Protocol for the simultaneous quantification of oxidative purine lesions in DNA using LC-MS/MS analysis

Summary

Most DNA damages induced through oxidative metabolism are single lesions which can accumulate in tissues. Here, we present a protocol for the simultaneous quantification of oxidative purine lesions (cPu and 8-oxo-Pu) in DNA. We describe steps for enzymatic digestion of DNA and sample pre-purification, followed by quantification through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We optimized this protocol in commercially available calf thymus DNA and used genomic and mitochondrial DNA extracted from cell cultures and animal and human tissues.

Graphical abstract

Highlights

• •Quantification of purine lesions in DNA from biological samples by LC-MS/MS
• •Synthesis of ¹⁵N-labeled 5′,8-cyclopurines and 8-oxo-purines for DNA lesions detection
• •Tailored enzymatic digestion of DNA for purine lesions detection
• •Oxidation-resistant procedure of sample cleanup, enrichment, and analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Most DNA damages induced through oxidative metabolism are single lesions which can accumulate in tissues. Here, we present a protocol for the simultaneous quantification of oxidative purine lesions (cPu and 8-oxo-Pu) in DNA. We describe steps for enzymatic digestion of DNA and sample pre-purification, followed by quantification through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We optimized this protocol in commercially available calf thymus DNA and used genomic and mitochondrial DNA extracted from cell cultures and animal and human tissues.

Before you begin

The protocol below describes the step sequence starting from DNA extraction from harvested tissues or lysed cells for the quantification of six purine lesions reported in Figure 1, i.e., the two 8-oxo-purines (8-oxo-Pu) and the four 5′,8-cyclopurines (cPu).

Figure 1.

Purine lesions formed after oxidatively induced DNA damage in vivo and in vitro

(upper part) structures of 8-oxo-2′-deoxyguanosine (8-oxo-dG) and 8-oxo-2′-deoxyadenosine (8-oxo-dA) (lower part) structures of 5′,8-cyclo-2′-deoxyguanosine (cdG) and 5′,8-cyclo-2′-deoxyadenosine (cdA) in their 5′S and 5′R diastereomeric forms.

Protocol optimization

The protocol was optimized using model experiments of HO^· radical reaction with double-stranded oligonucleotides or commercially available calf thymus DNA (ct-DNA).^1,2,3,4,5 We used this protocol to ascertain the levels of the six DNA lesions in wild type (wt) Cockayne Syndrome cells, CSA (CS3BE–wtCSA), wtCSB (CS1AN–wtCSB) cell lines, and their defective counterparts CS3BE and CS1AN,^6,7 in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines,⁸ as well as in estrogen receptor-alpha positive (ER-α) MCF-7 and triple negative MDA-MB-231 breast cancer cell lines.⁹ We also compared a murine model of immunodeficient (SCID) xenografted mice monitored at different ages (4- and 17-weeks) with the corresponding controls without tumor implantation, and carried out an assessment of lesions across different tissues such as liver, kidney and brain tissues.^10,11 Furthermore, we applied this protocol in genetic material isolated from tissue biopsies collected from inflammatory bowel diseases (IBD)-affected patients, and severely obese subjects versus control specimens,¹² as well as on neurons from XP patients with and without neurodegeneration.¹³

Endogenous and exogenous formation of cPu and 8-oxo-Pu

The cPu lesions are generated from the reaction of HO^· radicals with the genetic material via the C5′ radical chemistry of purine moieties,^1,2 whereas 8-oxo-Pu lesions are generated by oxidation at the C8 position by a variety of radical and non-radical reactive oxygen species (ROS).¹

Note: The cPu lesions are substrates of the nucleotide excision repair (NER) pathway. The 8-oxo-Pu lesions are substrates of the base excision repair (BER) pathway.

CRITICAL: The two 8-oxo-Pu are commercially available, while among the four cPu only 5′S-cdA can be purchased. Thus, for the complete quantification of all four cPu (5′S-cdA, 5′R-cdA, 5′S-cdG and 5′R-cdG), their synthetic preparation is needed to be used as analytical ref. ^14,15

CRITICAL: The preparation of ¹⁵N-labeled cPu and 8-oxo-Pu is needed for utilizing the liquid chromatography (LC) isotope dilution tandem mass spectrometry MS/MS) technique for quantitative analysis.

Note: The ¹⁵N-labeled materials are prepared starting from [¹⁵N₅]-2′-deoxyguanosine and [¹⁵N₅]-2′-deoxyadenosine that contain five ¹⁵N atoms in the base moiety following our synthetic protocols.¹⁶

Well-known methods are used for DNA isolation from harvested tissues or lysed cells, followed by an ameliorated enzymatic digestion to obtain the mixture of modified and unmodified 2′-deoxynucleosides.¹⁶ The quantification of the unmodified 2′-deoxynucleosides is made by HPLC-UV at 260 nm, while in parallel the lesions present in each sample, purified, are collected during the analysis by an automated fraction collector and the resulting enriched samples are subjected to LC-MS/MS analysis for quantifying the modified nucleosides, thus completing the protocol.

Institutional permissions

All experiments employing vertebrate animals must be approved by an appropriate institutional ethics committee on animal care and performed according to the relevant guidelines and regulations.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
Calf thymus	Sigma-Aldrich	73049-39-5
Butylated hydroxytoluene (BHT)	Sigma-Aldrich	W218405
Deferoxamine mesylate salt	Merck	138-14-7
Pentostatin	Merck	SML0508
[15N5]-2′-deoxyadenosine monohydrated	Cambridge Isotope Laboratories (Andover, USA)	CNLM-3896-CA-PK
[15N5]-2′- deoxyguanosine:H2O	Cambridge Isotope Laboratories (Andover, USA)	NLM-3899-CA-PK
Nuclease P1	Merck	N8630
Phosphodiesterase II	Merck	P9041
Phosphodiesterase I	Merck	P3243
DNase Ι	Merck	AMPD1
DNase II	Merck	D8764
Alkaline phosphatase	Merck	P0114
Benzonase	Merck	E1014
Ammonium formate	Merck	540-69-2
Acetonitrile	Merck	1.00029
Methanol	Merck	1.06035
N-bromosuccinimide	Merck	B81255
Formic acid 98%–100%	Merck	10264
Other
Finnigan TSQ Quantum Discovery MAX triple-stage quadrupole mass spectrometer	Thermo, USA	N/A
Waters Alliance HPLC system (Waters e2695 separations module) including a Waters 2998 photodiode array (PDA)	Waters	e2695
Quartz photoreactor equipped with a 125 W medium pressure mercury lamp	Photochemical Reactors, UK	N/A
Centrifuge	N/A	N/A
NanoDrop UV-vis spectrophotometer	Thermo Fisher Scientific	ND-2000
Amicon Ultra-0.5 centrifugal filter 3 kDa MWCO	Millipore	UFC5003
Glass inserts volume 150 μL	N/A	N/A
Freeze drier	N/A	N/A
Deep freezer (−80°C)	N/A	N/A

Step-by-step method details

Setting up the HPLC-UV method of analysis

Timing: 2 days

The HPLC-UV method setup is based on the separation of ten 2′-deoxynucleosides, i.e., the four unmodified DNA nucleosides (dC, dG, dTand dA), the four cPu (5′R-cdA, 5′S-cdA, 5′R-cdG and 5′S-cdG) and the two 8-oxo-Pu (8-oxo-dA, 8-oxo-dG).

• 1.Perform the analysis with an HPLC equipped with a 250 μL loop, using 4.6 mm × 150 mm Atlantis dC18, 100 Å column (5 μm particle size, Waters) guarded with a 4.6 mm × 20 mm Guard Column 2pK (Atlantis dC18 5 μm, Waters).

Note: The instrument is the Waters Alliance HPLC System (Waters e2695 Separations Module) including a Waters 2998 Photodiode Array (PDA) detector set at 260 nm; mobile phase: 2 mM ammonium formate, acetonitrile and methanol.

• 2.Use the following gradient program of the eluents [2 mM ammonium formate (solvent A), acetonitrile (solvent B) and methanol (solvent C)] : start with 99% solvent A, increasing solvent B from 1% to 30% within 35 min; then, solvent C from 0% to 40% in 5 min; return to initial conditions and wait 5 min for re-equilibration. Flow rate at 1 mL/min.
• 3.
  Prepare a 1000 ppm standards mixture solution of the four unmodified nucleosides dC, dG, dT and dA by weighting the standards in a 10 mL volumetric flask.

