Analysis of Oxidative Modification of Proteins

Liang‐Jun Yan1, Rajindar S. Sohal1

1 Southern Methodist University, Dallas, Texas
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.9
DOI:  10.1002/0471143030.cb0709s14
Online Posting Date:  May, 2002
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Abstract

Reactions between protein molecules and reactive oxygen species (ROS) often lead to the modification of certain amino acid residues such as histidine, lysine, arginine, proline, and threonine, forming carbonyl derivatives. Carbonylation of proteins has thus often been employed for the quantification of generalized protein oxidation. Besides carbonylation, other types of oxidative damage that have been investigated in depth are the modifications of cysteine, tyrosine, and aspartate, or asparagine residues. Except for cysteine residues, whose oxidation is often determined by the loss of protein thiol groups, quantification of oxidative damage to tyrosine, and aspartate residues is usually carried out by the measurement of specific oxidation products such as dityrosine, nitrotyrosine (when nitrogen species are the oxidants), and isoaspartate. Methods described in this unit include spectrophotometry, immunoblotting, radiolabeling, GC/MS, ELISA adapted for analysis of oxidative modification.

     
 
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Table of Contents

  • Basic Protocol 1: Spectrophotometric Quantitation of Protein Carbonyls Using 2,4‐Dinitrophenylhydrazine
  • Support Protocol 1: Immunoblot Detection of Protein Carbonyls
  • Basic Protocol 2: Quantitation of Protein Carbonyls Derivatized with Tritiated Sodium Borohydride
  • Support Protocol 2: Gel Electrophoretic Quantitation of Protein Carbonyls Derivatized with Tritiated Sodium Borohydride
  • Basic Protocol 3: Gel Electrophoretic Analysis of Protein Thiol Groups Labeled with [14C] Iodoacetamide
  • Basic Protocol 4: Quantification of Protein Dityrosine Residues by Mass Spectrometry
  • Support Protocol 3: Preparation of o,o′‐Dityrosine standard
  • Support Protocol 4: Analysis of Protein‐Bound Notrotyrosine by a Competitive ELISA Method
  • Basic Protocol 5: Enzymatic Analysis of Isoaspartate Formation
  • Support Protocol 5: Gel Electrophoretic Analysis of Isoaspartate Formation
  • Reagents and Solutions
  • Commentary
  • Acknowledgements
  • Figures
     
 
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Materials

Basic Protocol 1: Spectrophotometric Quantitation of Protein Carbonyls Using 2,4‐Dinitrophenylhydrazine

  Materials
  • DNPH solution (see recipe)
  • Protein solution
  • 2 M HCl
  • 20% (v/v) trichloroacetic acid solution, ice‐cold (TCA; see recipe)
  • 1:1 (v/v) ethanol/ethyl acetate
  • 0.2% (w/v) SDS/20 mM Tris⋅Cl, pH 6.8 ( appendix 2A)
  • Bicinchoninic acid protein assay kit (BCA; Pierce Co.)
  • Bovine serum albumin (BSA)
  • Benchtop centrifuge
  • Branson 2200 sonicator

Support Protocol 1: Immunoblot Detection of Protein Carbonyls

  Materials
  • DNPH‐treated proteins ( protocol 1)
  • 5% (w/v) nonfat dry milk in Tris‐buffed saline with and without Tween‐20 (TBST; see recipe)
  • Primary antibody (anti‐DNP antibody; Sigma)
  • Secondary antibody: may be horseradish peroxidase–conjugated; select on the basis of nature of primary antibody
  • Tris‐buffed saline with Tween‐20 (TBS and TBST; see recipe)
  • ECL detection solution (Amersham)
  • Minigel electrophoresis unit and transfer unit (Bio‐Rad; also see recipe for minigel recipes in unit 6.1)
  • Immobilon‐P membranes (Millipore)
  • UV‐transparent plastic wrap
  • X‐ray film
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), staining gels with Coomassie blue (unit 6.6), and electroblotting proteins onto membranes (unit 6.2)

Basic Protocol 2: Quantitation of Protein Carbonyls Derivatized with Tritiated Sodium Borohydride

  Materials
  • Protein solution
  • 3 M Tris⋅Cl, pH 8.6 ( appendix 2A)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • [3H]NaBH 4 working solution (see recipe)
  • 2 M HCl
  • 20% (v/v) trichloroacetic acid solution, ice‐cold (TCA; see recipe)
  • 1:1 (v/v) ethanol/ethyl acetate
  • 0.2% (w/v) SDS/20 mM Tris⋅Cl , pH 6.8 ( appendix 2A)
  • 0.5% (w/v) SDS/0.1 M NaOH
  • BCA protein assay kit (Pierce)
  • Scintisafe Plus 50% cocktail (Fisher Scientific.)
  • Benchtop centrifuge
  • Scintillation vials
CAUTION: Perform all incubations in a hood as tritium gas may be released during the reaction. When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D).

Support Protocol 2: Gel Electrophoretic Quantitation of Protein Carbonyls Derivatized with Tritiated Sodium Borohydride

  • Tritiated protein sample (see protocol 3)
  • 30% (v/v) hydrogen peroxide
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and staining gels with Coomassie blue R‐250 (unit 6.6).
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D).

