CN105911163B - A kind of assay method of acetaldehyde DNA adduct - Google Patents
A kind of assay method of acetaldehyde DNA adduct Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
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Abstract
A kind of assay method of acetaldehyde DNA adduct, that is Ethylidene dG and Propano dG assay method in DNA, including exposing calf thymus DNA using acetaldehyde solution and adding catalyst of the arginine as reaction, DNA solution carries out enzyme hydrolysis and simultaneously sequentially adds stable isotope internal standard, reducing agent NaBH3CN and DNA hydrolases.Hydrolyzate collects eluent and introduces LC MS/MS network analyses, can accurately detect Et dG and Propano dG contents level through the small column solid phase extractions of Strata X.The present invention is the assay method of acetaldehyde DNA adduct in brand-new DNA, caused error in sample pretreatment process can be reduced as uantitative analytical thing using stable isotope, tandem mass spectrometry preferably improves the selectivity, accuracy and sensitivity of method.By chromatographic column and the selection and optimization of elution requirement, chromatographic separation process is preferably improved, shortens the time of chromatography, reduces the consumption of organic solvent.
Description
Technical field
The invention belongs to the physical and chemical inspection technical field of DNA sample, N in DNA is related generally to2- ethylidene-guanine
（Ethylidene-dG）With 1, N2- propyl alcohol-guanine（Propano-dG）The determination techniques field of adduct, specifically one
Kind uses liquid chromatography-tandem mass spectrometry instrument（LC- MS/MS）The method for determining acetaldehyde-DNA adduct in DNA.
Background technology
Acetaldehyde is a kind of pollutant being widely present in environmental matrices.The imperfect combustion of organic matter is occurred mainly with,
Such as industrial production, cigarette smoke, motor-vehicle tail-gas.Active aldehyde radical, which needs not move through organism metabolism, just can directly attack life
Nucleophilic group in object, as the guanine in nucleic acid, adenine, cytimidine and thymidine form DNA adduct.Acetaldehyde is formed
Main DNA adduct include N2- ethylidene-guanine（Ethylidene-dG）With 1, N2- propyl alcohol-guanine（Propano-
dG）, wherein 1, N2- propyl alcohol-guanine（Propano-dG）It is that gained is further reacted by Ethylidene-dG and acetaldehyde, should
Process needs the catalysis of amino acid.N2- ethylidene-guanine（Ethylidene-dG）It is unstable under mononucleotide state, need
It is reduced to N in DNA enzymatic hydrolysis stage2- ethyl-guanine（Et-dG）It can just be detected.
At present, the analyzing detecting method on DNA adduct is reported more, is mainly had32Labelling technique after P-, enzyme-linked exempt from
Epidemic disease technology（ELISA）, high performance liquid chromatography（HPLC-UV）With liquid chromatography-tandem mass spectrometry technology（LC-MS/MS）.Wherein
LC-MS/MS is widely used in the detection and analysis of DNA adduct due to its higher sensitivity and selectivity.
The content of the invention
The purpose of the present invention is based on above-mentioned prior art situation and uses liquid chromatography-tandem mass spectrometry（LC-MS/MS）
Acetaldehyde-DNA adduct that technology is established（Ethylidene-dG and Propano-dG）Assay method.This method have it is quick,
Accurately, the features such as high sensitivity, available for qualitative and quantitative analysis while acetaldehyde-DNA adduct in DNA.
The purpose of the present invention is achieved through the following technical solutions：
A kind of assay method of acetaldehyde-DNA adduct, i.e. Ethylidene-dG and Propano-dG measure side in DNA
Method, comprise the following steps that：
A, acetaldehyde exposure calf thymus DNA：400 μ g calf thymus DNAs are taken, add the acetaldehyde solution prepared（Acetaldehyde is molten
Liquid is prepared with 0.1 M PBS solution, concentration 0.001-1 mM）, the PBS solution of control group addition equal volume.In above-mentioned solution
Each arginine for adding 8.0 mg, is put into 24 h in 37 DEG C of constant incubators；Use the ice ethanol precipitation DNA of 2 times of volumes and whole
Only react, supernatant is removed in centrifugation, cleans DNA sample with 70% ethanol water, obtained DNA agglomerates dry and add 200 μ L
Ultra-pure water redissolves, and DNA content is detected at 260nm with the ultramicrospectrophotometers of NanoDrop 2000, for double-stranded DNA
One absorbance units（OD）Equivalent to 50 μ g/mL.