  • a.
    Use another 10 mL flask and dilute to 10 ppm by adding also the required quantities from the stock solutions of known concentrations of the four cPu and the two 8-oxo-Pu (see Figure 1 for their formula).
  • b.
    Inject this mixture in the HPLC and record the chromatographic separation.

Note:Figure 2 shows a representative chromatogram with optimal separation of the ten compounds.

Figure 2.

A representative HPLC run showing the optimal separation of the ten 2′-deoxynucleosides (monitored at λ = 260 nm)

Preparation of ¹⁵N-labeled cPu and 8-oxo-Pu as internal standards

Timing: 2–3 days

Timing: 3–5 days (for step 5)

Timing: 2–3 days (for step 6)

The preparation of ¹⁵N isotopically enriched cPu and 8-oxo-Pu is a necessary step for the quantification of the purine lesions. The synthesis and purification of the products from the reaction mixture need a very careful approach for achieving high chemical and isotopic purity products. The steps of this preparation include: synthetic methods, photochemistry, γ-radiolysis and Fenton type reaction followed by normal or reverse phase chromatography.

• 4.
  Preparation of [¹⁵N₅]-5′R-cdA, [¹⁵N₅]-5′S-cdA and [¹⁵N₅]-8-oxo-dA internal standards.¹⁵

  • a.
    Charge a 25 mL glass vial with 4.1 mg of [¹⁵N₅]-dA monohydrate and add 10 mL of demineralized (DM) water to get a final concentration of 1.5 mM.
  • b.
    Flush it with N₂O for 15 min and then γ-irradiate it with a total dose of 2 kGy (dose rate used: 4.5 Gy min⁻¹).
  • c.
    Submit the crude reaction mixture to HPLC purification and collect the peaks at the appropriate elution times that correspond to [¹⁵N₅]-5′R-cdA, [¹⁵N₅]-5′S-cdA, and [¹⁵N₅]-8-oxo-dA.
  • d.
    Quench the resulting acidity by adding 5% NaHCO₃ aq. solution (pH ∼ 7, tested by pH indicator paper).
  • e.
    Check that the spectroscopic and analytical data are identical with the previously described in the literature for the isotopically labeled compounds.¹⁵
  • f.
    Prepare aqueous solutions and calculate the concentration of [¹⁵N₅]-5′R-cdA or [¹⁵N₅]-5′S-cdA by UV spectroscopy using ε = 14,900 M⁻¹ cm⁻¹ at λ_max = 266 nm or ε = 12,764 M⁻¹ cm⁻¹ at λ_max = 270 nm for [¹⁵N₅]-8-oxo-dA, and subject to LC-MS/MS analysis for calculating the isotopic purity.

CRITICAL: pH neutralization of the crude reaction mixture after gamma radiolysis

• 5.
  Preparation of [¹⁵N₅]-5′R-cdG, [¹⁵N₅]-5′S-cdG internal standards.¹⁵

  • a.
    Charge a 500 μL microcentrifuge tube equipped with a magnet stirring bar, with 1 mg (3.5 μmol) [¹⁵N₅]-2′-deoxyguanosine monohydrate followed by in 0.5 mL of water/acetonitrile 4:1 mixture. Add 1.3 mg (7.3 μmol) N-bromosuccinimide (7.3 μmol) to the suspension under stirring and leave at room temperature for 2 h.
  • b.
    Remove the solvent under a stream of argon, and add 0.1 mL acetone.
  • c.
    Leave stirring at room temperature for 4 h and then store it at −20°C overnight. Centrifuge the mixture at 1000 g for 1 min and remove the supernatant with a pipette.
  • d.
    Wash the precipitate with 60 μL of cold acetone (−18°C), centrifuge again and remove the liquid with a pipette.
  • e.
    Dry the residue under vacuum for 20 min to get the [¹⁵N₅]-8-bromo-2′-deoxyguanosine as light-brown powder.
  • f.
    Charge a 1 mL glass vial equipped with a magnet stirring bar, with [¹⁵N₅]-8-bromo-2′-deoxyguanosine 0.9 mg followed by in 2.6 mL of water, and stir at ambient temperature to get a 1 mM solution.
  • g.
    Transfer the solution into the immersing well quartz photoreactor equipped with a 125 W medium pressure mercury lamp.
  • h.
    Deoxygenate by gentle bubbling with argon for 10 min.
  • i.
    Irradiate the solution for 7 h (cdG in a 5′R/5′S ratio ∼6:1).
  • j.
    Quench the resulting acidity by adding 5% NaHCO3 aq. solution (pH ∼ 7, tested by pH indicator paper).
  • k.
    Submit the crude reaction mixture to HPLC purification and collect the peaks at the appropriate elution times that correspond to [¹⁵N₅]-5′R-cdG and [¹⁵N₅]-5′S-cdG.
  • l.
    Check that the spectroscopic and analytical data are identical with the previously described in the literature for the isotopically labeled compounds.¹⁵
  • m.
    Prepare aqueous solutions and calculate the concentration of [¹⁵N₅]-5′R- or [¹⁵N₅]-5′S-cdG by UV spectroscopy using ε = 13,850 M⁻¹ cm⁻¹ at λ_max = 258 nm, subject to LC-MS/MS analysis for calculating the isotopic purity.

CRITICAL:(i) UV lamp emission spectrum and power could affect the reaction yield. (ii) Quartz photoreactor is needed for having the products formed. (iii) pH neutralization of the crude reaction mixture after UV irradiation.

• 6.
  Preparation of [¹⁵N₅]-8-oxo-dG internal standard.¹⁵

  • a.
    Charge a 1 mL glass vial equipped with a magnet stirring bar, with [¹⁵N₅]-2′-deoxyguanosine monohydrate 0.24 mg (0.82 μmol) followed by in 164 μL of DM water.
  • b.
    Add while stirring 5.45 μL (2.7 μmol) of 0.5 M freshly prepared ascorbic acid followed by 3.3 μL (3.3 μmol) of 0.1 M CuSO₄ and 9.4 μL of 30% hydrogen peroxide.
  • c.
    Leave the reaction mixture stirring at room temperature for 2 h.
  • d.
    Quench the reaction by adding 100 μL of 5% Na₂SO₃ solution. Add 5% NaHCO₃ aq. solution till pH 7.
  • e.
    Submit the crude mixture to HPLC purification and collect the peaks at the appropriate elution time that correspond to [¹⁵N₅]-8-oxo-dG.
  • f.
    Check that the spectroscopic and analytical data are identical with the previously described in the literature for the isotopically labeled compounds.¹⁵
  • g.
    Prepare aqueous solutions and calculate the concentration of [¹⁵N₅]-8-oxo-dG by UV spectroscopy using ε = 10,300 M⁻¹ cm⁻¹ at λ_max = 293 nm, and subject to LC-MS/MS analysis for calculating the isotopic purity.

CRITICAL: Reaction quenching time and pH neutralization of the crude reaction mixture after quenching

Genomic DNA isolation (tissue harvesting or cells lysis)

Timing: 16 h

CRITICAL: For each procedure using stock solutions, let all nucleoside and salt dilutions thaw and equilibrate at room temperature and vortex them gently for at least 1 min). Perform all pipetting steps with well-calibrated pipettes.

Tissue harvesting

CRITICAL: Ensure sterile work in order to avoid cross-contamination of the extracted tissue with bacteria or other organisms from the environment.

• 7.Animal tissues are promptly removed using sterile instruments, chopped into small pieces (100–200 mg pieces), with each aliquot placed in a polypropylene microcentrifuge tube and immediately flashed frozen in liquid nitrogen.
• 8.Transfer the previously snap-frozen tissue into a 50 mL conical polypropylene tube filled with 5 mL of Tissue Lysis Buffer containing all appropriate inhibitors and antioxidants to minimize adventitious damage.

CRITICAL: Ensure frozen material remain frozen until samples are mixed with Tissue Lysis Buffer. Ensure tissue particles are able to move freely in the lysis mix and do not remain stuck on the bottom of the tube.

Cells lysis

CRITICAL: The cell culture used in your research should be regularly checked to ensure they are not infected with mycoplasma. Ensure sterile work in order to avoid cross-contamination of the extracted material with bacteria or other organisms from the environment.

• 9.Start with a cell pellet containing 1 × 10⁴–1 × 10⁶ cells. For frozen cells, thaw cell pellet slowly on ice and resuspend in 100 μL cold PBS (phosphate-buffered saline, sterile filtered, suitable for cell culture) by pipetting up and down.

CRITICAL: For fresh cells, pellet by centrifugation at 1,000 × g for 1 min and resuspend in 100 μL cold PBS by pipetting up and down.

• 10.Transfer the previously resuspended pellet into a 15 mL conical polypropylene tube filled with 2 mL of Cell Lysis Buffer A containing all appropriate inhibitors and antioxidants to minimize adventitious damage.

Tissue Lysis Buffer or Cell Lysis Buffer

Component	Concentration
Tris pH 7.5	10 mM
Ethylenediaminetetraacetic acid	2 mM
Sodium chloride	400 mM
Sodium dodecyl sulfate	1% (w/v)
Proteinase K	200 μg/mL
Deferoxamine	100 μΜ
Butylated hydroxytoluene	100 μΜ

CRITICAL: The addition of appropriate deaminase inhibitors and antioxidants minimizes artifacts arising during sample workup, which could lead to an over-estimation of adduct levels.

Carryover is the same for tissue harvesting or cells lysis

• 11.Mix thoroughly by pulse-vortexing for 10–20 s. Thorough mixing is essential for optimal results.

CRITICAL: Process the sample using a mechanical homogenizer is not recommended for avoiding oxidation of the sample.

• 12.Incubation of mixture at 55°C overnight in a water bath, incubator or temperature-controlled heating block.

CRITICAL: Overnight incubation will not negatively affect the quality of the purified gDNA and ensure rapid complete lysis and high yields.

CRITICAL: A water bath or temperature-controlled heating block with agitation is recommended for shaking. Otherwise, vortex samples every 20–30 min to speed up lysis).

• 13.Add 1 mL of saturated NaCl solution to the digestion mixture and incubate at 55°C for 15 min.
• 14.Remove from heat source cool the tube to 25°C.
• 15.Centrifuge at 10,000 rpm at 25°C for 30 min.
• 16.Precipitate nucleic acids in the supernatant using 2.5 volumes of absolute ethanol (molecular biology grade, Merck-Millipore).
• 17.Add RNase A (10 mg/mL) and RNase T1 (25 units/ μL) to the nucleic acid mixture and incubate at 37°C for 1 h.

CRITICAL: The presence of RNase A and RNase T1 used and the incubation time ensure complete digestion of the RNA to avoid interference with the UV quantification of total DNA prior to analysis and high DNA purity.

• 18.Add an equal volume of chloroform/isoamyl alcohol (24:1, v/v) and 2.5 volumes of absolute ethanol (molecular biology grade, Merck-Millipore).
• 19.Centrifuge at 10,000 rpm at 25°C for 10 min and wash the resulting DNA pellet twice with 70% cold ethanol.
• 20.Allow to dry in a fume hood for 20–30 min to completely evaporate ethanol.
• 21.Resuspend the genomic material in 500 mL of (sterile) nuclease-free water.

CRITICAL: Autoclaved water or water that has been stored for a long time might contain dust or additional contaminations that could impair the quality of the measurements.

CRITICAL: The DNA must be completely dissolved in the water before proceeding and complete solvation of quantities of DNA greater than 100 μg may require several h of incubation at ambient temperature (or 37°C) with shaking.

• 22.Measure the gDNA concentration by absorbance at 230, 260 and 280 nm by using a small volume quartz cuvette in photometer or a nanodrop UV-Vis Spectrophotometer (Thermo-Fischer). The optical density OD₂₆₀/OD₂₈₀ ratio should be between 1.85 and 1.90, and the OD₂₆₀/OD₂₃₀ ratio should be > 2, ideally between 2.3 and 2.5.

CRITICAL: : If a sample is contaminated with RNA, the OD₂₆₀/OD₂₈₀ ratio is often >1.9. A lower absorbance ratio may indicate the presence of protein, phenol or other contaminants that have an absorbance close to 208 nm. If the OD₂₆₀/OD₂₃₀ ratio is too low, the sample probably contains salt impurities.

Pause point: Isolated gDNA sample can be stored at the recommended temperature of −80°C for an infinite amount of time. For short-time storage, the sample can be kept at −20°C (at least 1 week) or even 4°C (up to 1 day).

