Analysis of Oxidative Modification of Proteins

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 )
[³ H]NaBH₄ 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 [¹⁴ C] 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 ).

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 NaHPO₄ /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

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 [¹³ C₆ ] L‐tyrosine (Cambridge Isotope Laboratories) in 0.1 M borate buffer, pH 9.1
30% (v/v) H₂ O₂
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 H₂ O
2.75 × 19.5–cm DEAE cellulose chromatography column (Bio‐Rad)
20 µM NaHCO₃ , pH 8.8 (see recipe )
Concentrated and 100 mM formic acid
100 mM NH₄ HCO₃

Benchtop centrifuge
4 × 34.5–cm BioGel P‐2 column (200‐4‐mesh; Bio‐Rad)

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 [³ H]methyl‐S‐adenosyl‐L‐methionine (5 to 15 Ci/mmol; [³ H] 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: [³ H]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

Figure 7.9.1 Generation of protein carbonyls by glycation and glycoxidation and by reactions with lipid peroxidation products of polyunsaturated fatty acids. (A ) Reactions of protein amino groups (PNH₂ ) with the lipid peroxidation product, malondialdehyde. (B ) Michael addition of 4‐hydroxy‐2‐nonenal to protein lysine (P‐NH₂ ), histidine (P‐His), or cysteine (PSH) residues. (C ) Reactions of sugars with protein lysyl amino groups (P‐NH₂ ). “Me” represents “metal ions.” Abbreviation: ROS, reactive oxygen species.

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Figure 7.9.2 Methods for labeling protein carbonyls: (1) Derivatization of protein carbonyls with 2,4‐ dinitrophenylhydrozine (DNPH), forming protein conjugated dinitrophenylhydrozones. (2) Derivatization of protein carbonyls with tritiated sodium borohydride.

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Figure 7.9.3 Radiolabeling of protein thiol groups with [¹⁴ C]iodoacetamide.

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Figure 7.9.4 Formation of one molecule of o,o′‐dityrosine from two molecules of tyrosine via tyrosyl radical intermediates.

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Figure 7.9.5 Formation of nitrotyrosine via reactive nitrogen species‐mediated nitration of tyrosine.

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Figure 7.9.6 Formation of isoaspartate by the deamidation of asparagine or the isomerization of aspartate.

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Figure 7.9.7 Pathway for the PIMT‐catalyzed methylation of isoaspartate used for quantitation.

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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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Analysis of Oxidative Modification of Proteins

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited