Skip to main content
SpringerLink
Log in

Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Since methylation at the N-7 and O6 positions of guanine and the N-3 position of adenine in DNA are the predominant reaction sites, N 7-methylguanine (N 7-MeG), O 6-methylguanine (O 6-MeG), and N 3-methyladenine (N 3-MeA) have been suggested as good biomarkers for assessing exposure to methylating agents. Here, we report the development of a sensitive and selective assay based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) to simultaneously measure N 7-MeG, O 6-MeG, and N 3-MeA in DNA hydrolysates. With the use of isotope internal standards (15N5-N 7-MeG, d 3-O 6-MeG, and d 3-N 3-MeA) and online solid-phase extraction, DNA hydrolysates can be directly analyzed within 12 min without prior sample purification. The limits of detection were 0.02, 0.002, and 0.01 ng/mL on-column (6.1, 0.6, and 3.4 fmol) for N 7-MeG, O 6-MeG, and N 3-MeA, respectively. Inter- and intraday imprecision (CV) were 3.6–9.6% and 2.7–13.6%, respectively. Mean recoveries were 96–109%. This method was then applied to quantitate the amounts of methylated purines in calf thymus DNA treated with methyl methanesulfonate (MMS). The levels of N 7-MeG, O 6-MeG, and N 3-MeA in calf thymus DNA increase with MMS concentration and incubation time. The ratio of relative yields of N 7-MeG, O 6-MeG, and N 3-MeA in MMS-treated DNA was found to be 1.00:0.0032:0.119, respectively. This LC-MS/MS assay provides the sensitivity and high throughput required to evaluate the extent of methylated lesions in DNA induced by methylating agents.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Beranek DT (1990) Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutat Res 231:11–30

    Article  CAS  Google Scholar 

  2. Barrows LR, Magee PN (1982) Nonenzymatic methylation of DNA by S-adenosylmethionine in vitro. Carcinogenesis 3:349–351

    Article  CAS  Google Scholar 

  3. Drablos F, Feyzi E, Aas PA, Vaagbo CB, Kavli B, Bratlie MS, Pena-Diaz J, Otterlei M, Slupphaug G, Krokan HE (2004) Alkylation damage in DNA and RNA—repair mechanisms and medical significance. DNA Repair (Amst) 3:1389–1407

    Article  CAS  Google Scholar 

  4. Shrivastav N, Li D, Essigmann JM (2010) Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation. Carcinogenesis 31:59–70

    Article  CAS  Google Scholar 

  5. Lawrence JF, Leduc R, Ryan JJ (1981) The determination of O 6-methyl- and 7-methylguanine in mouse liver DNA from dimethylnitrosamine-treated mice by high-performance liquid chromatography with UV absorbance detection. Anal Biochem 116:433–438

    Article  CAS  Google Scholar 

  6. Kaur H, Halliwell B (1996) Measurement of oxidized and methylated DNA bases by HPLC with electrochemical detection. Biochem J 318(Pt 1):21–23

    CAS  Google Scholar 

  7. Shuker DE, Durand MJ, Molko D (1993) Fluorescent postlabelling of modified DNA bases. IARC Scientific Publication, pp 227–232

  8. van Delft JH, Steenwinkel MJ, de Groot AJ, van Zeeland AA, Eberle-Adamkiewicz G, Rajewsky MF, Thomale J, Baan RA (1997) Determination of N 7- and O 6-methylguanine in rat liver DNA after oral exposure to hydrazine by use of immunochemical and electrochemical detection methods. Fundam Appl Toxicol 35:131–137

    Article  Google Scholar 

  9. Shuker DE, Friesen MD, Garren L, Prevost V (1991) A rapid gas chromatography–mass spectrometry method for the determination of urinary 3-methyladenine: application in human subjects. IARC Scientific Publication, pp 102–106

  10. Farmer PB, Bailey E, Naylor S, Anderson D, Brooks A, Cushnir J, Lamb JH, Sepai O, Tang YS (1993) Identification of endogenous electrophiles by means of mass spectrometric determination of protein and DNA adducts. Environ Health Perspect 99:19–24

    Article  CAS  Google Scholar 

  11. Haque K, Cooper DP, van Delft JH, Lee SM, Povey AC (1997) Accurate and sensitive quantitation of N 7-methyldeoxyguanosine-3′-monophosphate by 32P-postlabeling and storage-phosphor imaging. Chem Res Toxicol 10:660–666

    Article  CAS  Google Scholar 

  12. Singh R, Farmer PB (2006) Liquid chromatography–electrospray ionization–mass spectrometry: the future of DNA adduct detection. Carcinogenesis 27:178–196

    Article  CAS  Google Scholar 

  13. Hu CW, Chao MR, Sie CH (2010) Urinary analysis of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction: study of 8-oxo-7,8-dihydroguanine stability. Free Radic Biol Med 48:89–97

    Article  CAS  Google Scholar 

  14. Chao MR, Wang CJ, Yang HH, Chang LW, Hu CW (2005) Rapid and sensitive quantification of urinary N 7-methylguanine by isotope-dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on-line solid-phase extraction. Rapid Commun Mass Spectrom 19:2427–2432

    Article  CAS  Google Scholar 

  15. Hu CW, Yen CC, Huang YL, Pan CH, Lu FJ, Chao MR (2010) Oxidatively damaged DNA induced by humic acid and arsenic in maternal and neonatal mice. Chemosphere 79:93–99

    Article  CAS  Google Scholar 

  16. De Bont R, van Larebeke N (2004) Endogenous DNA damage in humans: a review of quantitative data. Mutagenesis 19:169–185

    Article  Google Scholar 

  17. Stokvis E, Rosing H, Beijnen JH (2005) Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun Mass Spectrom 19:401–407