CRITICAL: If you freeze your isolated gDNA, thaw your samples on ice before starting the DNA digestion steps. After thawing, pulse vortex the samples thoroughly and do a quick spin (5,000 g, 23°C, 5 s) in order to collect the solution at the bottom.

Enzymatic digestion of DNA in the presence of ¹⁵N-labeled internal standards

Timing: 42 h

CRITICAL: Use fresh (sterile) deionized water for each digestion. Autoclaved water or water that has been stored for a long time might contain dust or additional contaminations that could impair the digestion or the measurements.

• 23.Prepare samples containing 50 μg of genomic DNA (isolated from tissue or cell culture) in 1.5 mL polypropylene microcentrifuge tubes.

CRITICAL: Note that this quantity of DNA is more than actually needed for quantification of DNA lesions, but it ensures that all damage products will be present at levels exceeding the limits of quantification.

• 24.Dry the DNA completely under vacuum taking care to minimize drying time to avoid artifacts.

CRITICAL: Water removal is necessary as water is active in hydrolytic reactions and can degrade DNA. DNA samples should be stored without water of buffer impurities avoiding further oxidation and lesion artifacts. Heating during the drying step can create artifacts due to oxidation.

• 25.Add 84 μL of Ar flushed Solution A to each DNA sample and mix gently by pulse-vortexing for 5–10 s.

Solution A

Component	Concentration
Tris pH 7.9	10 mM
Magnesium chloride	10 mM
Sodium chloride	50 mM
Pentostatin	0.2 mM
Butylated hydroxytoluene	5 μΜ
Deferoxamine	3 mΜ

CRITICAL: Prepare a master mix divided among all of the samples as its use minimizes pipetting errors that could ultimately affect quantification of the damage products.

• 26.Add 6 μL of isotopically labeled internal standard mixture to each mixture and mix gently by pulse-vortexing for 5–10 s.

Isotopically labeled internal standard mixture

Isotopically labeled internal standard	Concentration
[15N5]-5′S-cdA	1 μL of 1200 ng/mL
[15N5]-5′R-cdA	1 μL of 1200 ng/mL
[15N5]-5′S-cdG	1 μL of 1200 ng/mL
[15N5]-5′R-cdG	1 μL of 1200 ng/mL
[15N5]-8-oxo-dG	1 μL of 2500 ng/mL
[15N5]-8-oxo-dA	1 μL of 1500 ng/mL

CRITICAL: Prepare a master mix of isotopically labeled internal standards divided among all of the samples.

• 27.Add 10 μL of a cocktail for DNA hydrolysis (Digestion Solution A) and mix gently by gentle pipetting for 5–10 s.

Digestion Solution A

Component	Concentration
Benzonase in 20 mM Tris-HCl pH 8.0	3 U
Magnesium chloride	2 mM
Sodium chloride	20 mM
phosphodiesterase I	4 mU
DNase I	3 U
phosphodiesterase II	2 mU
alkaline phosphatase	2 U

CRITICAL: Prepare a master mix divided among all of the samples.

CRITICAL: Addition of phosphodiesterase I at this step, along with the alkaline phosphatase ensures complete hydrolysis of the DNA sample to nucleosides.

• 28.Incubate the samples overnight at 37°C.
• 29.Add 35 μL of a s cocktail for DNA hydrolysis (Digestion Solution B) and mix gently by gentle pipetting for 5–10 s.

Digestion Solution B

Component	Concentration
Sodium acetate pH 5.6	0.3 M
Zinc chloride	10 mM
Nuclease P1 (in 30 mM sodium acetate pH 5.3, 5 mM zinc chloride and 50 mM sodium chloride)	0.5 U
phosphodiesterase II	4 mU
DNase II	125 mU

• 30.Incubate the mixture overnight at 37°C.
• 31.Quench the digestion mixture with 1% formic acid solution (final pH∼7).
• 32.After the digestion, pipette the total volume of the digestion mixture into a microspin filter (3 kDa).
• 33.Filter off the enzymes by centrifugation at 14,000 × g at 4°C for 20 min, rinse water, spin filter and centrifuge again at 14,000 × g at 4°C for 20 min.
• 34.Transfer the filtrate into a 2-mL screw top vial (Agilent or Waters), cap the vial with a parafilm and make few holes.
• 35.Freeze-dry the filtrate before HPLC analysis using a lyophilizer under a vacuum in order to remove water from the sample.

