CN105911164A - Determination method of formaldehyde-DNA adduct - Google Patents

Determination method of formaldehyde-DNA adduct Download PDF

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CN105911164A
CN105911164A CN201610226542.XA CN201610226542A CN105911164A CN 105911164 A CN105911164 A CN 105911164A CN 201610226542 A CN201610226542 A CN 201610226542A CN 105911164 A CN105911164 A CN 105911164A
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dna
formaldehyde
solution
home
assay method
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陈欢
侯宏卫
胡清源
刘鲁娟
张小涛
朱贝贝
王红娟
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National Tobacco Quality Supervision and Inspection Center
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National Tobacco Quality Supervision and Inspection Center
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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Abstract

A determination method of a formaldehyde-DNA adduct is a determination method of HOMe-dA and HOMe-dG in DNA, and concretely comprises the following steps: exposing calf thymus DNA to a formaldehyde solution, carrying out enzyme hydrolysis on the obtained DNA solution, and sequentially adding a reducing agent NaBH3CN, a stable isotope internal standard substance and DNA hydrolase; and carrying out Strata-X column solid phase extraction on the obtained hydrolysate, collecting the obtained eluate, blowing the collected eluate with nitrogen at room temperature until the eluate is dry, re-dissolving the dried eluate in 100[mu]L of a 30% methanol-water solution, introducing the obtained new solution to an LC-MS/MS system, and accurately detecting the content levels of Me-dA and Me-dG. The stable isotope is adopted as an internal standard quantitative analysis substance in order to reduce the error caused by a sample pretreatment process, and tandem mass spectrometry well improves the selectivity, the accuracy and the sensitivity of the method. The method well improves the chromatographic separation process, shortens the chromatography time and reduces the consumption of an organic solvent through selecting a chromatographic column and optimizing the eluting conditions.

Description

A kind of assay method of formaldehyde-DNA adduct
Technical field
The invention belongs to the physical and chemical inspection technical field of DNA sample, relate generally to N in DNA6-methylol-adenine And N (HOMe-dA)2The determination techniques field of-methylol-guanine (HOMe-dG) adduct, specifically one uses liquid phase Chromatographic tandem mass spectrograph (LC-MS/MS) measures the method for formaldehyde-DNA adduct in DNA.
Background technology
Formaldehyde is a kind of pollutant being widely present in environmental matrices, such as commercial production, motor-vehicle tail-gas and indoor dress Pool etc.；Formaldehyde has human inheritance's toxicity, is classified as a class carcinogen by international cancer research institution (IARC).Active aldehyde radical can With the nucleophilic group on attack DNA, form DNA adduct, wherein N6-methylol-adenine (HOMe-dA) and N2-methylol- Guanine (HOMe-dG) is main formaldehyde-DNA adduct.Both version is unstable under monokaryon glycosides state, with also Former dose is converted into N6-methyl-adenine (Me-dA) and N2-methyl-guanine (Me-dG) just can be detected.
Currently, with respect to DNA adduct analyzing detecting method report more, mainly have32After P-, labelling technique, enzyme connection are exempted from Epidemic disease technology (ELISA), high performance liquid chromatography (HPLC-UV) and liquid chromatography-tandem mass spectrometry technology (LC-MS/MS).Wherein LC-MS/MS is widely used in the detection analysis of DNA adduct due to its higher sensitivity and selectivity.
Summary 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) The assay method of formaldehyde-DNA adduct (HOMe-dA and HOMe-dG) that technology is set up.The method has quick, accurate, sensitive Degree high, can be used for the qualitative and quantitative analysis of formaldehyde-DNA adduct in DNA.
It is an object of the invention to be achieved through the following technical solutions:
The assay method of a kind of formaldehyde-DNA adduct, i.e. the assay method of HOMe-dA and HOMe-dG, including walking in detail below Rapid:
A, formaldehyde exposure calf thymus DNA: take 400 μ g calf thymus DNAs, add formalin (the formalin use prepared The PBS solution preparation of 0.1 M, concentration 0.001-1 mM), matched group adds the PBS solution of equal volume.Above-mentioned solution is put into 24 h in 37 DEG C of constant incubators；Use the ice ethanol precipitation DNA of 2 times of volumes and terminate reaction, being centrifuged and remove supernatant, use The ethanol water of 70% cleans DNA sample, and the DNA agglomerate obtained dries and adds 200 μ L ultra-pure waters and redissolves, and uses NanoDrop 2000 ultramicrospectrophotometers detect the content of DNA at 260nm, are equivalent to for one absorbance units (OD) of double-stranded DNA 50 μg/mL。
The enzyme hydrolysis of b, DNA and purification enrichment: above-mentioned DNA is dissolved in the 10 mM PIPES/5 mM MgCl of 500 μ L2Slow Rush in liquid (pH=7), in DNA solution, add 30 mg NaBH3CN, 20 μ L mixing internal standard and 60U deoxyribonuclease Ⅰs, In 37 DEG C, hatch 2h, be subsequently added 0.008U Phosphodiesterase I and 30U alkali phosphatase continues to hatch 2h in 37 DEG C.Water Solution liquid, through Strata-X Solid-Phase Extraction, is collected eluent liquid nitrogen and is blown to do, redissolve in 100 μ L 30% methanol aqueous solutions, be sample Product liquid to be measured.The purpose of this process is to make double-stranded DNA enzymolysis become mononucleotide state, blowing concentration by Solid-Phase Extraction and nitrogen DNA adduct needed for purifies and separates enrichment.Be designated as in described mixing 10 ng/mL [15N5] Me-dA and 10 ng/mL [13C10 15N5]Me-dG。
C, the preparation of standard working solution: with Me-dA and the Me-dG standard working solution of methanol preparation variable concentrations.
D, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measure, and draw the variable concentrations hybrid standard work prepared molten Liquid, injects LC-MS/MS system, obtains equation of linear regression, and liquid to be measured to sample is measured, and records analyte and internal standard peak The ratio of area, substitutes into unary linear regression equation, tries to achieve the content of analyte in sample liquid to be measured.
Chromatographic condition: Thermo AcclaimTMPolar Advantage II C18 chromatographic column (4.6 × 150 mm, 3 μm), flow visualizing chooses A: methanol and B: pure water, and elution requirement is isocratic elution: 0-8 min:50% A；Flow velocity is 0.5 ML/min, 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 dry gas is 75 psi, and collision gas is 9 Psi, injects voltage: 10 V, impact energy: 9 eV, residence time: 200 ms.Monitoring ion pair is Me-dA:266.1 → 150.2 Quota ion pair, 266.1 → 117.2 qualitative ion pairs；Internal standard [15N5] Me-dA is 271.2 → 155.2；Me-dG:282.2 → 166.2 quota ions pair, 282.2 → 117.1 qualitative ion pairs；Internal standard [13C10 15N5] Me-dG is 297.2 → 176.2.
Each analyte and interior target MRM parameter are shown in Table 1.Analysis process is by Applied Biosystems Analyst Version 1.5.1 software controls.
Analyte and interior target MRM parameter thereof under table 1 multiple-reaction monitoring pattern
* it is quota ion
The range of linearity of the inventive method and detection limit:
Series standard working solution is injected LC-MS/MS, obtains standard working curve, equation of linear regression and correlation coefficient. The point of Me-dA and Me-dG standard curve is 0.02,0.05,0.1,0.2,0.5,1.0,1.5 and 2.0 ng/mL, two kinds of internal standards [15N5] Me-dA and [13C10 15N5] Me-dG concentration is 2 ng/mL.Object linear good, correlation coefficient is all higher than 0.9999.Utilizing mark-on dilution to obtain quantitative limit and the detection limit of method, concentration corresponding to ten times of signal to noise ratios of target peak is quantitative Limit, concentration corresponding to three times of signal to noise ratios is detection limit.The standard curve of analyte and quantitative limit and detection limit are shown in Table 2.