Basic Protocol 3: Gel Electrophoretic Analysis of Protein Thiol Groups Labeled with [14C] Iodoacetamide

  Materials
  • Protein sample
  • 1% (w/v) SDS/0.6 mM Tris⋅Cl buffer, pH 8.6 ( appendix 2A)
  • 2‐mercaptoethanol, neat
  • Nitrogen gas
  • 500 mM [14C] iodoacetamide, 1 µCi/ml (Amersham)
  • 500 mM nonradiolabeled iodoacetamide
  • SDS‐PAGE gels for Bio‐Rad Mini gel system (see recipe):
  •  10% resolving gel
  •  4% stacking gel
  • 10% (v/v) trichloroacetic acid (see recipe)
  • Whatman 3MM filter paper
  • X‐ray film
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and staining gels with Coomassie blue R‐250 (unit 6.1).
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D).

Basic Protocol 4: Quantification of Protein Dityrosine Residues by Mass Spectrometry

  Materials
  • Tissue sample
  • o,o′‐dityrosine internal standards, labeled and unlabeled (see protocol 7)
  • Nitrogen gas
  • 6 M HCl/1% (v/v) benzoic acid/1% (v/v) phenol
  • Argon
  • 10% and 0.1% (v/v) TCA solution (see recipe)
  • 50 mM NaHPO 4/100 µM diethylenetriamine pentaacetic acid (DTPA), pH 7.4
  • 25% methanol
  • 1:3 (v/v) HCl/n‐propyl alcohol
  • 1:4 (v/v) pentafluoropropionic anhydride/ethyl acetate
  • Ethyl acetate
  • n‐propanol
  • 0.1% (w/v) trifluoroacetic acid (TFA)
  • Supelclean SPE reversed‐phase C‐18 column (Supelco)
  • Hewlett Packard 5890 gas chromatography equipped with a 12‐m DB‐1 capillary column interfaced with Hewlett‐Packard 5988A mass spectrometer
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D).

Support Protocol 3: Preparation of o,o′‐Dityrosine standard

  Materials
  • Horseradish peroxidase (grade I; Boehringer Mannheim)
  • 0.1 M borate buffer, pH 9.1 (see recipe)
  • 5 mM L‐tyrosine (Sigma) or [13C 6] L‐tyrosine (Cambridge Isotope Laboratories) in 0.1 M borate buffer, pH 9.1
  • 30% (v/v) H 2O 2
  • 2‐mercaptoethanol
  • 0.01 M NaOH ( appendix 2A)
  • 200 µM borate buffer, pH 8.8: diluted from 0.2 M borate buffer (see recipe) with H 2O
  • 2.75 × 19.5–cm DEAE cellulose chromatography column (Bio‐Rad)
  • 20 µM NaHCO 3, pH 8.8 (see recipe)
  • Concentrated and 100 mM formic acid
  • 100 mM NH 4HCO 3
  • Benchtop centrifuge
  • 4 × 34.5–cm BioGel P‐2 column (200‐4‐mesh; Bio‐Rad)
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D).

Support Protocol 4: Analysis of Protein‐Bound Notrotyrosine by a Competitive ELISA Method

  Materials
  • 10 µg/ml nitro‐bovine serum albumin (nitro‐BSA; Alexis Biochemicals) in plate coating buffer
  • Nitro‐BSA standard (see recipe)
  • ELISA buffers (see recipe):
  •  Plate coating buffer
  •  1× phosphate‐buffered saline/Tween 20 (PBST)
  •  Blocking buffer
  •  1× diethanolamine (DEA) buffer
  • Protein sample
  • Primary antibody: mouse anti‐nitrotyrosine antibodies (Upstate Biotechnology)
  • Secondary antibody: rabbit anti‐mouse IgG conjugated with alkaline phosphatase
  • Tris‐buffered saline/Tween‐20 (TBST; see recipe)
  • 1 mg/ml p‐nitrophenyl phosphate (5‐mg tablets; Sigma) in DEA buffer (see recipe for ELISA buffer)
  • 96‐well ELISA plates
  • Plastic wrap
  • Plate reader

Basic Protocol 5: Enzymatic Analysis of Isoaspartate Formation

  Materials
  • 0.2 M Bis‐Tris buffer, pH 6.0 (see recipe)
  • 10 µM [3H]methyl‐S‐adenosyl‐L‐methionine (5 to 15 Ci/mmol; [3H] SAM; NEN)
  • Protein‐L‐isoaspartyl methyltransferase (PIMT; Promega Corporation or purified from a known source)
  • 0.2 M NaOH ( appendix 2A)
  • Safety‐Solve II counting fluor (Research Products International)
  • Sponge plugs (Jaece Industries): cut into small pieces
  • Scintillation vials with extra caps
CAUTION: [3H]methanol is volatile at room temperature. Perform all reactions under a hood. When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D).
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Figures