B, DNA enzyme hydrolysis and SPE（SPE）：Above-mentioned DNA is dissolved in 500 μ L 10 mM Tris-HCl/5 mM
MgCl2Buffer solution（pH=7）In, 30 mg of addition reducing agent NaBH3CN, Ethylidene-dG is reduced to Et-dG；Add
20 μ L mix internal standard, add 60U deoxyribonuclease Ⅰs, 2h is incubated in 37 DEG C, then add 0.008U Phosphodiesterase Is
Continue incubation 2h in 37 DEG C with 30U alkaline phosphatases.Hydrolyzate is through the activation of 1 mL methanol and the ultrapure water balances of 1 mL
Strata-X solid phase extraction columns, with 1 mL ultra-pure waters and 1 mL 10% methanol water wash, finally with 1 mL 50% methanol
Water elution, it is sample prepare liquid to collect eluent.The purpose of this process is in order that double-stranded DNA is digested into mononucleotide shape
State, concentrating and purifying separation is blown by SPE and nitrogen and is enriched with required DNA adduct.100 ng/ are designated as in the mixing
ML [15N5] Et-dG and [15N5]Propano-dG。
C, the preparation of standard working solution：Prepare Et-dG the and Propano-dG standards work of various concentrations respectively with methanol
Solution.
D, liquid chromatography-tandem mass spectrometry（LC-MS/MS）Measure, it is molten to draw the various concentrations hybrid standard work prepared
Liquid, LC-MS/MS systems are injected, equation of linear regression is obtained, sample prepare liquid is measured, measure analyte and internal standard peak
The ratio of area, unary linear regression equation is substituted into, try to achieve the content of analyte in sample prepare liquid.
Chromatographic condition：Choose Thermo AcclaimTMPolar Advantage II C18 chromatographic columns（4.6×150
Mm, 3 μm）, flow visualizing selection methanol（A）And pure water（B）, elution requirement is isocratic elution：0-15 min：35% A；Stream
Speed is 0.40 mL/min, and sample size is 5 μ L.
Mass Spectrometry Conditions：Electric spray ion source（ESI）, multiple-reaction monitoring cation scan mode；Spray voltage：5500 V,
Ion source temperature：550 DEG C, the amount of gas curtain gas is 20 psi, and atomization gas is 70 psi, and it is 75 psi to dry gas, collision gas 9
Psi, inject voltage：10 V, impact energy：9 eV, residence time：200 ms.Monitoring ion pair is Et-dG：296.2→180.2
Quota ion pair, 296.2 → 117.2 qualitative ion pairs；Internal standard [15N5] Et-dG be 271.2 → 155.2；Propano-dG：
338.3 → 222.1 quota ion pairs, 338.3 → 117.2 qualitative ion pairs；Internal standard [15N5] Propano-dG be 343.2 →
227.2。
Analyte and interior target MRM parameters are shown in Table 1.Whole analysis process is by Applied Biosystems Analyst
Version 1.5.1 softwares control.
Analyte and its interior target MRM parameters under the multiple-reaction monitoring pattern of table 1
* it is quota ion
The range of linearity and detection limit of the inventive method：
Series standard working solution is injected into LC-MS/MS, obtains standard working curve, equation of linear regression and correlation
Coefficient.The point of Et-dG and Propano-dG standard curves is 0.02,0.05,0.1,0.2,1.0,1.5 and 2.0 ng/mL.Target
Thing it is linear good, coefficient correlation is all higher than 0.9999.Dilute to obtain the quantitative limit of method and detection limit, target peak using mark-on
Concentration is quantitative limit corresponding to ten times of signal to noise ratio, and concentration corresponding to three times signal to noise ratio is detection limit.The standard curve of analyte and
Quantitative limit and detection limit are shown in Table 2.
The standard working curve and LOD and LOQ of the object of table 2
The inventive method recovery of standard addition and repeatability：
The recovery of standard addition for adding calibration method preparation method in calf thymus DNA sample is taken, three is have chosen altogether and adds
Add level, each pitch-based sphere replication 3 times, the rate of recovery and repeatability of obtained method, the results are shown in Table 3.Object
The rate of recovery is between 97.2%-101.6%, and RSD is less than 10%, it was demonstrated that the accuracy of method and repeated result are preferable.
The recovery of standard addition and repeatability of the analyte of table 3
The method of the present invention overcomes the deficiency of prior art sample treatment, for acetaldehyde-DNA adduct in DNA
Chromatographic condition be optimized, and the coherent detection condition to LC-MS/MS is optimized.This hair compared with prior art
Bright method has following excellent results：
1. compared with traditional liquid-phase chromatography method, due to have chosen tandem mass spectrum so that the selectivity of method and sensitive
Degree improves, and is more beneficial for the measure of acetaldehyde-DNA adduct of low content in DNA.
