Measuring Electrophile Stress

Keri A. Tallman1, Andrew Vila1, Ned A. Porter1, Lawrence J. Marnett1

1 Vanderbilt University Medical Center, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.11
DOI:  10.1002/0471140856.tx1711s40
Online Posting Date:  May, 2009
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Polyunsaturated fatty acids are primary targets during oxidative stress. Diffusible electrophilic α,β‐unsaturated aldehydes, such as 4‐hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling and modify gene expression pathways. We describe a global strategy for identifying the protein targets of HNE modification. A similar approach can be used for any electrophiles derived from an oxidized lipid. Curr. Protoc. Toxicol. 40:17.11.1‐17.11.13. © 2009 by John Wiley & Sons, Inc.

Keywords: electrophile; click chemistry; protein modification

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Table of Contents

  • Introduction
  • Basic Protocol 1: Biotin Tagging of Peptides
  • Support Protocol 1: Preparation of Neutravidin Column
  • Basic Protocol 2: Biotin Tagging of Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Biotin Tagging of Peptides

  • 1 mM peptide in 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • 50 mM Al‐HNE in DMSO (see Fig. ; synthesize according to Vila et al., )
  • 2.0 M sodium borohydride (NaBH 4) in triethyleneglycol dimethyl ether
  • 10% (v/v) HCl
  • Acetonitrile (CH 3CN)
  • 100 mM Az‐Biotin in DMSO (see Fig. ; synthesize according to Vila et al., )
  • 100 mM ligand in DMSO (see Fig. ; synthesize according to Chan et al., )
  • 500 mM sodium ascorbate
  • 500 mM CuSO 4
  • 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • Neutravidin column ( protocol 2)
  • 70% (v/v) acetonitrile (CH 3CN)/25% (v/v) H 2O/5% (v/v) formic acid (HCO 2H)
  • 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2A) containing 0.02% (w/v) NaN 3

Support Protocol 1: Preparation of Neutravidin Column

  • 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2A)
  • Neutravidin (Pierce, cat. no. 29200)
  • 50 mM sodium phosphate buffer, pH 7.4 ( appendix 2A) containing 0.02% (w/v) NaN 3
  • Polypropylene column, 1‐ to 5‐ml capacity (Pierce, #29925)
  • Appropriate diameter filter discs (supplied with column)

Basic Protocol 2: Biotin Tagging of Proteins

  • 5 mM Al‐HNE in DMSO (see Fig. ; synthesize according to Vila et al., )
  • 10 mg/ml bovine serum albumin (BSA)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 30 mM Az‐Biotin in DMSO (see Fig. ; synthesize according to Vila et al., )
  • 300 mM tris‐(2‐carboxyethyl)‐phosphine⋅HCl (TCEP) (Pierce, cat. no. 20491)
  • 12.75 mM ligand (see Fig. ; synthesize according to Chan et al., ) in 4:1 (v/v) t‐butyl alcohol:DMSO
  • 300 mM CuSO 4
  • 500 mM sodium cyanoborohydride (NaCNBH 3) (in PBS (see appendix 2A for PBS)
  • Laemmli electrophoresis sample buffer (Pierce) containing 5% β‐mercaptoethanol
  • Precast Tris‐glycine minigel with 10% separating gel and 4% stacking gel (BioRad, cat no. 161‐1101EDU)
  • Streptavidin‐peroxidase from Streptomyces avidinii (HRP‐SA) (Sigma, cat. no. S5512)
  • RKO cells (ATCC, cat. no. CRL‐2577)
  • Complete DMEM medium (see recipe) and serum‐free DMEM
  • DMSO
  • M‐PER mammalian cell lysis buffer (Pierce) with 1% protease inhibitor cocktail (PIC) for mammalian cell culture (Sigma, cat. no P8340) or alternative cell lysis buffer as described in Vila et al. ( ); see recipe
  • 500 mM EDTA stock, pH 8.0
  • 10 mM BHA stock in methanol
  • Methanol, ice cold
  • 0.1 M ammonium bicarbonate (mass spectrometry grade/0.2% (w/v) SDS
  • Streptavidin Sepharose High Performance beads (SA beads; GE Healthcare, cat. no. 17‐5113‐01)
  • 5 M and 1 M NaCl
  • 0.1 M ammonium bicarbonate (mass spectrometry grade)
  • 1% (w/v) SDS
  • 4 M urea
  • Elution solvent: 70% (v/v) acetonitrile (CH 3CN)/25% (v/v) H 2O/5% (v/v) formic acid (HCO 2H)
  • Novex 4% to 20%, Tris‐glycine gradient gel (Invitrogen, cat. no. EC6025)
  • Colloidal Blue Staining Kit (Invitrogen, cat. no. LC6025)
  • 95° and 70°C water bath or heat block
  • Nitrocellulose membrane
  • 15‐cm tissue culture dishes
  • Cell scrapers
  • Centrifuge
  • End‐over‐end rotator
  • SpeedVac evaporator (or equivalent centrifugal vacuum evaporator)
  • Additional reagents and equipment for gel electrophoresis ( appendix 3F) and transfer of proteins from gel to nitrocellulose membrane (unit 2.3)
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Literature Cited