The standard working curve of table 2 analyte and LOD and LOQ
The inventive method recovery of standard addition and repeatability:
Take calf thymus DNA sample adds the recovery of standard addition of calibration method preparation method, altogether have chosen three and add water Flat, each pitch-based sphere replication 3 times, the response rate of the method obtained and repeatability, the results are shown in Table 3.The recovery of object Rate is between 91.8%-107.0%, and RSD is less than 5%, it was demonstrated that the accuracy of method and repeatability result are preferable.
The recovery of standard addition of table 3 analyte and repeatability
The method of the present invention overcomes the deficiency of prior art sample treatment, for the color of formaldehyde-DNA adduct in DNA Spectral condition is optimized, and is optimized the coherent detection condition of LC-MS/MS.Side the most of the present invention Method has a following excellent results:
1. compare with traditional liquid-phase chromatography method, owing to have chosen tandem mass spectrum so that selectivity and the sensitivity of method carry The mensuration of the formaldehyde-DNA adduct of low content in height, beneficially DNA.
2. this method has easy and simple to handle, quick, accurate, highly sensitive and reproducible advantage.
3. the inventive method uses stable isotope can reduce in sample pretreatment process as uantitative analytical thing The error caused, tandem mass spectrometry preferably improves 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, decreases organic The consumption of solvent.
Accompanying drawing explanation
The total ion current figure of Fig. 1 formaldehyde-DNA adduct.
Under Fig. 2 difference formaldehyde exposure dosage, the content of Me-dA and Me-dG in calf thymus DNA.
Detailed description of the invention
The present invention is described further below in conjunction with example, but is not to limit the present invention.
The assay method of formaldehyde-DNA adduct in a kind of DNA, its test process is to calf thymus DNA by formalin Expose, DNA solution is carried out enzyme hydrolysis and adds reducing agent, stable isotope internal standard and DNA hydrolytic enzyme.Hydrolyzed solution warp The little column solid phase extraction of Strata-X, collects eluting liquid nitrogen and blows concentration, introduce LC-MS/MS systematic analysis.
Example 1:
1. instrument and reagent:
AB SCIEX triple quadrupole rods tandem mass spectrometry instrument (California, USA Applied Biosystems, Inc.), Agilent 1200 is efficient Liquid chromatograph (Agilent company of the U.S.), NanoDrop 2000 ultramicrospectrophotometer (Sai Mofei scientific & technical corporation of the U.S.), permanent Temperature incubator (Sai Mofei scientific & technical corporation of the U.S.), Milli-Q water purification system (Merck KGaA group), Analyst 1.5.1 number According to gathering and processing software.
Deoxyribonuclease Ⅰ, alkali phosphatase (U.S.'s knob Great Britain biotech company), Phosphodiesterase I and calf Thymic DNA (Sigma of the U.S.), methanol (De Shan Reagent Company of Korea S), NaBH3CN(Shanghai Aladdin Reagent Company), Strata-X polymer posts (33 μm, 30 mg/1mL, Guangdong Féraud door scientific instrument company limited).Standard substance Me-dA, Me-dG and internal standard substance [15N5]Me-dA、[13C10 15N5]Me-dG.Reagent is chromatographically pure.
2. sample treatment:
Formaldehyde exposure calf thymus DNA: take 400 μ g calf thymus DNAs, add 1 mL variable concentrations formalin (0.001, 0.01,0.05,0.1,0.5 and 1.0 mM), matched group adds the PBS solution of equal volume.Above-mentioned solution is put into 37 DEG C of constant temperature 24 h in incubator；Use the ice ethanol precipitation DNA of 2 times of volumes and terminate reaction, being centrifuged and remove supernatant, with the ethanol of 70% Water cleans DNA sample, and the DNA agglomerate obtained dries and adds 200 μ L ultra-pure waters and redissolves, and divides by NanoDrop 2000 ultramicron Light photometer detects the content of DNA at 260nm, is equivalent to 50 μ g/mL for one absorbance units of double-stranded DNA.
The enzyme hydrolysis of DNA and purification enrichment: above-mentioned DNA is dissolved in the 10 mM PIPES/5 mM MgCl of 500 μ L2Buffering In liquid (pH=7), add 20 μ L and mix internal standard, add 60U deoxyribonuclease Ⅰ, in 37 DEG C, hatch 2h, be subsequently added 0.008U Phosphodiesterase I and 30U alkali phosphatase continue to hatch 2h in 37 DEG C.Hydrolyzed solution is through 1 activation of mL methanol and 1 mL The Strata-X solid phase extraction column of pure water equilibrium, with 1 mL ultra-pure water and the methanol-water drip washing of 1 mL 10%, finally uses 1 mL The methanol-water eluting of 50%, collects eluent liquid nitrogen and is blown to do, redissolve in 100 μ L 30% methanol aqueous solutions, be sample to be measured Liquid.
3. assay method:
Each 5 L of calf thymus DNA sample after the standard solution of absorption variable concentrations and pre-treatment, inject LC-MS/MS system System carries out separating to be analyzed, and records the content of formaldehyde-DNA adduct in sample.
Example 2: as described in Example 1, utilize method that this research sets up to variable concentrations formaldehyde (0.001,0.01, 0.05,0.1,0.5 with 1.0 mM) formaldehyde-DNA adduct has carried out detecting that (each sample is parallel in the calf thymus DNA that exposes Detect three times).Establishing criteria curve and peak area draw the concentration of DNA adduct in each sample, according to formula
In calculating each sample of acquisition, DNA adduct is relative to the content of nucleotide, and result is multiplied by 108Be converted into DNA adduct number/ 108Individual nucleotide.The amount of data result display Me-dA with Me-dG becomes effect relation (SPSS, P ＜ with formaldehyde exposure dosage 0.004, Fig. 2).