2. this method have easy to operate, quick, accurate, high sensitivity and it is reproducible the advantages of.
3. the inventive method can be reduced in sample pretreatment process using stable isotope as uantitative analytical thing
Caused error, tandem mass spectrometry preferably improve selectivity and accuracy and the sensitivity of method.By chromatographic column and
The selection and optimization of elution requirement, preferably improves chromatographic separation process, shortens the time of chromatography, reduces organic
The consumption of solvent.
Brief description of the drawings
The total ion current figure of Fig. 1 acetaldehyde-DNA adduct.
Under Fig. 2 difference acetaldehyde reconditionings, Et-dG and Propano-dG contents in calf thymus DNA.
Embodiment
The present invention is described further below in conjunction with example, but is not the limitation present invention.
The assay method of acetaldehyde-DNA adduct in a kind of DNA, its test process are to calf thymus DNA with acetaldehyde solution
Exposed（Arginine is as catalyst）, enzyme hydrolysis is carried out to DNA solution and adds reducing agent NaBH3CN, stable isotope
Internal standard and DNA hydrolases.Hydrolyzate collects eluent and introduces LC-MS/MS network analyses through the small column solid phase extractions of Strata-X.
Example 1：
1. instrument and reagent：
The triple quadrupole rods tandem mass spectrometry instrument of AB SCIEX（California, USA Applied Biosystems, Inc.）, Agilent 1200
High performance liquid chromatography（Agilent company of the U.S.）, the ultramicrospectrophotometers of NanoDrop 2000（U.S.'s match is silent to fly science and technology public affairs
Department）, constant incubator（Sai Mofei scientific ＆ technical corporation of the U.S.）, nitrogen concentrate drying instrument（Bai Taiqi companies of Sweden）, Milli-Q water is pure
Change system（Merck KGaA group）, Analyst 1.5.1 data acquisition and procession softwares.
Deoxyribonuclease Ⅰ, alkaline phosphatase（U.S. knob Great Britain biotech company）, Phosphodiesterase I and calf
Thymic DNA（Sigma of the U.S.）, methanol（De Shan Reagent Companies of South Korea）, NaBH3CN（Shanghai Aladdin Reagent Company）, first
Alcohol（De Shan Reagent Companies of South Korea）, Strata-X polymer posts（33 μm, 30 mg/1mL, Guangdong Féraud door scientific instrument have
Limit company）, standard items Et-dG and Propano-dG and its internal standard compound [15N5] Et-dG and [15N5]Propano-dG.Reagent is
Chromatographically pure.
2. sample treatment：
Acetaldehyde exposes calf thymus DNA：400 μ g calf thymus DNAs are taken, add the acetaldehyde solution of 1 mL various concentrations
（0.001,0.01,0.05,0.1,0.5 and 1.0 mM）, control group adds the PBS solution of equal volume, in above-mentioned solution respectively plus
Enter 8.0 mg arginine, be put into 24 h in 37 DEG C of constant incubators；It is anti-using the ice ethanol precipitation DNA and termination of 2 times of volumes
Should, supernatant is removed in centrifugation, and DNA sample is cleaned with 70% ethanol water, and obtained DNA agglomerates dry and to add 200 μ L ultrapure
Water redissolves, and detects DNA content at 260 nm with the ultramicrospectrophotometers of NanoDrop 2000, for double-stranded DNA one
Absorbance units（OD）Equivalent to 50 μ g/mL.
DNA enzyme hydrolysis and SPE（SPE）：Above-mentioned DNA is dissolved in 500 μ L 10 mM Tris-HCl/5 mM
MgCl2Buffer solution（pH=7）In, sequentially add 30 mg NaBH3CN, 20 μ L mix internal standard and 60U deoxyribonuclease Ⅰs,
2h is incubated in 37 DEG C, 0.008U Phosphodiesterase Is is then added and 30U alkaline phosphatases continues to be incubated 2h in 37 DEG C.Hydrolysis
Liquid collects eluent as prepare liquid through Strata-X SPEs.Be designated as in the mixing 100 ng/mL [15N5]Et-dG
[15N5]Propano-dG。
3. assay method：
Each 5 μ L of calf thymus DNA sample after the standard liquid of absorption various concentrations and pre-treatment, inject LC-MS/
MS systems carry out separation analysis, measure the content of acetaldehyde-DNA adduct in sample.