   Baskin, J.M., Prescher, J.A., Laughlin, S.T., Agard, N.J., Chang, P.V., Miller, I.A., Lo, A., Codelli, J.A., and Bertozzi, C.R. 2007. Copper‐free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. U.S.A.. 104:16793‐16797.
   Bennaars‐Eiden, A., Higgins, L., Hertzel, A.V., Kapphahn, R.J., Ferrington, D.A., and Bernlohr, D.A. 2002. Covalent modification of epithelial fatty acid‐binding protein by 4‐hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology. J. Biol. Chem. 277:50693‐50702.
   Carbone, D.L., Doorn, J.A., Kiebler, Z., Sampey, B.P., and Petersen, D.R. 2004. Inhibition of Hsp72‐mediated protein refolding by 4‐hydroxy‐2‐nonenal. Chem. Res. Toxicol. 17:1459‐1467.
   Carbone, D.L., Doorn, J.A., Kiebler, Z., Ickes, B.R., and Petersen, D.R. 2005a. Modification of heat shock protein 90 by 4‐hydroxynonenal in a rat model of chronic alcoholic liver disease. J. Pharmacol. Exp. Ther. 315:8‐15.
   Carbone, D.L., Doorn, J.A., Kiebler, Z., and Petersen, D.R. 2005b. Cysteine modification by lipid peroxidation products inhibits protein disulfide isomerase. Chem. Res. Toxicol. 18:1324‐1331.
   Chan, T.R., Hilgraf, R., Sharpless, K.B., and Fokin, V.V. 2004. Polytriazoles as copper(I)‐stabilizing ligands in catalysis. Org. Lett. 6:2853‐2855.
   Dennehy, M.K., Richards, K.A., Wernke, G.R., Shyr, Y., and Liebler, D.C. 2006. Cytosolic and nuclear protein targets of thiol‐reactive electrophiles. Chem. Res. Toxicol. 19:20‐29.
   Dinkova‐Kostova, A.T., Holtzclaw, W.D., Cole, R.N., Itoh, K., Wakabayashi, N., Katoh, Y., Yamamoto, M., and Talalay, P. 2002. Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc. Natl. Acad. Sci. U.S.A. 99:11908‐11913.
   Grimsrud, P.A., Picklo, M.J., Griffin, T.J., and Bernlohr, D.A. 2007. Carbonylation of adipose proteins in obesity and insulin resistance: Identification of adipocyte fatty acid‐binding protein as a cellular target of 4‐hydroxynonenal. Mol. Cell Proteomics 6:624‐637.
   Grune, T., Siems, W.G., Schonheit, K., and Blasig, I.E., 1993. Release of 4‐hydroxynonenal, an aldehydic mediator of inflammation, during postischaemic reperfusion of the myocardium. Int. J. Tissue React. 15:145‐150.
   Hang, H.C., Yu, C., Kato, D.L., and Bertozzi, C.R. 2003. A metabolic labeling approach toward proteomic analysis of mucin‐type O‐linked glycosylation. Proc. Natl. Acad. Sci. U.S.A. 100:14846‐14851.
   Hartley, D.P., Kroll, D.J., and Petersen, D.R. 1997. Prooxidant‐initiated lipid peroxidation in isolated rat hepatocytes: Detection of 4‐hydroxynonenal‐ and malondialdehyde‐protein adducts. Chem. Res. Toxicol. 10:895‐905.
   Hartley, D.P., Kolaja, K.L., Reichard, J., and Petersen, D.R. 1999. 4‐Hydroxynonenal and malondialdehyde hepatic protein adducts in rats treated with carbon tetrachloride: Immunochemical detection and lobular localization. Toxicol. Applied Pharmacol. 161:23‐33.
   Ji, C., Kozak, K.R., and Marnett, L.J. 2001. IkappaB kinase, a molecular target for inhibition by 4‐hydroxy‐2‐nonenal. J. Biol. Chem. 276:18223‐18228.
   Koen, Y.M., Yue, W., Galeva, N.A., Williams, T.D., and Hanzlik, R.P. 2006. Site‐specific arylation of rat glutathione‐S‐transferase A1 and A2 by bromobenzene metabolites in vivo. Chem. Res. Toxicol. 19:1426‐1434.
   Levonen, A.L., Landar, A., Ramachandran, A., Ceaser, E.K., Dickinson, D.A., Zanoni, G., Morrow, J.D., and Darley‐Usmar, V.M. 2004. Cellular mechanisms of redox cell signalling: Role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products. Biochem. J. 378:373‐382.
   Lin, D., Lee, H.G., Liu, Q., Perry, G., Smith, M.A., and Sayre, L.M. 2005. 4‐Oxo‐2‐nonenal is both more neurotoxic and more protein reactive than 4‐hydroxy‐2‐nonenal. Chem. Res. Toxicol. 18:1219‐1231.