Pause point: Cap the vial. The lyophilized sample can be stored at the recommended temperature of −80°C. The sample can be stored at −20°C for at least 2 weeks.

HPLC-UV quantification, pre-purification and sample enrichment for LC-MS/MS analysis

Timing: 12 h

HPLC-UV clean-up and enrichment of the enzyme free samples were performed under conditions reported in the section setting up the HPLC-UV method of analysis. The fractions containing the lesions were collected. The collected fractions were freeze-dried, pooled, freeze-dried again and re-dissolved in Milli-Q water before been injected for LC-MS/MS analysis. The quantification of the unmodified nucleosides was based on their absorbance at 260 nm.

CRITICAL: perform a thorough cleanup of the HPLC system and check by LC-MS/MS analysis of blanks that the system is clean prior any sample analysis particularly if the same system is used in the purification of the standards and internal standards after synthesis.

Measurement of modified nucleosides by LC-MS/MS

Timing: 2–3 days

A LC-MS/MS system Finnigan TSQ Quantum Discovery Max triple-stage quadrupole mass spectrometer (Thermo, USA), equipped with electrospray ionization (ESI) source in positive mode was employed for the detection and quantification of the lesions in the enzymatically digested DNA samples.

• 36.Reconstitute the collected fractions after HPLC analysis in 100 μL DM water.
• 37.Placed the samples in a 2-mL vial equipped with a 150 μL glass insert.
• 38.Use an ESI LC-MS/MS system, equipped with a 100 μL loop, a 2.1 mm × 150 mm Atlantis dC18, 100 Å column (3 μm particle size, Waters) guarded by a 2.1 mm × 10 mm Guard Column (Atlantis dC18 3 μm, Waters).
• 39.Use the following gradient elution program used for the chromatographic separation of the DNA lesions initiated with 99% of 2 mM ammonium formate (solvent A) and 1% acetonitrile (solvent B) (keep for 1 min), increase solvent B from 1% to 9.8% within 20 min and then immediately to 15% solvent B (keep for 5 min). Return to initial conditions and wait for 10 min to re-equilibrate.
• 40.Keep the flow rate remained constant at 0.2 mL/min, and the injection volume was 30 μL and column temperature was set at 30°C.
• 41.Use the following calibration parameters to fill in the schedule parameters of the triple quadrupole tandem mass spectrometer. Set the TurboIonSpray probe operating in positive ion mode with an ion spray voltage of 5.5 kV (IS) and nitrogen as collision gas (CAD) at medium. Set the probe temperature to 650°C, curtain gas to 15 psi, and nebulizing gas 1 and 2 to 80 psi and 60 psi, respectively.

Lesion	Rt (min.)	Precursor ion (m/z)	Product ions (m/z)	Declustering potential (V)	Entrance potential	Collision energy (eV)	Cell exit potential (V)
5′R-cdG	5.1	266.2	180.1	60	10	25	10
			202.1	60	10	35	12
5′R-cdA	9.0	250.1	164.2	65	10	20	9
			136.2	65	10	40	6
5′S-cdG	10.7	266.2	180.1	60	10	30	10
			202.1	60	10	35	11
8-oxo-dG	13.2	284.1	168.2	50	10	20	8
			140.1	50	10	45	7
5′S-cdA	15.2	250.2	164.1	65	10	25	9
			136.2	65	10	40	6
8-oxo-dA	18.1	250.2	164.1	65	10	25	9
			136.2	65	10	40	6

• 42.Use the previously prepared solutions of known concentration (Step: setting up the HPLC-UV method of analysis) containing the lesions for preparing 10 ppb aqueous solution of each lesion and inject for checking the mass detector sensitivity and chromatography.
• 43.Prepare diluted solutions of the lesions for calculating the lower limit of detection (LOD) and the lower limit of quantification (LOQ).
• 44.Check the response by changing the injection volumes from 5 μL up to 30 μL in a no wastes mode. A linear response is expected.
• 45.Inject replicates of the lesions solutions and record the chromatograms. Prepare the response curves and check linearity and reproducibility.
• 46.Use the previously prepared, concentrated, solutions containing the ¹⁵N labeled lesions to make diluted solutions and subject to LC-MS/MS analysis for evaluating the isotopic purity and calculating their concentration.
• 47.Prepare the calibration solutions containing both the unlabeled and ¹⁵N labeled lesions.
• 48.Prepare the response curves and check again linearity, reproducibility and carryover.