Claims (6)

1. N in an assay method for formaldehyde-DNA adduct, i.e. DNA6-methylol-adenine (HOMe-dA) and N2-hydroxyl first The assay method of base-guanine (HOMe-dG), it is characterised in that: include step in detail below:
A, formaldehyde exposure calf thymus DNA: take 400 μ g calf thymus DNAs, add the formalin prepared, and matched group adds The PBS solution of equal volume；Above-mentioned solution is put into 24 h in 37 DEG C of constant incubators；Use the ice ethanol precipitation of 2 times of volumes DNA also terminates reaction, is centrifuged and removes supernatant, and the ethanol water with 70% cleans DNA sample, and the DNA agglomerate obtained dries and adds 200 μ L ultra-pure waters redissolve, and detect the content of DNA with NanoDrop 2000 ultramicrospectrophotometer, for double at 260nm One absorbance units (OD) of chain DNA is equivalent to 50 μ g/mL；
The enzyme hydrolysis of b, DNA and purification enrichment: above-mentioned DNA is dissolved in the 10 mM PIPES/5 mM MgCl of 500 μ L2Buffer In, in DNA solution, add the NaBH of 30 mg3CN, by N6-methylol-adenine (HOMe-dA) and N2-methylol-bird is fast Purine (HOMe-dG) is reduced into N6-methyl-adenine (Me-dA) and N2-methyl-guanine (Me-dG) detects, and adds 20 μ L mixes internal standard, adds 60U deoxyribonuclease Ⅰ, hatches 2h in 37 DEG C, be subsequently added 0.008U Phosphodiesterase I and 30U alkali phosphatase continues to hatch 2h in 37 DEG C, and hydrolyzed solution, through Strata-X Solid-Phase Extraction, is collected eluent liquid nitrogen and is blown to Dry, redissolve in 100 μ L 30% methanol aqueous solutions, be sample liquid to be measured；
C, the preparation of standard working solution: with methanol preparation variable concentrations Me-dA and Me-dG standard working solution；
D, liquid chromatography-tandem mass spectrometry (LC-MS/MS) measure, and draw the variable concentrations hybrid standard working solution prepared, note Entering LC-MS/MS system, obtain equation of linear regression, liquid to be measured to sample is measured, and records analyte and internal standard peak area Ratio, substitutes into unary linear regression equation, tries to achieve the content of analyte in sample liquid to be measured.
The assay method of formaldehyde-DNA adduct the most according to claim 1, it is characterised in that: the formaldehyde in step a is molten The liquid PBS solution of 0.1 M is prepared, concentration 0.001-1 mM.
The assay method of formaldehyde-DNA adduct the most according to claim 1, it is characterised in that: mixing described in step b Be designated as in conjunction 10 ng/mL [15N5] Me-dA and 10 ng/mL [13C10 15N5]Me-dG。
The assay method of formaldehyde-DNA adduct the most according to claim 1, it is characterised in that: step d is chosen Thermo AcclaimTMPolar Advantage II C18 chromatographic column, specification 4.6 × 150 mm, 3 μm, flow visualizing Choosing methanol A and pure water B, isocratic elution, analysis time is 8 min, and sample size is 5 μ L.
The assay method of formaldehyde-DNA adduct the most according to claim 4, it is characterised in that: isocratic condition is: 0-8 min:50% A, flow velocity is 0.5 mL/min.
The assay method of formaldehyde-DNA adduct the most according to claim 1, it is characterised in that: tandem mass spectrum in step d The condition of detector: 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 dry gas is 75 psi, and collision gas is 9 psi, penetrates Enter voltage: 10 V, impact energy: 9 eV, residence time: 200 ms；Monitoring ion pair be Me-dA:266.1 → 150.2 quantitatively from Son is right, 266.1 → 117.2 qualitative ion pairs；Internal standard [15N5] Me-dA is 271.2 → 155.2；Me-dG:282.2 → 166.2 Quota ion pair, 282.2 → 117.1 qualitative ion pairs；Internal standard [13C10 15N5] Me-dG is 297.2 → 176.2.
CN201610226542.XA 2016-04-13 2016-04-13 Determination method of formaldehyde-DNA adduct Pending CN105911164A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140017672A1 (en) * 2007-10-31 2014-01-16 Rebecca HOLMBERG Method and kit for purifying nucleic acids
CN104502492A (en) * 2015-01-14 2015-04-08 武汉大学 Chemical derivatization method and application thereof to nucleic acid modification detection by liquid chromatogram-mass spectrometer (LC-MS) method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140017672A1 (en) * 2007-10-31 2014-01-16 Rebecca HOLMBERG Method and kit for purifying nucleic acids
CN104502492A (en) * 2015-01-14 2015-04-08 武汉大学 Chemical derivatization method and application thereof to nucleic acid modification detection by liquid chromatogram-mass spectrometer (LC-MS) method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
刘鲁娟 等: "液相色谱-串联质谱法检测甲醛暴露产生的两种DNA加合物", 《环境科学学报》 *

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Application publication date: 20160831