   Marnett, L.J., Riggins, J.N., and West, J.D. 2003. Endogenous generation of reactive oxidants and electrophiles and their reactions with DNA and protein. J. Clin. Invest. 111:583‐593.
   Montine, T.J., Huang, D.Y., Valentine, W.M., Amarnath, V., Saunders, A., Weisgraber, K.H., Graham, D.G., and Strittmatter, W.J. 1996. Crosslinking of apolipoprotein E by products of lipid peroxidation. J. Neuropathol. Exp. Neurol. 55:202‐210.
   Neely, M.D., Sidell, K.R., Graham, D.G., and Montine, T.J. 1999. The lipid peroxidation product 4‐hydroxynonenal inhibits neurite outgrowth, disrupts neuronal microtubules, and modifies cellular tubulin. J. Neurochem. 72:2323‐2333.
   Perluigi, M., Fai Poon, H., Hensley, K., Pierce, W.M., Klein, J.B., Calabrese, V., De Marco, C., and Butterfield, D.A. 2005. Proteomic analysis of 4‐hydroxy‐2‐nonenal‐modified proteins in G93A‐SOD1 transgenic mice: A model of familial amyotrophic lateral sclerosis. Free Radic. Biol. Med. 38:960‐968.
   Sayre, L.M., Zelasko, D.A., Harris, P.L., Perry, G., Salomon, R.G., and Smith, M.A. 1997. 4‐Hydroxynonenal‐derived advanced lipid peroxidation end products are increased in Alzheimer's disease. J. Neurochem. 68:2092‐2097.
   Schneider, C., Tallman, K.A., Porter, N.A., and Brash, A.R. 2001. Two distinct pathways of formation of 4‐hydroxynonenal: Mechanisms of nonenzymatic transformation of the 9‐ and 13‐hydroperoxides of linoleic acid to 4‐hydroxyalkenals. J. Biol. Chem. 276:20831‐20838.
   Simona, S.G., Zhang, B., Sobecki, S.M., Billheimer, D.D., and Liebler, D.C. 2008. Global analysis of protein damage by the lipid electrophile 4‐hydroxy‐2‐nonenal. Mol. Cell. Prot. 8:670‐680.
   Soreghan, B.A., Yang, F., Thomas, S.N., Hsu, J., and Yang, A.J. 2003. High‐throughput proteomic‐based identification of oxidatively induced protein carbonylation in mouse brain. Pharmaceut. Res. 20:1713‐1720.
   Speers, A.E. and Cravatt, B.F. 2004. Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11:535‐546.
   Speers, A.E. and Cravatt, B.F. 2005. A tandem orthogonal proteolysis strategy for high‐content chemical proteomics. J. Am. Chem. Soc. 127:10018‐10019.
   Uchida, K., Szweda, L.I., Chae, H.Z., and Stadtman, E.R. 1993. Immunochemical detection of 4‐hydroxynonenal protein adducts in oxidized hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90:8742‐8746.
   Uchida, K., Toyokuni, S., Nishikawa, K., Kawakishi, S., Oda, H., Hiai, H., and Stadtman, E.R. 1994. Michael addition‐type 4‐hydroxy‐2‐nonenal adducts in modified low‐density lipoproteins: Markers for atherosclerosis. Biochemistry 33:12487‐12494.
   Uchida, K., Itakura, K., Kawakishi, S., Hiai, H., Toyokuni, S., and Stadtman, E.R. 1995. Characterization of epitopes recognized by 4‐hydroxy‐2‐nonenal specific antibodies. Arch. Biochem. Biophys. 324:241‐248.
   Vila, A., Tallman, K.A., Jacobs, A.T., Liebler, D.C., Porter, N.A., and Marnett, L.J. 2008. Identification of protein targets of 4‐hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem. Res. Toxicol. 21:432‐444.
   Wang, Q., Chan, T.R., Hilgraf, R., Fokin, V.V., Sharpless, K.B., and Finn, M.G. 2003. Bioconjugation by copper(I)‐catalyzed azide‐alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125:3192‐3193.
   West, J.D. and Marnett, L.J. 2006. Endogenous reactive intermediates as modulators of cell signaling and cell death. Chem. Res. Toxicol. 19:173‐194.
   Zhang, B., Chambers, M.C., and Tabb, D.L. 2007. Proteomic parsimony through bipartite graph analysis improves accuracy and transparency. J. Proteome Res. 6:3549‐3557.
Key References
   Chan et al., 2004. See above.
  Describes the synthesis of the ligand.
   Vila et al., 2008. See above.
  Describes the synthesis of reagents used in this protocol.
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