CRITICAL: evaluate carry over effects periodically by using blanks.

Expected outcomes

Successfully running the protocol will result in setting up the HPLC-UV method of analysis of ten 2′-deoxynucleosides, i.e., the four unmodified DNA nucleosides, the four cPu and the two 8-oxo-Pu (Figure 2). Initially, the preparation of ¹⁵N isotopically enriched cPu and 8-oxo-Pu is executed. The stepwise procedure for the quantification of 8-oxo-Pu and cPu lesions includes the isolation of genomic DNA from the biological samples, either by tissue harvesting or cell lysis, and the appropriate digestion to single nucleosides by an enzymatic cocktail containing nucleases. Afterward, HPLC-UV clean-up and enrichment of the enzyme free samples is carried out while last steps describe the determination and quantification of modified nucleosides by liquid chromatography coupled with tandem mass spectrometry including calibration solutions and response curves related to linearity, reproducibility and carryover.

Lesions quantification and statistical analysis

All measurements were performed in triplicate and the data are expressed as mean ± standard deviation (SD). The unpaired t-test is used for statistical analysis and a two-tailed p-value < 0.05 and p-value < 0.01 are considered to indicate a statistical significant difference.

Limitations

• •
  The lower level of detection (LLOD) and lower level of quantification (LLOQ) of the LC-MS/MS method dictates the minimum amount of DNA input per sample that can be analyzed.

  Note: The LC-MS/MS system stability regarding the sensitivity and reproducibility should be evaluated periodically for re-evaluation of the LLOD and LLOQ.

• •The protocol cannot be applied in DNA extracted with other than the described method and protocol that do not take into consideration the artifactual oxidation during workup and the action of deaminase enzymes.
• •Partial application of the protocol or modifications (e.g., in enzymes used for enzymatic digestion, incubation times, use of antioxidants, etc.) may jeopardize the reliability of the data received.

Troubleshooting

Problem 1

Cross contamination of isotopically enriched nucleosides during synthesis and purification from the glassware, instrumentation and everything used previously also for the synthesis of the non-isotopically enriched standards.

Potential solution

In case of the mass spectrometry analysis indicate contamination of the isotopically labeled nucleosides with the unlabeled ones, the material has to be discarded and the synthesis should be repeated. Ensure all the glassware and equipment to be used is thoroughly cleaned or they haven’t been used before for the synthesis of the unlabeled nucleosides.

Problem 2

Retention time of analytes are drifting during the sample purification and enrichment.

Potential solution

In case of small changes in the retention time of the four major analytes (dC, dG, dT and dA) during the analysis of the samples, make a small adjustment to the collection time windows of the lesions. If the changes in the elution time are severe, then perform extensive column wash and consider to involve a column wash step after a small number of analysis performed.

Problem 3

If DNA is going to be used for experiments with ROS etc. then be sure you have it free of salts, EDTA, thiols and antioxidants before use since it’s going to give you different reactivity.

Potential solution

EtOH precipitation or 100 kDa centrifuge filters can be used to wash out the unwanted molecules etc.

Problem 4

Commercially available ctDNA is shipped in buffers and EDTA that you need to take into consideration before using it in reactions. Needs 1 day to be gently shaking in solution to achieve maximum solubilization.

Potential solution

It takes 1 day under gentle shaking to achieve maximum solubilization.

Problem 5

The pH of the sample after removal of the enzymes is not within the acceptable range pH 6–7.

Potential solution

When you finish with the centrifuge then take a small drop and check the pH with a pH indicator paper. If not close to ∼ pH = 6–7 then adjust it with few drops of 10% formic acid solution you have prepared in advance. Note: the formic acid needs to be as pure a possible (99%) when preparing the stock 1% solution. The pH should be brought to ∼ pH 6–7 in order to protect the HPLC column from damaging and also for reproducibly and accuracy reasons.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chryssostomos Chatgilialoglu (chrys@isof.cnr.it).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Michael A. Terzidis (mterzidis@ihu.gr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The published article includes all datasets generated or analyzed during this study.^{4,5,6,7,8,9,10,11,12,13} This study did not generate new datasets.