Characterization of Thioether‐Linked Protein Adducts of DNA Using a Raney‐Ni‐Mediated Desulfurization Method and Liquid Chromatography‐Electrospray‐Tandem Mass Spectrometry

Goutam Chowdhury1, F. Peter Guengerich2

1 Department of Chemistry, School of Natural Sciences, Shiv Nadar University, G. B. Nagar, 2 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.15
DOI:  10.1002/0471142700.nc1015s60
Online Posting Date:  March, 2015
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Abstract

This unit contains a complete procedure for the detection and structural characterization of DNA protein crosslinks (DPCs). The procedure also describes an approach for the quantitation of the various structurally distinct DPCs. Although various methods have been described in the literature for labile DPCs, characterization of nonlabile adducts remain a challenge. Here we present a novel approach for characterization of both labile and non‐labile adducts by the use of a combination of chemical, enzymatic, and mass spectrometric approaches. A Raney Ni‐catalyzed reductive desulfurization method was used for removal of the bulky peptide adducts, enzymatic digestion was used to digest the protein to smaller peptides and DNA to nucleosides, and finally LC‐ESI‐tandem mass spectrometry (MS) was utilized for detection and characterization of nucleoside adducts. © 2015 by John Wiley & Sons, Inc.

Keywords: DNA‐protein crosslinks; desulfurization; structural characterization; Raney Ni; LC‐MS

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Characterization of Thioether‐Linked Protein Adducts of DNA Using a Raney NI‐Mediated Desulfurization Method and Liquid Chromatography‐Electrospray‐Tandem Mass Spectrometry
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Characterization of Thioether‐Linked Protein Adducts of DNA Using a Raney NI‐Mediated Desulfurization Method and Liquid Chromatography‐Electrospray‐Tandem Mass Spectrometry

  Materials
  • Calf thymus DNA (Sigma‐Aldrich)
  • DNase‐free water
  • Authentic standards (from commercial suppliers or by synthesis)
  • Cross‐linking agent [in this case ethylene dibromide (DBE)]
  • Cross‐linking protein [in this case O6‐alkylguanine DNA alkyltransferase (AGT)]
  • Ethyl iodide
  • Potassium carbonate in DMF
  • Raney Ni (Sigma‐Aldrich)
  • Potassium phosphate buffer, pH 7.7 and 7.4
  • Ethylenediaminetetraacetic acid (EDTA)
  • Ethanol (100%, absolute)
  • Sodium acetate
  • Tris·Cl (Trizma HCl)
  • Calcium chloride
  • Sodium dodecyl sulfate (SDS)
  • Proteinase K
  • Scintillation cocktail
  • Nitrogen
  • Phosphodiesterase I
  • Nuclease P1
  • Alkaline phosphatase
  • DNase I
  • Formic acid (HCO 2H)
  • Acetonitrile (CH 3CN)
  • Sonicator
  • Acquity ultra performance liquid chromatography (UPLC) system (Waters Associates)
  • LTQ Orbitrap mass spectrometer (Thermo Fisher)
  • Acquity BEH octadecylsilane (C18) UPLC column (2.1 mm × 100 mm, 1.7 μm)
  • Octadecylsilane (C18) semi‐prep HPLC column
  • 37°C incubator
  • Benchtop centrifuge
  • Scintillation vials
  • Scintillation counter
  • Heating block
  • Vials (size will be such that the stir bar will properly fit)
  • Stir bar with a plastic extension (see Fig.  )
  • Agilent C18 (octadecylsilane) SPE HPLC column
  • Amicon ultra filter (MWCO of 3000)
  • UV‐vis detector and flow counter
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Figures

Videos

Literature Cited

Literature Cited
  Cho, S.‐H. and Guengerich, F.P. 2012. Conjugation of butadiene diepoxide with glutathione yields DNA adducts in vitro and in vivo. Chem. Res. Toxicol. 25:706‐712.
  Chowdhury, G., Cho, S.‐H., Pegg, A.E., and Guengerich, F.P. 2013. Detection and characterization of 1,2‐dibromoethane‐derived DNA crosslinks formed with O6‐alkylguanine‐DNA alkyltransferase. Angew. Chem. Int. Ed. 52:12879‐12882.
  Cmarik, J.L., Humphreys, W.G., Bruner, K.L., Lloyd, R.S., Tibbetts, C., and Guengerich, F.P. 1992. Mutation spectrum and sequence alkylation selectivity resulting from modification of bacteriophage M13mp18 DNA with S‐(2‐chloroethyl)glutathione. Evidence for a role of S‐(2‐N7‐guanyl)ethyl)glutathione as a mutagenic lesion formed from ethylene dibromide. J. Biol. Chem. 267:6672‐6679.
  Hecht, S.M., Kirkegaard, L.H., and Bock, R.M. 1971. Chemical modifications of transfer RNA species. Desulfurization with Raney nickel. Proc. Natl. Acad. Scie. U. S. A. 68:48‐51.
  Humphreys, W.G., Kim, D.‐H., Cmarik, J.L., Shimada, T., and Guengerich, F.P. 1990. Comparison of the DNA‐alkylating properties and mutagenic responses of a series of S‐(2‐haloethyl)‐substituted cysteine and glutathione derivatives. Biochemistry 29:10342‐10350.
  Liu, L., Pegg, A.E., Williams, K.M., and Guengerich, F.P. 2002. Paradoxical enhancement of the toxicity of 1,2‐dibromoethane by O6‐alkylguanine‐DNA alkyltransferase. J. Biol. Chem. 277:37920‐37928.
  Ozawa, N. and Guengerich, F.P. 1983. Evidence for formation of an S‐[2‐(N7‐guanyl)ethyl]glutathione adduct in glutathione‐mediated binding of the carcinogen 1,2‐dibromoethane to DNA. Proc. Natl. Acad. Sci. U.S.A. 80:5266‐5270.
  Reardon, J.T. and Sancar, A. 2006a. Purification and characterization of Escherichia coli and human nucleotide excision repair enzyme systems. Methods Enzymol. 408:189‐213.
  Reardon, J.T. and Sancar, A. 2006b. Repair of DNA‐polypeptide crosslinks by human excision nuclease. Proc. Natl. Acad. Sci. U.S.A. 103:4056‐4061.
  Taghizadeh, K, McFaline, J.L., Pang, B., Sullivan, M., Dong, M., Plummer, E., and Dedon, P.C. 2008. Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry. Nat. Protoc. 3:1287‐1298.
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