Example 2：As described in Example 1, the method established using this research is to various concentrations acetaldehyde（0.001,0.01,
0.05,0.1,0.5 and 1.0 mM）Acetaldehyde-DNA adduct is detected in exposed calf thymus DNA（Each sample is parallel
Detection is three times）.Establishing criteria curve and peak area draw the concentration of DNA adduct in each sample, according to formula
Calculate DNA adduct in each sample of acquisition and, relative to the content of nucleotides, be as a result multiplied by 108Be converted into DNA adduct number/
108Individual nucleotides.Data result shows Et-dG and Propano-dG amount and acetaldehyde reconditioning into effect relation（SPSS, P
＜ 0.003, Fig. 2）.
Claims (3)
1. a kind of assay method of acetaldehyde-DNA adduct, the acetaldehyde-DNA adduct is N2- ethylidene-guanine
（Ethylidene-dG）、1,N2- propyl alcohol-guanine（Propano-dG）, it is characterised in that：Including step in detail below：
A, acetaldehyde exposure calf thymus DNA：400 μ g calf thymus DNAs are taken, add the acetaldehyde solution prepared, control group adds
The PBS solution of equal volume, each arginine for adding 8.0 mg, is put into 24 h in 37 DEG C of constant incubators in above-mentioned solution；Make
With the ice ethanol precipitation DNA and terminating reaction of 2 times of volumes, centrifugation removes supernatant, DNA samples is cleaned with 70% ethanol water
Product, obtained DNA agglomerates dry and add the redissolution of 200 μ L ultra-pure waters, existed with the ultramicrospectrophotometers of NanoDrop 2000
DNA content is detected at 260nm, for one absorbance units of double-stranded DNA（OD）Equivalent to 50 μ g/mL；
B, DNA enzyme hydrolysis and SPE（SPE）：Above-mentioned DNA is dissolved in 500 μ L 10 mM Tris-HCl/5 mM
MgCl2In buffer solution, 30 mg reducing agent NaBH is added3CN, by N2- ethylidene-guanine（Ethylidene-dG）Reduction
Into N2- ethyl-guanine（Et-dG）To detect, 20 μ L mixing internal standards are added, 60U deoxyribonuclease Ⅰs are added, at 37 DEG C
Middle incubation 2h, then adds 0.008U Phosphodiesterase Is and 30U alkaline phosphatases continue to be incubated 2h in 37 DEG C, and hydrolyzate is through 1
ML methanol activates and the Strata-X solid phase extraction columns of the ultrapure water balances of 1 mL, with 1 mL ultra-pure waters and 1 mL 10% methanol
Water wash, finally with 1 mL 50% methanol water elution, it is sample prepare liquid to collect eluent；
C, the preparation of standard working solution：The N of various concentrations is prepared with methanol2- ethyl-guanine（Et-dG）、1,N2- propyl alcohol-bird
Purine（Propano-dG）Standard working solution；
D, liquid chromatography-tandem mass spectrometry（LC-MS/MS）Measure, draws the various concentrations hybrid standard working solution prepared, notes
Enter LC-MS/MS systems, obtain equation of linear regression, sample prepare liquid is measured, measure analyte and internal standard peak area
Ratio, unary linear regression equation is substituted into, try to achieve the content of analyte in sample prepare liquid；In LC-MS/MS measure, choose
Thermo AcclaimTMPolar Advantage II C18 chromatographic columns, specification 4.6 × 150 mm, 3 μm, flow visualizing
Choose methanol A and pure water solution B, isocratic condition are：0-15 min：35% A；Flow velocity is 0.40 mL/min, analysis time
For 15 min, sample size is 5 μ L；
Tandem mass spectrum detector condition is as follows：Electric spray ion source（ESI）, multiple-reaction monitoring cation scan mode；Spraying electricity
Pressure：5500 V, ion source temperature：550 DEG C, the amount of gas curtain gas is 20 psi, and atomization gas is 70 psi, and it is 75 psi to dry gas,
Collision gas is 9 psi, injects voltage：10 V, impact energy：9 eV, residence time：200 ms；Monitoring ion pair is Et-dG：
296.2 → 180.2 quota ion pairs, 296.2 → 117.2 qualitative ion pairs；Internal standard [15N5] Et-dG be 271.2 → 155.2；
Propano-dG：338.3 → 222.1 quota ion pairs, 338.3 → 117.2 qualitative ion pairs；Internal standard [15N5]Propano-dG
For 343.2 → 227.2.
2. the assay method of acetaldehyde-DNA adduct according to claim 1, it is characterised in that：Acetaldehyde in step a is molten
Liquid is prepared with 0.1 M PBS solution, concentration 0.00-1 mM.
3. the assay method of acetaldehyde-DNA adduct according to claim 1, it is characterised in that：Mixed described in step b
Inside be designated as 100 ng/mL [15N5] Et-dG and [15N5]Propano-dG。
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