2AX was 442, and the daughter ions m / z were 182 and 136; the parent ion m / z of the γ-H2AX was 482, and the daughter ions m / z were 433 and 182.

3. The method according to claim 1, characterized in that The linear ranges of H2AX and γ-H2AX in the method are both 1-250 ng / mL.

4. The method according to claim 1, characterized in that The minimum quantification limits of H2AX and γ-H2AX of the method were both 1 ng / mL.

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