Videos

Literature Cited

Literature Cited
   Agarwal, S. and Sohal, R.S. 1994. Aging and proteolysis of oxidized proteins. Arch. Biochem. Biophys. 309:24‐28.
   Aswad, D.W. (ed.). 1995. Deamidation and isoaspartate formation in peptides and proteins. CRC, Boca Raton, Fla.
   Buss, H., Chan, T.P., Sluis, K.B., Domigan, N.M., and Winterbourn, C.C. 1997. Protein carbonyl measurement by a sensitive ELISA method [published errata appear in Free Radic Biol Med 1998 May; 24:1352]. Free Radic. Biol. Med. 23:361‐366.
   Clark, S. 1985. Protein carboxyl methyltransferase: Two distinct classes of enzymes. Annu. Rev. Biochem. 54:479‐506.
   Davies, K.J., Lin, S.W., and Pacifici, R.E. 1987. Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein. J. Biol. Chem. 262:9914‐9920.
   Ellman, G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70‐77.
   Giulivi, C. and Davies, K.J. 1993. Dityrosine and tyrosine oxidation products are endogenous markers for the selective proteolysis of oxidatively modified red blood cell hemoglobin by (the 19 S) proteasome. J. Biol. Chem. 268:8752‐8759.
   Graf, L., Bajusz, S., Patthy, A., Barat, E., and Cseh, G. 1971. Revised amide location for porcine and human adrenocorticotropic hormone. Acta Biochim. Biophys. Acad. Sci. Hung. 6:415‐418.
   Hughes, B.A., Roth, G.S., and Pitha, J. 1980. Age‐related decrease in repair of oxidative damage to surface sulfhydryl groups on rat adipocytes. J. Cell. Physiol. 103:349‐33.
   Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J.C., Smith, C.D., and Beckman, J.S. 1992. Peroxynitrite‐mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophys. 298:431‐437.
   Kim, E., Lowenson, J.D., MacLaren, D.C., Clarke, S., and Young, S.G. 1997. Deficiency of a protein‐repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in mice. Proc. Natl. Acad. Sci. U.S.A. 94:6132‐6137.
   Leeuwenburgh, C., Wagner, P., Holloszy, J.O., Sohal, R.S., and Heinecke, J.W. 1997. Caloric restriction attenuates dityrosine cross‐linking of cardiac and skeletal muscle proteins in aging mice. Arch. Biochem. Biophys. 346:74‐80.
   Levine, R.L. 1983. Oxidative modification of glutamine synthetase. J. Biol. Chem. 258:11823‐11827.
   Levine, R.L., Williams, J.A., Stadtman, E.R., and Shacter, E. 1994. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol. 233:346‐357.
   McKenzie, S.J., Baker, M.S., Buffinton, G.D., and Doe, W.F. 1996. Evidence of oxidant‐induced injury to epithelial cells during inflammatory bowel disease. J. Clin. Invest. 98:136‐141.
   Reanick, A.Z. and Packer, L. 1994. Oxidative damage to proteins: Spectrophotometric method for carbonyl assay. Methods Enzymol. 233:357‐363.
   Stadtman, E.R. 1992. Protein oxidation and aging. Science. 257:1220‐1224.
   Stadtman, E.R. and Berlett, B.S. 1997. Reactive oxygen‐mediated protein oxidation in aging and disease. Chem. Res. Toxicol. 10:485‐494.
   ter Steege, J.C., Koster‐Kamphuis, L., van Straaten, E.A., Forget, P.P., and Buurman, W.A. 1998. Nitrotyrosine in plasma of celiac disease patients as detected by a new sandwich ELISA. Free Radic. Biol. Med. 25:953‐963.
   Yan, L.J., Levine, R.L., and Sohal, R.S. 1997. Oxidative damage during aging targets mitochondrial aconitase. Proc. Natl. Acad. Sci. USA. 94:11168‐11172.
   Yan, L.J. and Sohal, R.S. 1998a. Gel electrophoretic quantitation of protein carbonyls derivatized with tritiated sodium borohydride. Anal. Biochem. 265:176‐82.
   Yan, L.J. and Sohal, R.S. 1998b. Mitochondrial adenine nucleotide translocase is modified oxidatively during aging. Proc. Natl. Acad. Sci U.S.A. 95:12896‐12901.
Key References
   Berlett, B.S. and Stadtman, E.R. 1997. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272:20313‐20316.
  Detailed biochemical mechanisms of protein oxidation, general principles of intracellular accumulation of oxidized proteins and implication of oxidized proteins in aging and disease.
   Heinecke, J.W., Hsu, F.F., Crowley, J.R., Hazen, S.L., Leeuwenburgh, C., Mueller, D.M., Rasmussen, J.E. and Turk, J. 1999. Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: Sensitive and quantitative assays for oxidized amino acids in proteins and tissues. Methods Enzymol. 300:124‐144.
  Extensive description of tyrosine modified products, including dityrosine, nitrotyrosine, and chlorotyrosine, by GC/MS method.
   Aswad, 1995. See above
  Extensive coverage of methods for the quantification of isoaspartate formation in proteins/peptides. A useful collection of examples of isoaspartate formation in individual proteins.
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