    Article  CAS  Google Scholar 

  18. Chadt J, Sykora D, Nilsson R, Vodicka P (2008) Monitoring of dimethyl sulphate-induced N 3-methyladenine, N 7-methylguanine and O(6)-methylguanine DNA adducts using reversed-phase high performance liquid chromatography and mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 867:43–48

    Article  CAS  Google Scholar 

  19. Yang Y, Nikolic D, Swanson SM, van Breemen RB (2002) Quantitative determination of N 7-methyldeoxyguanosine and O 6-methyldeoxyguanosine in DNA by LC-UV-MS-MS. Anal Chem 74:5376–5382

    Article  CAS  Google Scholar 

  20. Zhang F, Bartels MJ, Pottenger LH, Gollapudi BB, Schisler MR (2006) Simultaneous quantitation of 7-methyl- and O 6-methylguanine adducts in DNA by liquid chromatography–positive electrospray tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 833:141–148

    Article  CAS  Google Scholar 

  21. Feng S, Roethig HJ, Liang Q, Kinser R, Jin Y, Scherer G, Urban M, Engl J, Riedel K (2006) Evaluation of urinary 1-hydroxypyrene, S-phenylmercapturic acid, trans, trans-muconic acid, 3-methyladenine, 3-ethyladenine, 8-hydroxy-2′-deoxyguanosine and thioethers as biomarkers of exposure to cigarette smoke. Biomarkers 11:28–52

    Article  CAS  Google Scholar 

  22. Mustonen R, Hemminki K (1992) 7-Methylguanine levels in DNA of smokers’ and non-smokers’ total white blood cells, granulocytes and lymphocytes. Carcinogenesis 13:1951–1955

    Article  CAS  Google Scholar 

  23. Mustonen R, Schoket B, Hemminki K (1993) Smoking-related DNA adducts: 32P-postlabeling analysis of 7-methylguanine in human bronchial and lymphocyte DNA. Carcinogenesis 14:151–154

    Article  CAS  Google Scholar 

  24. Zhao C, Tyndyk M, Eide I, Hemminki K (1999) Endogenous and background DNA adducts by methylating and 2-hydroxyethylating agents. Mutat Res 424:117–125

    Article  CAS  Google Scholar 

  25. Hecht SS (1999) DNA adduct formation from tobacco-specific N-nitrosamines. Mutat Res 424:127–142

    Article  CAS  Google Scholar 

  26. Wilson VL, Weston A, Manchester DK, Trivers GE, Roberts DW, Kadlubar FF, Wild CP, Montesano R, Willey JC, Mann DL, Harris CC (1989) Alkyl and aryl carcinogen adducts detected in human peripheral lung. Carcinogenesis 10:2149–2153

    Article  CAS  Google Scholar 

  27. Shields PG, Povey AC, Wilson VL, Weston A, Harris CC (1990) Combined high-performance liquid chromatography/32P-postlabeling assay of N 7-methyldeoxyguanosine. Cancer Res 50:6580–6584

    CAS  Google Scholar 

  28. Lawley PD, Warren W (1976) Removal of minor methylation products 7-methyladenine and 3-methylguanine from DNA of Escherichia coli treated with dimethyl sulphate. Chem Biol Interact 12:211–220

    Article  CAS  Google Scholar 

  29. Gates KS, Nooner T, Dutta S (2004) Biologically relevant chemical reactions of N 7-alkylguanine residues in DNA. Chem Res Toxicol 17:839–856

    Article  CAS  Google Scholar 

  30. Osborne MR, Phillips DH (2000) Preparation of a methylated DNA standard, and its stability on storage. Chem Res Toxicol 13:257–261

    Article  CAS  Google Scholar 

  31. Muller M, Belas FJ, Blair IA, Guengerich FP (1997) Analysis of 1, N 2-ethenoguanine and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in DNA treated with 2-chlorooxirane by high performance liquid chromatography/electrospray mass spectrometry and comparison of amounts to other DNA adducts. Chem Res Toxicol 10:242–247

    Article  CAS  Google Scholar 

  32. Yang YD (1998) Simultaneous determination of creatine, uric acid, creatinine and hippuric acid in urine by high performance liquid chromatography. Biomed Chromatogr 12:47–49

    Article  CAS  Google Scholar 

  33. Beranek DT, Weis CC, Swenson DH (1980) A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis 1:595–606

    Article  CAS  Google Scholar 

  34. Encell L, Shuker DE, Foiles PG, Gold B (1996) The in vitro methylation of DNA by a minor groove binding methyl sulfonate ester. Chem Res Toxicol 9:563–567

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by a grant from the National Science Council, Republic of China (grants NSC 97-2314-B-040-015-MY3 and NSC 100-2628-B-040-001-MY4).

Author information

Authors and Affiliations

  1. Department of Public Health, Chung Shan Medical University, Taichung, 402, Taiwan

    Chiung-Wen Hu

  2. Department of Family and Community Medicine, Chung Shan Medical University Hospital, Taichung, 402, Taiwan

    Chiung-Wen Hu

  3. Department of Occupational Safety and Health, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N Road, Taichung, 402, Taiwan

    Chih-Ming Chen, Hsin Hui Ho & Mu-Rong Chao

Authors
  1. Chiung-Wen Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chih-Ming Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hsin Hui Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mu-Rong Chao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mu-Rong Chao.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hu, CW., Chen, CM., Ho, H.H. et al. Simultaneous quantification of methylated purines in DNA by isotope dilution LC-MS/MS coupled with automated solid-phase extraction. Anal Bioanal Chem 402, 1199–1208 (2012). https://doi.org/10.1007/s00216-011-5559-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5559-1

Keywords

Search

Navigation