CN117907491B - Double-derivatization technology-based abasic site LC-MS/MS analysis method - Google Patents

Double-derivatization technology-based abasic site LC-MS/MS analysis method Download PDF

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CN117907491B
CN117907491B CN202410275409.8A CN202410275409A CN117907491B CN 117907491 B CN117907491 B CN 117907491B CN 202410275409 A CN202410275409 A CN 202410275409A CN 117907491 B CN117907491 B CN 117907491B
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nbha
pmoa
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CN117907491A (en
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谢剑炜
李治
袁舒妍
武海江
徐华
徐斌
陈佳
吴剑峰
马波
刘玉龙
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Academy of Military Medical Sciences AMMS of PLA
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

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Abstract

The invention discloses an abasic site LC-MS/MS analysis method based on a double-derivatization technology, which 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 tested; (6) Carrying out 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 concentrations of the AP-NBHA, the AP-PMOA and the dG in the sample to be detected according to an internal standard-standard curve method; (8) calculating the content of abasic sites. The invention solves the technical problems that the AP site detection in the biological sample can be spontaneously generated, false positive is easy to occur, and the accurate quantification of the AP site in the biological sample can not be realized only by a single derivatization liquid method.

Description

Double-derivatization technology-based abasic site LC-MS/MS analysis method
Technical Field
The invention relates to a method for quantitatively detecting abasic sites in human cells by liquid chromatography-tandem mass spectrometry based on a double-derivatization technology, and belongs to the technical field of medicine analysis.
Background
In daily life, the cell DNA is inevitably damaged by contacting with genotoxic substances, and if unrepaired or wrongly repaired cells inhibit transcription and translation, the cell DNA has potential threat of inducing serious diseases such as cancers, malformations and the like. In the face of the vast amount of genotoxic compounds, a common DNA damage evaluation method is necessary to develop, and the method has important practical significance in the aspects of establishing a genotoxic substance early detection, evaluation and prevention system, controlling the quality of genotoxic impurities of medicines, screening the toxicity of harmful compounds and environmental pollutants in foods, monitoring long-term low-dose occupational exposure crowd and the like.
The formation of abasic (Apurinic/APYRIMIDINIC SITES, AP) sites is one of the most widespread manifestations of DNA damage results, so quantitative analysis thereof can indicate and evaluate a variety of DNA damage types; when nucleotides spontaneously hydrolyze or are damaged by genotoxic compounds, the AP site is generated through a base excision repair pathway, and the AP site is used as a DNA damage marker for evaluating DNA damage in animals and people caused by genotoxic substances.
At present, the detection methods of the AP locus mainly comprise four methods, namely a 32P labeling method, an Elisa-like analysis method, a fluorescence analysis method and a liquid method, wherein the 32P labeling method can quantify the AP locus in DNA at the fmol level, but the method is time-consuming and labor-consuming and involves the problems of radioactive pollution and the like; the Elisa-like method is easy to perform, but has many drawbacks due to lack of specificity in antibody cross-reactions; the fluorescence analysis method can detect the AP locus or other aldehyde group-containing compounds, but besides considering proper fluorescent labels, special reaction conditions are considered to evaluate and optimize the reaction efficiency of fluorescent marker molecules and target objects; the liquid chromatography-mass spectrometry technology is becoming the mainstream development method for AP site detection due to high sensitivity and high specificity, but AP sites have the characteristic of spontaneous generation, and accurate quantification [1] of the AP sites of biological samples cannot be realized by a single liquid chromatography-mass spectrometry method.
Identification and application research [2] on novel abasic site crosslinked adducts in cells reported: the third chapter is the identification of adducts in cells and the construction of quantitative detection methods for AP-base adducts. The effect of two different DNA hydrolysis methods on DNA adduct analysis and identification was compared using mass spectrometry techniques. The method is high-efficiency and stable, is suitable for mass spectrum quantitative analysis of DNA adducts with known structures, can generate products in various forms such as bases, nucleosides and the like, is suitable for detection of adducts with new structures and unknown structures, and is determined and selected for screening and identifying the AP-base adducts by an enzymolysis method (scheme III). The cell samples after enzymolysis are screened by a high resolution mass spectrometry screening technology (LC-HRMS), and three adducts which are AP-dA, AP-dG and AP-dC crosslinked adducts respectively are formed by the AP site and deoxyribonucleobases in the cells for the first time. In a multi-reaction monitoring (MRM) mode, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for detecting the AP-base adduct in the cells with high sensitivity and high specificity is established, and the method comprises the following steps of examining the methodology content including selectivity, linear range, matrix effect, accuracy and precision, and the result shows that the established method meets the detection requirement. The detection object of the document is an adduct formed by crosslinking an abasic site with a DNA base, and because the reactive sites of deoxyguanosine in one of the synthetic raw materials are more in the synthesis process of the crosslinked adduct, the method is not a method for directly detecting and directly reacting the objective condition of the abasic site in a biological sample by derivatizing the deoxyguanosine.
"Determination of Apurinic/Apyrimidinic Lesions in DNA with High-Performance Liquid Chromatography and Tandem Mass Spectrometry"[3] Reported are: AP detection is currently accomplished by an immunoassay method using aldehyde-reactive probes; however, these methods lack the specificity required to specifically recognize the AP site. Thus, an accurate method for detecting AP sites based on mass spectrometry was developed, which is derived from AP DNA that had been pre-labeled with O-4-nitrobenzyl hydroxylamine (NBHA). The problem with this reference to the detection of AP-NBHA using the "singly derivatised method" is that the method of removing excess derivatised reagent is only three washes with 70% ethanol, which has two disadvantages: firstly, whether excessive derivatization reagent is completely removed or not cannot be judged, and secondly, abasic sites can be generated in the washing process, so that false positives exist in experimental results.
The detection method of DNA abasic site and the progress of biological research thereof [4] report that: however, when the quantitative method for the liquid quality of the AP site is constructed, besides the derivatization efficiency of a derivatization reagent, an important influence factor to be considered is the instability of the self structure of the AP site, and the quantitative result is easy to deviate because the AP site can be formed or lost in the DNA cleavage, digestion or derivatization process, so that the chemical stability of the AP site needs to be focused when the AP site is measured at present. The following sample processing conditions are generally considered to avoid false positive or false negative results as much as possible: ① The conditions are as mild as possible, such as the enzymolysis temperature cannot be too high, the sample is gently oscillated when being mixed uniformly, and the proper pH value is selected; ② The selection of the additive during enzymolysis, such as adding the deferoxamine mesylate, can effectively prevent the sample from being oxidized; ③ The timing of derivatization, e.g., the earlier the derivatizing agent is added, the more the AP sites are monitored for background levels in the organism. This document proposes "the chemical stability is important for the measurement of the AP site", but does not give any specific proposal. In addition, the article mentions that the methanesulfonic acid deferoxamine can effectively prevent the sample from being oxidized, and the methanesulfonic acid deferoxamine is an antioxidant, so that the generation of abasic sites can be reduced as much as possible, but the problem that false positives are caused by the generation of abasic sites in the subsequent enzymolysis and other processes cannot be solved; the article also mentions "the earlier the derivatizing agent is added, the more the AP site is able to monitor the background level of the organism. The problem that researchers are aware of when detecting abasic sites by the single derivatization method reported at present is that the latest literature chooses derivatization immediately after proteolysis, but cannot solve the problem that the number of abasic sites caused in the subsequent treatment process is accurately given at the same time, and cannot be monitored.
