Analysis of Oxidative Modification of Proteins

Liang‐Jun Yan1

1 Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.4
DOI:  10.1002/0471140864.ps1404s56
Online Posting Date:  April, 2009
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Abstract

Proteins are targets of oxidative modification. This unit describes detailed procedures for the analysis of popular indices of protein oxidation including protein carbonyl formation, loss of protein thiols, and nitrotyrosine and dityrosine formation, as well as isoaspartate formation. Procedures are detailed for the analysis of protein carbonyls labeled with 2,4‐dinitrophenylhydrazine, tritiated sodium borohydride, and biotin‐hydrazide, followed by detection measurements that are based on the distinguishing feature of each labeling chemical. Methods are outlined for the determination of protein cysteine oxidation by quantifying the loss of free protein thiols using radiolabeled [14C]‐iodoacetamide. Protocols are described for the measurement of protein dityrosine by gas chromatography/mass spectrometry, as are the details for the detection of protein nitrotyrosine by a competitive ELISA approach. Finally, methods are described for the quantification of protein‐bound isoaspartate using protein‐L‐isoaspartyl methyltransferase that converts aberrant L‐isoaspartyl residues in peptides and proteins to normal aspartyl residues. Curr. Protoc. Protein Sci. 56:14.4.1‐14.4.28. © 2009 by John Wiley & Sons, Inc.

Keywords: protein oxidative modification; carbonylation; cysteine; dityrosine; isoaspartate; nitrotyrosine

     
 
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Table of Contents

  • Introduction
  • 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: Derivatization of Protein Carbonyls with Biotin‐Hydrazide
  • Basic Protocol 4: Gel Electrophoretic Analysis of Protein Thiol Groups Labeled with [14C]Iodoacetamide
  • Basic Protocol 5: Quantification of Protein Dityrosine Residues by Mass Spectrometry
  • Support Protocol 3: Preparation of o,o′‐Dityrosine Standard
  • Support Protocol 4: Analysis of Protein‐Bound Nitrotyrosine by a Competitive Elisa Method
  • Basic Protocol 6: Enzymatic Analysis of Isoaspartate Formation
  • Support Protocol 5: Gel Electrophoretic Analysis of Isoaspartate Formation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • 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 2E)
  • Bicinchoninic acid protein assay kit (BCA; Pierce)
  • 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‐buffered saline with 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‐buffered saline with and without 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)
  • Immobilon‐P membranes (Millipore)
  • UV‐transparent plastic wrap
  • X‐ray film
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), staining gels with Coomassie blue (unit 10.5), and electroblotting proteins onto membranes (unit 10.7)

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

  Materials
  • Protein solution
  • 3 M Tris⋅Cl, pH 8.6 ( appendix 2E)
  • 0.5 M EDTA, pH 8.0 ( appendix 2E)
  • [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 2E)
  • 0.5% (w/v) SDS/0.1 M NaOH
  • BCA protein assay kit (Pierce)
  • Scintisafe Plus 50% cocktail (Fisher Scientific.)
  • Benchtop centrifuge
  • Scintillation vials
  • Additional reagents and equipment for determination of protein concentration (unit 3.4)
CAUTION: Perform all incubations in a hood as tritium gas may be released during the reaction. Follow standard guidelines for handling, storage, and disposal of radioactive materials ( appendix 2B).

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 10.1) and staining gels with Coomassie blue R‐250 (unit 10.5).
CAUTION: Radioactive materials require special handling. See appendix 2B concerning safe use of radioisotopes.

Basic Protocol 3: Derivatization of Protein Carbonyls with Biotin‐Hydrazide

  Materials
  • Protein mixture
  • Derivatizing buffer (100 mM sodium acetate, 20 mM NaCl, pH 6.0 containing 1% SDS; store at room temperature.
  • 60 mM biotin‐hydrazide prepared in DMSO (aliquot and store at −20°C)
  • 30 mM sodium cyanoborohydride (prepare fresh in PBS before use)
  • 100% trichloroacetic acid
  • 1:1 (v/v) ethanol:ethyl acetate
  • 2‐D gel sample buffer (see recipe)
  • Rotator
  • Additional reagents and equipment for 2‐D procedures (unit 10.4) and immunblot method (unit 10.10)

Basic Protocol 4: 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 2E)
  • 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 10.1) and staining gels with Coomassie blue R‐250 (unit 10.5).
CAUTION: Radioactive materials require special handling. See appendix 2B concerning safe use of radioisotopes.

Basic Protocol 5: Quantification of Protein Dityrosine Residues by Mass Spectrometry

  Materials
  • Tissue sample
  • o,o′‐dityrosine internal standards, labeled and unlabeled (see protocol 8)
  • 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 (see Chapter 16 for details of mass spectrometry)
CAUTION: Radioactive materials require special handling. See appendix 2B concerning safe use of radioisotopes.

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 2E)
  • 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: Radioactive materials require special handling. See appendix 2B concerning safe use of radioisotopes.

Support Protocol 4: Analysis of Protein‐Bound Nitrotyrosine by a Competitive Elisa Method

  Materials
  • 10 µg/ml nitro‐bovine serum albumin (nitro‐BSA; Alexis Biochemicals) in plate coating buffer
  • ELISA buffers (see reciperecipes):
    • 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
  • 96‐well ELISA plates
  • Plastic wrap
  • Plate reader

Basic Protocol 6: Enzymatic Analysis of Isoaspartate Formation

  Materials
  • 0.2 M Bis‐Tris buffer, pH 6.0 (see recipe)
  • 10 uM [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 2E)
  • 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 and follow guidelines on handling, storage, and disposal of radioactive materials ( appendix 2B).
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Figures

Videos

Literature Cited

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   Rogers, L.K., Leinweber, B.L., and Smith, C.V. 2006. Detection of reversible protein thiol modification. Anal. Biochem. 358:171‐184.
   Sethuraman, M., McComb, M.E., Heibeck, T., Costello, C.E., and Cohen, R.A. 2004. Isotope‐coded affinity tag approach to identify and quantify oxidant‐sensitive protein thiols. Mol. Cell Proteomics. 3:273‐278.
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   Tyther, R., Ahmeda, A., Johns, E., and Sheehan, D. 2007. Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats. Proteomics 7:4555‐4564.
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Key References
   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.
   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.
   Reissner, K.J. and Aswad, D.W. 2003. Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals? Cell Mol. Life Sci. 60:1281‐1295.
  Extensive coverage of the mechanisms of isoaspartate formation and its removal by PIMT. Evidence provided for a potential role of isoaspartate formation in regulating normal protein function.
   Stadman, E.R. and Levine, R.L. 2003. Free radical‐mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25:207‐218.
  Detailed biochemical mechanisms of protein oxidation, general principles of intracellular accumulation of oxidized proteins and implication of oxidized proteins in aging and disease.
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