U.S. patent application Ser. No. 16/935,447, METHODS FOR DETERMINING BASE LOCATIONS IN A POLYNUCLEOTIDE, discloses a method of polynucleotide sequencing that detects the position of a selected nucleobase with greater accuracy. The method can be used to determine the location and nature of modified bases (i.e., non-canonical bases) in a polynucleotide, or to improve the sequencing accuracy of a DNA sequencing "problem" region (e.g., such as a synchronization polymer, GC-rich region, etc.). The exemption sequencing method is nanopore sequencing. Nanopore sequencing is used to generate unique signals at points in the polynucleotide sequence where base sites (AP sites or no purine or pyrimidine sites) are present. As part of this method, a base site is created specifically enzymatically, leaving an AP site in its place, using a DNA glycosidase that recognizes a predetermined nucleobase species and cleaves the N-glycosidic bond releasing only that base. The method is a 'polynucleotide sequencing technology', which is only used for determining the position and the property of a modified base in a polynucleotide, wherein the sequencing technology is verified by using a special base modification of an abasic site as an example, and the abasic site is not quantitatively detected.
Reference is made to:
[1]Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).
[2] Wu Haijiang identification of novel abasic site crosslinked adducts in cells and applied research [ D ]. Proc. Military sciences, 2023.DOI:10.27193/d.cnki. Gjsky.2022.000074.
[3]Roberts K P , Sobrino J A , Payton J ,et al.Determination of Apurinic/Apyrimidinic Lesions in DNA with High-Performance Liquid Chromatography and Tandem Mass Spectrometry[J].Chemical Research in Toxicology, 2006, 19(2):300.DOI:10.1021/tx0502589.
[4] Wu Haijiang, zhang Yajiao, yuan Shuyan, et al, methods for detecting DNA abasic sites and their biological research advances [ J ]. Analytical test report, 2022 (009): 041.
Disclosure of Invention
Problems to be solved by the invention:
Aiming at the defects in the prior art, namely spontaneous generation and easy occurrence of false positive in AP site detection, accurate quantification of the AP site in a biological sample cannot be realized only by a single derivatization liquid method, the application aims to establish a liquid mass spectrometry combined analysis method for quantitatively detecting the abasic site in the biological sample based on a double derivatization technology so as to solve the problem.
In order to achieve the above purpose, the specific technical scheme of the invention is as follows:
In the first aspect, an analysis method for quantitatively detecting abasic sites in biological samples based on double-derivatization technology by liquid-phase mass spectrometry is established
The method comprises the following steps:
1. Preparing standard curve working solution
Preparing 1000ng/mL of AP-NBHA, AP-PMOA and dG standard curve working solutions respectively by using ultrapure water;
2. Preparing an internal standard working solution
Preparing an internal standard working solution containing 6-mdA-d3 of 1000ng/mL by using ultrapure water;
3. preparing standard curve sample
Mixing ultrapure water and an AP-NBHA standard curve working solution in a proper proportion to prepare an AP-NBHA standard curve sample, wherein the concentration of the AP-NBHA in the AP-NBHA standard curve sample is 0.01, 0.10, 0.20, 1.00 and 10.00ng/mL respectively;
mixing ultrapure water and an AP-PMOA standard curve working solution in a proper proportion to prepare an AP-PMOA standard curve sample, wherein the concentration of the AP-PMOA in the AP-PMOA standard curve sample is 0.01, 0.05, 0.10 and 1.00ng/mL respectively;
Mixing ultrapure water and dG standard curve working solution in a proper proportion to prepare dG standard curve samples, wherein the concentration of dG in the dG standard curve samples is 1.00, 5.00, 10.00 and 50.00ng/mL respectively;
4. Preparing standard curve sample containing internal standard
The internal standard working solution is respectively added into the AP-NBHA, the AP-PMOA and the dG standard curve samples to prepare a standard curve sample containing an internal standard, and the concentration of 6-mdA-d3 in the standard curve sample containing the internal standard is 0.10ng/mL;
5. sample processing to be tested
The method comprises the following steps: extracting DNA from HepG2 cells, performing primary derivatization (a derivatization reaction of deoxyribose ring-opening aldehyde), performing secondary derivatization, precipitating DNA, performing enzymolysis and preparing a sample solution to be detected;
6. sample solution to be tested and standard curve sample solution containing internal standard are subjected to LC-MS/MS analysis
7. Quantitatively analyzing the concentrations of AP-NBHA, AP-PMOA and dG in the sample to be tested according to an internal standard-standard curve method
8. Calculation of the content of abasic sites
A second aspect of methodological verification of the method established in the first aspect
The specificity, linear range, accuracy and precision and recovery rate of the detection of AP-NBHA, AP-PMOA and dG by the method established in the first aspect are verified.
Specialization: by adopting the method, the determination of the AP-NBHA, the AP-PMOA, the dG and the internal standard 6-mdA-d3 is not interfered;
linear range:
The linear range of the AP-NBHA is 0.02-100ng/mL, the lowest quantitative limit is 0.02ng/mL, the lowest detection limit is 0.01ng/mL,
The linear range of the AP-PMOA is 0.01-1ng/mL, the lowest quantitative limit is 0.01ng/mL, the lowest detection limit is 0.005ng/mL,
DG linear range is 1-100ng/mL, the lowest quantitative limit is 1ng/mL, and the lowest detection limit is 0.5ng/mL;
Accuracy and precision:
The accuracy and the variation coefficient of the AP-NBHA in the batch are respectively 100 to 105 percent and 1.9 to 5.8 percent; the accuracy and the variation coefficient between batches of the AP-NBHA are respectively 99 to 102 percent and 3.0 to 4.8 percent;
the accuracy and the variation coefficient of the AP-PMOA in the batch are 93 to 107 percent and 1.2 to 8.3 percent respectively; the accuracy and the variation coefficient between batches of the AP-PMOA are respectively 95 to 107 percent and 1.1 to 9.0 percent
The accuracy and the variation coefficient of dG in the batch are respectively 96-108 percent and 5.8-10.2 percent; the accuracy and the variation coefficient of dG are 93 to 105 percent and 3.8 to 8.5 percent respectively;
The method provided by the application has the advantages that the accuracy and precision between batches of detecting the abasic site in the HepG2 cell meet the requirements of Chinese pharmacopoeia of 2020 edition.
Recovery rate:
the average recovery rate of the AP-NBHA is 92.6 to 107.7 percent, and the relative standard deviation is 5.3 to 6.7 percent; meets the Chinese pharmacopoeia regulations of 2020 edition
Third aspect, the method established in the first aspect is verified by artificially increasing abasic sites
1. Verification experiment 1 after artificially adding abasic site
Artificially increasing abasic sites by heat treatment before enzymolysis, and as a result: the background content of abasic sites in HepG2 cells is low, the AP-NBHA is only 1.08-2.38 AP/10 7 nts, and after the abasic sites are artificially added by heating treatment before enzymolysis, the AP-PMOA reaches 2351-3405 AP/10 7 nts.
2. Verification experiment 2 after artificially increasing abasic site
The dealkalization site is artificially increased by prolonging the time of pre-cooling isopropanol precipitation of nucleic acid, and the result proves that the double-derivatization method can react with the generation of the dealkalization site in the enzymolysis process; the double derivatization method also more reflects the number of abasic sites in the cell background by comparison to the single derivatization method.
Compared with the prior art, the application has the beneficial effects that:
The invention provides a quantitative analysis method based on double derivatization technology and combining liquid phase mass spectrometry of abasic sites, which solves the technical problems that false positives are easy to occur in AP site detection in biological samples, and accurate quantification of the AP sites in the biological samples cannot be realized only by a single derivatization liquid quality method.
Drawings
FIG. 1, schematic flow diagrams of abasic sites and NBHA and PMOA derivatization mechanisms in the process of the invention and the process of the invention, wherein,
A is the abasic site and PMOA derivatization mechanism in the method of the invention,
B is the abasic site and NBHA derivatization mechanism in the method of the invention,
C is a flow diagram of the method of the invention;
FIG. 2, mass spectral responses of blank matrix +AP-NBHA and blank matrix +6-mdA-d3, wherein,
A is the mass spectral response of blank matrix + AP-NBHA,
B is the mass spectrum response of blank matrix +6-mdA-d 3;
FIG. 3, the method of the present invention detects the linear range of AP-NBHA;
FIG. 4, mass spectral response of blank matrix +AP-PMOA;
FIG. 5, the method of the present invention detects the linear range of AP-PMOA;
FIG. 6, mass spectral response of blank matrix +dG;
FIG. 7, the method of the present invention detects the linear range of dG;
FIG. 8, background level of abasic sites in HepG2 cells and artificially increased abasic site levels by heating prior to enzymatic hydrolysis, wherein,
A is the background content of abasic sites in HepG2 cells,
B is the content of abasic sites after artificially increasing abasic sites by heating before enzymolysis;
FIG. 9, background levels of abasic sites AP-NBHA in HepG2 cells measured by single and double derivatization methods, respectively, were artificially increased by extending the time of isopropanol precipitation of DNA prior to enzymatic hydrolysis.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
The experimental methods used in the examples below are conventional methods unless otherwise specified.
All materials, reagents, etc. in the examples described below are commercially available unless otherwise specified.
Experimental materials
1. Reference substance and internal standard
AP-NBHA: chinese name: (3S, 4R) -3,4, 5-trihydroxy valeraldehyde-O-4-nitro benzyl oxime, laboratory synthesis, purity > 92%, preservation: preserving at 4 ℃;
AP-PMOA: chinese name: (3S, 4R) -3,4, 5-trihydroxypentanal-O-pyridine-3-methyl oxime, laboratory synthesis, purity greater than 96%, preservation: preserving at 4 ℃;
dG: chinese name: deoxyguanosine, available from Ark Pharm company (U.S.), cat No.: 312693-72-4, purity: > 99%, save: preserving at 4 ℃;
6-mdA-d3: chinese name: deuterated 2' -deoxy-N-methyladenosine, internal standard, available from TRC company (canada), cat No.: 2002-35-9, purity: > 95%, save: preserving at 4 ℃;
2. Cells
Human hepatoma cells (HepG 2 cells), purchased from ATCC
3. Main reagent
Acetonitrile (chromatographic grade), the scientific and technological company of Baoling Wei (Beijing)
Formic acid (chromatographic grade), thermo company (united states)
Ethanol, national pharmaceutical group chemical reagent Co., ltd (Beijing)
NBHA Sigma Co., ltd (U.S.)
PMOA, sigma Co., ltd (U.S.)
Alkaline phosphatase (derived from Escherichia coli), engineering Co., ltd (Shanghai)
Phosphodiesterase I, biological engineering Co., ltd (Shanghai)
Nuclease P1, NEB Co., ltd (America)
4. Instrument for measuring and controlling the intensity of light
Instrument name Model number Manufacturer(s)
CO 2 incubator 3111 Thermo company (USA)
ACQUITY UPLC high performance liquid chromatograph ACQUITY UPLC Ⅰ-Class Waters company (USA)
TQ-XS triple quadrupole mass spectrometer Xevo TQ-XS Waters company (USA)
Inverted microscope Ckx41 Olympus Co (Japan)
Low-temperature centrifugal machine 3-18KS Sigma Co (USA)
Milli-Q purified water system Advantage A10 Miltiore (U.S.)
5. Commonly used buffer solution and reagent configuration
(1) Tris lysis buffer: 10mM Tris-base,0.32M sucrose, 5mM MgCl 2, 0.1mM deferoxamine mesylate, pH adjusted to 7.5, 1% Triton X-100 was added;
(2) Tris-HCl solution: (100 mM, pH 8.9): precisely weighing 1.21g of Tris-base into a volumetric flask with 100mL, adding sterilized ultrapure water to a volume of 100mL, and preparing a Tris-HCl solution with the concentration of 100mM and the pH of 8.9;
(3) 1 XPBS buffer: adding 20 XPBS buffer solution dry powder and ultrapure water to 2L, sterilizing under high pressure, and preserving at 4 ℃ for later use;
(4) Protease solution: (20 mg/mL): weighing 20mg of proteinase K, dissolving in 1mL of sterilized ultrapure water, and freezing at-20 ℃ for preservation;
(5) Nuclease P1 solution: 10 Xbuffer solution and sterilized ultrapure water are selected to prepare 10U/. Mu.L;
(6) Alkaline phosphatase solution: centrifuging at 14000rpm at 4deg.C for 5min, removing supernatant (ammonium sulfate solution), adding Tris-HCl solution with pH of 8.9 into the precipitate, and re-dissolving to obtain enzyme solution with concentration of 3U/20 μL;
Example 1, establishment of double derivatization technique-based abasic site LC-MS/MS analysis method
1. Preparing standard curve working solution
Taking an AP-NBHA reference substance, and preparing an AP-NBHA standard curve working solution with 1000ng/mL by using ultrapure water;
taking an AP-PMOA reference substance, and preparing an AP-PMOA standard curve working solution with the concentration of 1000ng/mL by using ultrapure water;
Preparing dG reference substance into a dG standard curve working solution of 1000ng/mL by using ultrapure water;
2. Preparing an internal standard working solution
6-MdA-d3, preparing an internal standard working solution of 1000ng/mL by using ultrapure water;
3. preparing standard curve sample
Mixing ultrapure water and an AP-NBHA standard curve working solution in a proper proportion to prepare an AP-NBHA standard curve sample, wherein the concentration of the AP-NBHA in the AP-NBHA standard curve sample is 0.01, 0.10, 0.20, 1.00 and 10.00ng/mL respectively;
mixing ultrapure water and an AP-PMOA standard curve working solution in a proper proportion to prepare an AP-PMOA standard curve sample, wherein the concentration of the AP-PMOA in the AP-PMOA standard curve sample is 0.01, 0.05, 0.10 and 1.00ng/mL respectively;
Mixing ultrapure water and dG standard curve working solution in a proper proportion to prepare dG standard curve samples, wherein the concentration of dG in the dG standard curve samples is 1, 5, 10, 50 and 100ng/mL respectively;
4. Preparing standard curve sample containing internal standard
The internal standard working solution is respectively added into the AP-NBHA, the AP-PMOA and the dG standard curve samples to prepare a standard curve sample containing an internal standard, and the concentration of 6-mdA-d3 in the standard curve sample containing the internal standard is 0.10ng/mL; standard curve samples containing internal standard are respectively: an AP-NBHA standard curve sample containing an internal standard, an AP-PMOA standard curve sample containing an internal standard and a dG standard curve sample containing an internal standard.
5. Sample processing to be tested
5.1 Extraction of DNA from HepG2 cells
5.1.1 Cell digestion
When the cells are grown to the logarithmic phase by adherence, taking one dish of HepG2 cells in the logarithmic phase, rinsing the cells with a precooled phosphate buffer solution, digesting the cells by pancreatin, and re-suspending to adjust the cell concentration to about 2-3 multiplied by 10 5/EP tubes;
5.1.2 precipitation of nuclei
Centrifuging the cell suspension at 4deg.C for 3min at 1000r/min, removing supernatant, precipitating cell nucleus in pre-cooled Tris lysis buffer, centrifuging at 4deg.C for 10min at 5000rpm, removing supernatant, and precipitating to obtain cell nucleus;
5.1.3 extraction of DNA from HepG2 cells
The obtained cell nucleus precipitate is added with 500 mu LPBS buffer solution, 30 mu L of 10% sodium dodecyl sulfate and 20 mu L of proteinase K, and the solution is incubated for 1.5h at 37 ℃ to obtain enzymatic hydrolysate containing HepG2 cell DNA.
5.2 Primary derivatization (derivatization of deoxyriboring-opened aldehydes)
Adding 29 mu L of 100mM derivatization reagent 1 (NBHA) into 550 mu L of enzymolysis liquid containing HepG2 cell DNA, and incubating for 1.5h at 37 ℃ to carry out derivatization reaction to obtain a primary derivatization sample solution;
5.3 Secondary derivatization
Adding 64 mu L of 50mM derivatization reagent 2 (PMOA) into the obtained primary derivatization sample solution, and incubating at 37 ℃ for 1.5h to carry out derivatization reaction to obtain a secondary derivatization sample solution;
5.4 precipitation of DNA
Adding 1.5mL of pre-cooled isopropanol to precipitate DNA at the temperature of minus 20 ℃ into the secondary derivatization sample solution, washing the precipitate (DNA) with 70% ethanol for three times, and uniformly collecting after natural volatilizing;
5.5 enzymolysis
Re-dissolving the DNA sample in 5.4 with 90 mu L of ultrapure water, incubating with nuclease P1 (1U) and derivatization reagent 2 for 2h at 37 ℃, and then incubating with phosphodiesterase I (0.5U) and alkaline phosphatase (3U) for 4h to obtain enzymolysis liquid;
5.6 preparation of sample solution to be measured
Adding 1-time volume methanol and 5-time volume acetonitrile into the enzymolysis solution in 5.5 to remove enzyme, vacuum centrifuging and drying (1500 rpm,40 ℃ C., vacuum degree 13.3 hpa), re-dissolving with 50 mu L of ultrapure water to obtain a sample solution to be detected, transferring the sample solution to a sample injection vial for LC-MS/MS analysis, and detecting the contents of AP-NBHA, AP-PMOA, dG and internal standard 6-mdA-d 3.
6. Sample solution to be tested and standard curve sample solution containing internal standard are subjected to LC-MS/MS analysis
6.1 Chromatographic conditions:
Chromatographic column: waters ACQUITY UPLC HSS T3, 100 mm. Times.2.1 mm,1.8 μm;
Mobile phase
Mobile phase a: formic acid aqueous solution with volume fraction of 0.1%;
Mobile phase B: formic acid-acetonitrile solution with the volume fraction of 0.1%;
Flow rate: 0.3mL/min;
Measuring three contents of AP-NBHA, AP-PMOA and dG, wherein each content of sample solution to be measured and each content of standard curve sample solution containing an internal standard are independently sampled, and the sample amount is 5 mu L;
AP-NBHA and dG gradient elution, elution procedure was as follows:
the percentages of the mobile phase A and the mobile phase B are volume percentages
AP-PMOA gradient elution was performed as follows:
the percentages of the mobile phase A and the mobile phase B are volume percentages
6.2 Mass Spectrometry conditions
Positive ion scanning, multi-reaction monitoring data acquisition mode, mass spectrum conditions are as follows:
6.3 test parameters are as follows:
7. quantitatively analyzing the concentrations of AP-NBHA, AP-PMOA and dG in the sample to be tested according to an internal standard-standard curve method
Taking the chromatographic peak-to-peak area ratio of AP-NBHA and 6-mdA-d3 in an AP-NBHA standard curve sample containing an internal standard as an ordinate, and taking the concentration of AP-NBHA in an AP-NBHA standard curve sample containing an internal standard as an abscissa, and carrying out linear regression to obtain an internal standard-AP-NBHA standard curve; substituting the chromatographic peak-to-peak area ratio of AP-NBHA and 6-mdA-d3 in the sample to be measured into an internal standard-AP-NBHA standard curve for calculation to obtain the concentration (C AP-NBHA) of the AP-NBHA in the sample to be measured,
Taking the chromatographic peak-to-peak area ratio of the AP-PMOA in the AP-PMOA standard curve sample containing the internal standard and 6-mdA-d3 as an ordinate, and taking the concentration of the AP-PMOA in the AP-PMOA standard curve sample containing the internal standard as an abscissa, and carrying out linear regression to obtain an internal standard-AP-PMOA standard curve; substituting the chromatographic peak-to-peak area ratio of the AP-PMOA and 6-mdA-d3 in the sample to be detected into an internal standard-AP-PMOA standard curve for calculation to obtain the concentration (C AP-PMOA) of the AP-PMOA in the sample to be detected,
Taking the area ratio of dG in the dG standard curve sample containing the internal standard and the chromatographic peak of 6-mdA-d3 as an ordinate, and taking the concentration of dG in the dG standard curve sample containing the internal standard as an abscissa, and carrying out linear regression to obtain an internal standard-dG standard curve; substituting the chromatographic peak-to-peak area ratio of dG and 6-mdA-d3 in the sample to be measured into an internal standard-dG standard curve for calculation to obtain the concentration of dG in the sample to be measured (C dG);
8. Calculation of the content of abasic sites
(1) Amount of total nucleoside in cellular DNA (n Nucleoside )
The amount of total nucleoside in cellular DNA was calculated as 21% dG of the total nucleoside in DNA as follows:
n Nucleoside =CdG×V Volume of Xdilution/M dG/0.21
Wherein:
c dG represents the dG concentration in the sample calculated by the internal standard-standard curve method,
M dG is the molar mass of dG, 267;
v Volume of represents the total volume of the sample after reconstitution;
(2) Amount of abasic site substance (n AP-NBHA or n AP-PMOA)
The formula is as follows:
n AP-NBHA=CAP-NBHA×V Volume of /MAP-NBHA, or
nAP-PMOA=CAP-PMOA×V Volume of /MAP-PMOA
Wherein: c AP-NBHA、CAP-PMOA respectively represents the concentrations of AP-NBHA and AP-PMOA in the sample calculated by an internal standard-standard curve method,
V Volume of represents the total volume after reconstitution of the sample,
M AP-NBHA is the molar mass of AP-NBHA, 284,
M AP-PMOA is the molar mass of AP-PMOA, 240;
(3) Content of abasic sites
The ratio of the amount of the substance of AP-NBHA or AP-PMOA to the amount of the substance of total nucleosides in the cellular DNA is expressed as abasic site content AP-NBHA/nts or AP-PMOA/nts as follows:
AP-NBHA/nts=n AP-NBHA/n Nucleoside , or
AP-PMOA/nts=nAP-PMOA/n Nucleoside
(4) Example
Assuming that the volume of the sample after the re-dissolution is 50 mu L, the concentration calculated by the AP-NBHA generation internal standard-standard curve is 0.1ng/mL, 2 mu L of the sample after the re-dissolution is taken for dilution 1000 times for sample injection when the dG sample is measured, and the dG concentration in the sample calculated by the dG generation internal standard-standard curve method is 100ng/mL, then:
nAP-NBHA=CAP-NBHA×V Volume of /MAP-NBHA
=0.1ng/mL×50μL/284g/moL
=1.76×10-14moL
n Nucleoside =CdG×V Volume of Xdilution/M dG/0.21
=100ng/mL×50μL×1000/267/0.21
=8.90×10-8mol，
Abasic site content AP-NBHA/nts=n AP-NBHA/n Nucleoside
=1.97×10-7AP-NBHA/nts
=1.97AP-NBHA/107nts。
The abasic site and NBHA and PMOA derivatization mechanisms in the method of the invention are shown in FIG. 1A and FIG. 1B respectively, and the flow diagram of the method of the invention is shown in FIG. 1C.
Example 2, AP-NBHA methodological verification
1. Specialization of
Preparation of blank matrix: a blank matrix was prepared from "5.4 precipitated DNA", "5.5 digested" and "5.6 test sample solution" following the procedure of "5.1.1 cell digest", "5.1.2 precipitated nuclei", "5.1.3 extract DNA from HepG 2" in example 1 without two derivatization reactions.
Taking a blank matrix, adding an AP-NBHA standard curve working solution, and preparing the AP-NBHA concentration of 1ng/mL;
Taking a blank substrate, adding 6-mdA-d3 internal standard working solution, and preparing a solution with the concentration of 6-mdA-d3 of 0.1 ng/mL;
LC-MS/MS analysis was performed as in example 1, 6, respectively, and the results were
FIG. 2A is a blank matrix +AP-NBHA mass spectral response, showing that the method of the application does not interfere with the AP-NBHA assay;
FIG. 2B is a response of a mass spectrum of blank matrix +6-mdA-d3, showing that the measurement of 6-mdA-d3 is not interfered with by the method of the application;
the specificity results show that: by adopting the method of the application, the determination of the AP-NBHA and the internal standard 6-mdA-d3 is not interfered.
2. Linear range and sensitivity
Adding AP-NBHA reference substances into blank matrix, respectively preparing AP-NBHA solutions with the concentrations of 0.02, 0.1, 1,2, 5, 10, 20, 50 and 100ng/mL, respectively adding internal standard 6-mdA-d3 to make the concentration of 6-mdA-d3 be 0.1ng/mL, and finally performing LC-MS/MS analysis according to the method '6, sample solution to be tested and standard curve sample solution containing internal standard' in example 1
The peak area ratio of the reference substance and the internal standard is taken as an ordinate (y), and the mass concentration of the reference substance is taken as an abscissa (x, mg/mL) to manufacture a working curve: y=ax+b; the lowest concentration of the signal to noise ratio S/N >3 of the spectrogram in the working curve is used as a detection limit, and the lowest concentration of the signal to noise ratio S/N > 10 is used as the lowest quantitative limit;
The results are shown in FIG. 3 and Table 1, linear equation: y=0.01037x+0.00722, r 2 =0.999, ap-NBHA linear range of 0.02-100ng/mL, minimum limit of quantitation 0.02ng/mL, minimum limit of detection 0.01ng/mL.
TABLE 1 detection limit, quantitative limit, and quality control sample concentrations
3. Accuracy and precision
AP-NBHA reference substances are respectively added into a blank matrix, AP-NBHA quality control samples with the concentrations of 0.02, 0.05, 50 and 75ng/mL are prepared according to the experimental results of the linear range, internal standards of 6-mdA-d3 are respectively added, the concentration of 6-mdA-d3 is 0.1ng/mL, 6 parts of each concentration quality control sample is repeatedly prepared, and analysis and detection are carried out according to the conditions of '5.3 secondary derivatization', '5.4 precipitation DNA', '5.5 enzymolysis', '5.6 sample solution preparation to be detected', '6 sample solution to be detected, and LC-MS/MS analysis' of standard curve sample solutions containing the internal standards in the embodiment 1, and the accuracy is calculated through measuring the concentration and the percentage of the true concentration. Precision was calculated by calculating the relative standard deviation of the measured concentrations of 6 quality control samples for each concentration, and the results are shown in table 2.
TABLE 2 accuracy and precision of AP-NBHA in abasic site LC-MS/MS quantitative analysis method
As can be seen from table 2:
The accuracy and the precision in the batch of the method AP-NBHA are respectively 100% -105% and 1.9% -5.8%, which indicate that the accuracy and the precision in the batch of the method for detecting the abasic site AP-NBHA in the HepG2 accord with the rule of Chinese pharmacopoeia of 2020 edition;
The accuracy and precision of the method AP-NBHA between batches are respectively 99% -102% and 3.0% -4.8%, which shows that the accuracy and precision of the method for detecting the abasic site AP-NBHA in HepG2 between batches accord with the rule of Chinese pharmacopoeia of 2020 edition.
4. Recovery rate
AP-NBHA reference substances are respectively added into a blank matrix to prepare AP-NBHA quality control samples with the concentrations of 0.02, 0.05, 50 and 75ng/mL, internal standards of 6-mdA-d3 are respectively added, the concentration of 6-mdA-d3 is 0.1ng/mL, 6 samples are taken for each concentration sample, the analysis and detection of the contents of AP-NBHA, AP-PMOA and dG in the samples to be detected are carried out according to the method of the standard curve in example 1, which comprises the steps of carrying out LC-MS/MS analysis on the sample solution to be detected, carrying out the sample solution to be detected and the standard curve containing the internal standard according to the method of the standard curve, and carrying out the quantitative analysis on the contents of the AP-NBHA, the AP-PMOA and the dG in the samples to be detected according to the method of the standard curve, wherein the results are shown in Table 3.
TABLE 3 recovery rate in the method of quantitative analysis of abasic site LC-MS/MS (n=6)
A: CV is the relative standard deviation, relative standard deviation (%) = standard deviation/mean x 100 (reserved to the decimal point one digit later)
The recovery rate results show that: the method for treating the sample to be tested has no influence on the determination of the AP-NBHA.
Example 3 AP-PMOA methodological verification
1. Specialization of
Preparation of blank matrix: "1, specificity" as in example 2.
Taking a blank substrate, adding an AP-PMOA standard curve working solution to prepare a solution with the concentration of the AP-PMOA of 0.05ng/mL, and carrying out LC-MS/MS analysis according to 6 in example 1 to obtain the result:
FIG. 4 is a blank matrix+AP-PMOA mass spectrum response showing that the method of the present application does not interfere with the AP-PMOA determination;
The specificity results show that: by adopting the method of the application, the determination of the AP-PMOA is not interfered.
2. Linear range and sensitivity
Adding AP-PMOA reference substances into the blank matrix, respectively preparing AP-PMOA solutions with the concentrations of 0.01, 0.05, 0.1, 0.5 and 1.0ng/mL, respectively adding internal standard 6-mdA-d3 to enable the concentration of 6-mdA-d3 to be 0.1ng/mL, and finally carrying out LC-MS/MS analysis according to the '6, sample solution to be tested and standard curve sample solution containing the internal standard' in the embodiment 1
The peak area ratio containing the standard substance and the internal standard is taken as an ordinate (y), and the mass concentration of the standard substance is taken as an abscissa (x, mg/mL) to manufacture a working curve: y=ax+b; the lowest concentration of the signal to noise ratio S/N >3 of the spectrogram in the working curve is used as a detection limit, and the lowest concentration of the signal to noise ratio S/N > 10 is used as the lowest quantitative limit;
The results are shown in fig. 5 and table 4, linear equation: y=0.722x+0.0398, r 2 =0.999, the linear range of the AP-PMOA is 0.01-1ng/mL, the lowest quantitative limit is 0.01ng/mL, and the lowest detection limit is 0.005ng/mL.
TABLE 4 detection limit, quantitative limit, and quality control sample concentrations
3. Accuracy and precision
AP-PMOA reference substances are respectively added into a blank matrix, AP-PMOA quality control samples with the concentrations of 0.05, 0.1, 0.5 and 1ng/mL are prepared according to the experimental results of the linear range, internal standards of 6-mdA-d3 are respectively added, the concentration of 6-mdA-d3 is 0.1ng/mL, 6 parts of each concentration quality control sample is repeatedly prepared, and analysis and detection are carried out according to the conditions of '5.3 secondary derivatization', '5.4 precipitation DNA', '5.5 enzymolysis', '5.6 sample solution preparation to be detected', '6, sample solution to be detected and standard curve sample solution containing the internal standard' in the embodiment 1, and the accuracy is calculated through measuring the concentration and the percentage of the true concentration. Precision was calculated by calculating the relative standard deviation of the measured concentrations of 6 quality control samples for each concentration, and the results are shown in table 5.
TABLE 5 accuracy and precision of AP-PMOA in LC-MS/MS quantitative analysis method
As can be seen from table 5:
The in-batch accuracy and the precision of the method for detecting the abasic site AP-PMOA in the HepG2 are respectively 93% -107% and 1.2% -8.3%, and are proved to be in accordance with the rule of the Chinese pharmacopoeia of 2020 edition;
the accuracy and precision of the method for detecting the abasic site AP-PMOA in the HepG2 are respectively 95% -107% and 1.1% -9.0%, and are consistent with the rule of Chinese pharmacopoeia of 2020 edition.
4. Recovery rate
In view of the similarity in nature of AP-PMOA and AP-NBHA, no recovery experiments were performed for AP-PMOA on the premise that the AP-NBHA methodological recovery verification meets the methodological standard.
Example 4 dG methodological validation
1. Specialization of
Preparation of blank matrix: "1, specificity" as in example 2.
A blank substrate was prepared by adding a dG standard curve working solution to prepare a solution having a dG concentration of 50ng/mL, and LC-MS/MS analysis was performed as in 6 of example 1, resulting in:
FIG. 6 is a blank matrix+dG mass spectrum response showing that the method of the application does not interfere with dG determination;
the specificity results show that: by adopting the method of the application, the determination of dG is not interfered.
2. Linear range and sensitivity
DG reference substances are added into a blank matrix to prepare dG solutions with the concentration of 1, 5, 10, 50 and 100ng/mL respectively, internal standards of 6-mdA-d3 are added respectively to ensure that the concentration of 6-mdA-d3 is 0.1ng/mL, and finally LC-MS/MS analysis is carried out according to the '6, sample solution to be detected and standard curve sample solution containing the internal standards' in the embodiment 1
The peak area ratio containing the standard substance and the internal standard is taken as an ordinate (y), and the mass concentration of the standard substance is taken as an abscissa (x, mg/mL) to manufacture a working curve: y=ax+b; the lowest concentration of the signal to noise ratio S/N >3 of the spectrogram in the working curve is used as a detection limit, and the lowest concentration of the signal to noise ratio S/N > 10 is used as the lowest quantitative limit;
the results are shown in fig. 7 and table 6, linear equation: y=0.7189 x+0.6354, r 2 =0.999, dg linear range 1-100ng/mL, lowest quantitation limit 1ng/mL, lowest detection limit 0.5ng/mL.
TABLE 6 detection limit, quantitative limit, and quality control sample concentrations
3. Accuracy and precision
DG reference substances are respectively added into a blank matrix, dG quality control samples with the concentrations of 5, 10, 50 and 100ng/mL are prepared according to the experimental results of the linear range, internal standards of 6-mdA-d3 are respectively added, the concentration of 6-mdA-d3 is 0.1ng/mL, 6 parts of each concentration quality control sample is repeatedly prepared, and analysis and detection are carried out according to the conditions of '5.3 secondary derivatization', '5.4 precipitation DNA', '5.5 enzymolysis', '5.6 sample solution preparation to be detected', '6, sample solution to be detected and standard curve sample solution containing the internal standards' in the embodiment 1, and the accuracy is calculated through the percentage of the measured concentration and the true concentration. Precision was calculated by calculating the relative standard deviation of the measured concentrations of 6 quality control samples for each concentration, and the results are shown in table 7.
TABLE 7 accuracy and precision of dG in abasic site LC-MS/MS quantitative analysis method
As can be seen from table 7:
The accuracy and the variation coefficient of dG in the batch of the method are respectively 96% -108% and 5.8% -10.2%, which shows that the accuracy and the precision of the method for detecting the abasic site in HepG2 in the batch of the method accord with the rule of Chinese pharmacopoeia of 2020 edition;
The accuracy and the variation coefficient of dG between batches are 93% -105% and 3.8% -8.5%, respectively, which shows that the accuracy and the precision between batches of detecting the abasic site in the HepG2 cell meet the rule of Chinese pharmacopoeia of 2020 edition.
4. Recovery rate
DG is generated after the final enzymolysis step, and the subsequent step is only dilution and sample injection, so that no recovery rate experiment is required.
Example 5 validation experiment 1 after artificial addition of abasic site
Other steps are the same as in example 1, only the 5.5 enzymolysis is different, the purpose is to artificially increase the abasic site by heating before enzymolysis, and the enzymolysis operation steps are as follows: the DNA sample in 5.4 was reconstituted with 90. Mu.L of ultrapure water, and the reconstituted DNA was incubated at 50℃for two hours after the addition of derivatization reagent 2, followed by incubation with nuclease P1 (1U) at 37℃for 2 hours.
As shown in FIG. 8 and Table 8, the background content of abasic sites in HepG2 cells is low, the AP-NBHA is only 1.08-2.38 AP/10 7 nts, and the AP-PMOA reaches 2351-3405 AP/10 7 nts after the abasic sites are artificially increased by heating treatment before enzymolysis.
Example 6 validation experiment 2 after artificial addition of abasic site
1. Dual derivatization samples
Other steps are the same as in example 1, except that only "5.4 precipitate DNA" is used, the purpose is to artificially increase the abasic site by prolonging the time of precipitating DNA with isopropanol, and the steps of precipitating DNA are as follows: adding 1.5mL of precooled isopropanol at-20 ℃ to precipitate DNA overnight, washing the precipitate (DNA) with 70% ethanol for three times, and collecting uniformly after natural volatilizing.
2. Singly derivatized samples
The difference from the above double derivatization sample step was that the "5.3 double derivatization" in example 1 was not performed for purposes of Shan Yan biochemical and double derivatization comparisons.
The background levels of abasic site AP-NBHA in HepG2 cells, as determined using the single and double derivatization methods, respectively, are shown in FIG. 9.
The results of measuring abasic site content of HepG2 cells cultured in the same time are shown in Table 8 as in examples 1,5 and 6.
TABLE 8 detection of AP-NBHA and AP-PMOA content in co-cultured HepG2 according to examples 1, 5 and 6
As can be seen from table 8: directly proving that abasic sites are generated in the sample treatment process, wherein the number of abasic sites is 1.55AP/10 7 nts; after artificially increasing abasic sites, the number of AP-PMOA is obviously increased; the number of the AP-NBHA is not increased, so that the double-derivatization method can be proved to be capable of reacting with the generation of abasic sites in the enzymolysis process; the double derivatization method also more reflects the number of abasic sites in the cell background by comparison with the single derivatization method, which in fact includes the number of AP sites generated in the enzymatic step, while the double derivatization method excludes abasic sites generated in the enzymatic step.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (8)

1. The method for analyzing the abasic site LC-MS/MS based on the double derivatization technology is characterized by comprising the following steps:
(1) Preparing standard curve working solution:
preparing 1000ng/mL of aqueous solution of AP-NBHA as an AP-NBHA standard curve working solution,
Preparing 1000ng/mL of aqueous solution of AP-PMOA as an AP-PMOA standard curve working solution,
Preparing an aqueous solution of dG with the concentration of 1000ng/mL as a dG standard curve working solution;
(2) Preparing an internal standard working solution:
preparing 1000ng/mL of a 6-mdA-d3 aqueous solution as an internal standard working solution;
(3) Preparing standard curve samples:
Preparing AP-NBHA standard curve samples with the concentrations of 0.01, 0.10, 0.20, 1.00 and 10.00ng/mL respectively by using the AP-NBHA standard curve working solution and ultrapure water,
Preparing AP-PMOA standard curve samples with the concentrations of 0.01, 0.05, 0.10 and 1.00ng/mL respectively by using the AP-PMOA standard curve working solution and ultrapure water,
Preparing dG standard curve samples with the concentrations of 1,5, 10, 50 and 100ng/mL by using the dG standard curve working solution and ultrapure water respectively;
(4) Preparing standard curve samples containing an internal standard:
The internal standard working solution is respectively added into the AP-NBHA, the AP-PMOA and the dG standard curve samples to prepare a standard curve sample containing an internal standard, wherein the concentration of 6-mdA-d3 is 0.10 ng/mL;
(5) Sample processing to be tested
A. extraction of DNA from HepG2 cells
A.1 cell digestion
When the cells are grown to the logarithmic phase by adherence, taking one dish of HepG2 cells in the logarithmic phase, rinsing the cells with a precooled phosphate buffer solution, digesting the cells by pancreatin, and re-suspending to adjust the cell concentration to about 2-3 multiplied by 10 5/EP tubes;
a.2 precipitation of nuclei
Centrifuging the cell suspension at 4 ℃ for 3min at 1000rpm, removing the supernatant, precipitating cell nuclei in a precooled Tris lysis buffer, centrifuging at 4 ℃ for 10min at 5000rpm, and removing the supernatant to obtain the precipitate cell nuclei;
A3 extraction of DNA from HepG2 cells
Adding 500 mu LPBS buffer solution, 30 mu L of 10% sodium dodecyl sulfate and 20 mu L of proteinase K into the obtained cell nucleus precipitate, and incubating the solution at 37 ℃ for 1.5h to obtain enzymatic hydrolysate containing HepG2 cell DNA;
B. Primary derivatization
Adding 29 mu L of 100mM derivatization reagent 1 NBHA into 550 mu L of enzymolysis liquid containing HepG2 cell DNA, and incubating for 1.5h at 37 ℃ to carry out derivatization reaction to obtain a primary derivatization sample solution;
C. Secondary derivatization
Adding 64 mu L of 50mM derivatization reagent 2 PMOA into the obtained primary derivatization sample solution, and incubating at 37 ℃ for 1.5h to carry out derivatization reaction to obtain a secondary derivatization sample solution;
D. Precipitation of DNA
Adding 1.5mL of pre-cooled isopropanol to precipitate DNA at the temperature of minus 20 ℃ into the secondary derivatization sample solution, washing the precipitated DNA with 70% ethanol for three times, and uniformly collecting after natural volatilizing;
E. Enzymolysis
Re-dissolving the DNA sample in the step D by using 90 mu L of ultrapure water, incubating the re-dissolved DNA sample with 1U of nuclease P1 and derivatization reagent 2 for 2 hours at 37 ℃, and then incubating the re-dissolved DNA sample with 0.5U of phosphodiesterase I and 3U of alkaline phosphatase for 4 hours to obtain enzymolysis liquid;
F. preparation of sample solution to be tested
Adding 1-time volume methanol and 5-time volume acetonitrile into the enzymolysis solution in the E to remove enzyme, carrying out vacuum centrifugal drying at 1500rpm and 40 ℃ under the vacuum degree of 13.3hpa, re-dissolving with 50 mu L of ultrapure water to obtain a sample solution to be tested, transferring the sample solution to a sample injection vial for LC-MS/MS analysis, and detecting the contents of AP-NBHA, AP-PMOA, dG and internal standard 6-mdA-d 3;
(6) The sample solution to be tested and the standard curve sample solution containing the internal standard are subjected to LC-MS/MS analysis:
The chromatographic conditions for the LC-MS/MS analysis are as follows:
Chromatographic column: waters ACQUITY UPLC HSS T3, 100 mm. Times.2.1 mm,1.8 μm;
Mobile phase a: formic acid aqueous solution with volume fraction of 0.1%;
Mobile phase B: formic acid-acetonitrile solution with the volume fraction of 0.1%;
Flow rate: 0.3mL/min;
Measuring three contents of AP-NBHA, AP-PMOA and dG, wherein each content of sample solution to be measured and each content of standard curve sample solution containing an internal standard are independently sampled, and the sample amount is 5 mu L;
the elution modes of AP-NBHA and dG are gradient elution, and the gradient elution program is as follows:
0-2 min, 96% of phase A and 4% of phase B;
2-5 min, the A phase is reduced from 96% to 10%, and the B phase is increased from 4% to 90%;
5-7 min, wherein the phase A is 10% and the phase B is 90%;
7-9 min, the A phase is increased from 10% to 96%, and the B phase is decreased from 90% to 4%;
The AP-PMOA elution mode is gradient elution, and the gradient elution program is as follows:
0-1 min, 95% of phase A and 5% of phase B;
1-5 min, the A phase is reduced from 95% to 80%, and the B phase is increased from 5% to 20%;
5-7 min, the phase A is 80% and is reduced to 50%, and the phase B is increased from 20% to 50%;
7-8 min, the phase A is increased from 50% to 95%, and the phase B is decreased from 50% to 5%;
the mass spectrometry conditions for the LC-MS/MS analysis are as follows:
positive ion scanning, multiple reaction monitoring data acquisition mode,
Mass spectrometry parameters:
capillary voltage: 2500V; ion source temperature: 150 ℃; desolventizing gas nitrogen temperature: 500 ℃; desolventizing gas nitrogen flow rate: 800L/h; collision gas argon flow rate: 0.15mL/min;
(7) Quantitatively analyzing the concentrations of AP-NBHA, AP-PMOA and dG in the sample to be tested according to an internal standard-standard curve method:
Taking the chromatographic peak-to-peak area ratio of AP-NBHA and 6-mdA-d3 in an AP-NBHA standard curve sample containing an internal standard as an ordinate, and taking the concentration of AP-NBHA in an AP-NBHA standard curve sample containing an internal standard as an abscissa, and carrying out linear regression to obtain an internal standard-AP-NBHA standard curve; substituting the chromatographic peak-to-peak area ratio of AP-NBHA and 6-mdA-d3 in the sample to be measured into an internal standard-AP-NBHA standard curve for calculation to obtain the concentration C AP-NBHA of the AP-NBHA in the sample to be measured,
Taking the chromatographic peak-to-peak area ratio of the AP-PMOA in the AP-PMOA standard curve sample containing the internal standard and 6-mdA-d3 as an ordinate, and taking the concentration of the AP-PMOA in the AP-PMOA standard curve sample containing the internal standard as an abscissa, and carrying out linear regression to obtain an internal standard-AP-PMOA standard curve; substituting the chromatographic peak-to-peak area ratio of the AP-PMOA and 6-mdA-d3 in the sample to be measured into an internal standard-AP-PMOA standard curve for calculation to obtain the concentration C AP-PMOA of the AP-PMOA in the sample to be measured,
Taking the area ratio of dG in the dG standard curve sample containing the internal standard and the chromatographic peak of 6-mdA-d3 as an ordinate, and taking the concentration of dG in the dG standard curve sample containing the internal standard as an abscissa, and carrying out linear regression to obtain an internal standard-dG standard curve; substituting the chromatographic peak-to-peak area ratio of dG and 6-mdA-d3 in the sample to be measured into an internal standard-dG standard curve for calculation to obtain the concentration C dG of dG in the sample to be measured;
(8) Calculation of the content of abasic sites
A. amount of total nucleoside in cellular DNA n Nucleoside
The amount of total nucleoside in cellular DNA was calculated as 21% dG of the total nucleoside in DNA as follows:
n Nucleoside =CdG×V Volume of Xdilution/M dG/0.21
Wherein:
c dG represents the dG concentration in the sample calculated by the internal standard-standard curve method,
M dG is the molar mass of dG, 267;
v Volume of represents the total volume of the sample after reconstitution;
B. Amount of abasic site material n AP-NBHA or n AP-PMOA
The formula is as follows:
n AP-NBHA=CAP-NBHA×V Volume of /MAP-NBHA, or
nAP-PMOA=CAP-PMOA×V Volume of /MAP-PMOA
Wherein: c AP-NBHA、CAP-PMOA respectively represents the concentrations of AP-NBHA and AP-PMOA in the sample calculated by an internal standard-standard curve method,
V Volume of represents the total volume after reconstitution of the sample,
M AP-NBHA is the molar mass of AP-NBHA, 284,
M AP-PMOA is the molar mass of AP-PMOA, 240;
C. content of abasic sites
The ratio of the amount of the substance of AP-NBHA or AP-PMOA to the amount of the substance of total nucleosides in the cellular DNA is expressed as abasic site content AP-NBHA/nts or AP-PMOA/nts as follows:
AP-NBHA/nts=n AP-NBHA/n Nucleoside , or
AP-PMOA/nts=nAP-PMOA/n Nucleoside ；
NBHA is O-4-nitrobenzyl hydroxylamine/O-4-nitrobenzylhydroxylamine;
The AP-NBHA is a product derived from (3S, 4R) -3,4,5 trihydroxy valeraldehyde-O-benzyl-4-nitro-methyl oxime, AP locus and NBHA;
The PMOA is O-3-pyridylmethyl hydroxylamine/O- (pyridine-3-yl-methyl) -hydroxylamine;
The AP-PMOA is a product of derivatization of (3S, 4R) -3,4, 5-trihydroxy valeraldehyde-O-pyridine-3-methyl oxime, an AP site and PMOA;
the dG is 2 '-deoxyguanosine/2' -Deoxyguanosine;
The 6-mdA-d3 is deuterated N 6 -Methyl-2 'deoxyadenosine/N 6 -Methyl-2' -deoxyadenosine-d3.
2. The method of claim 1, wherein the monitored ion pair of AP-NBHA during LC-MS/MS analysis is m/z285.0 → m/z117.0; the monitoring ion pair of the AP-PMOA is m/z 241.1- & gt m/z92.0; the monitored ion pair of dG is m/z 268.0-m/z 152.0; the ion pair for monitoring the ion of 6-mdA-d3 is m/z 269.0- & gt m/z153.0.
3. The method of claim 1, wherein the method AP-NBHA has a linear range of 0.02 to 100ng/mL; the linear range of the AP-PMOA is 0.01-1ng/mL; dG has a linear range of 1-100ng/mL.
4. The method of claim 1, wherein the minimum quantitative limit of method AP-NBHA is 0.02ng/mL; the lowest quantitative limit of the AP-PMOA is 0.01ng/mL; the minimum quantification limit for dG is 1ng/mL.
5. The method of claim 1, wherein: the step (5) further comprises the preparation of an internal standard-added quality control sample, wherein the preparation process of the internal standard-added quality control sample comprises the following steps:
Preparation of blank matrix:
Extracting DNA from the HepG2 cell containing 10 mug of DNA according to A, hepG cells in the step (5), carrying out enzymolysis on the DNA, D, precipitated DNA, E, enzymolysis on the DNA, F and preparing a sample solution to be tested to obtain a blank matrix;
adding AP-NBHA reference substances into the blank matrix respectively to prepare AP-NBHA quality control samples with the concentrations of 0.02, 0.05, 50 and 75ng/mL, adding internal standard 6-mdA-d3 respectively to enable the concentration of 6-mdA-d3 to be 0.1ng/mL, and obtaining an AP-NBHA quality control sample added with the internal standard;
Respectively adding AP-PMOA reference substances into a blank matrix, preparing AP-PMOA quality control samples with the concentrations of 0.05, 0.1, 0.5 and 1ng/mL, respectively adding internal standards of 6-mdA-d3 to enable the concentration of 6-mdA-d3 to be 0.1ng/mL, and obtaining an AP-PMOA quality control sample added with the internal standards;
dG reference substances are respectively added into the blank matrix, dG quality control samples with the concentrations of 5, 10, 50 and 100ng/mL are prepared, and internal standards of 6-mdA-d3 are respectively added, so that the concentration of 6-mdA-d3 is 0.1ng/mL, and the dG quality control sample with the internal standards is obtained.
6. The method of claim 5, wherein said internally-labeled AP-NBHA quality control sample is subjected to LC-MS/MS analysis using step (6), and the established method is validated for the peak to peak area ratio of AP-NBHA and 6-mdA-d3 chromatograms and its concentration data in said internally-labeled AP-NBHA quality control sample.
7. A method as in claim 5 wherein said internally-labeled AP-PMOA quality control sample is subjected to LC-MS/MS analysis using step (6), and the established method is validated for the ratio of AP-PMOA to 6-mdA-d3 chromatographic peak to peak area and its concentration data in said internally-labeled AP-PMOA quality control sample.
8. The method of claim 5, wherein said internally-labeled dG quality control sample is subjected to LC-MS/MS analysis using step (6), and the resulting ratio of dG to 6-mdA-d3 chromatographic peak to peak area and concentration data in said internally-labeled dG quality control sample is used to verify the established method.
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