Isotope Dilution Gas Chromatography-Mass Spectrometric Analysis of Tyrosine Oxidation Products in Proteins and Tissues

  • Jay W. Heinecke
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Oxidative reactions that modify proteins have been implicated in the pathogenesis of aging and disease (1). It has been difficult to identify the physiologically relevant pathways, however, because the reactive intermediates are short-lived. We attempt to determine which oxidative pathways damage proteins in vivo by first identifying stable end products of potential pathways through in vitro experiments. We then analyze normal and diseased tissues for those compounds. For example, two stable isomers of p-tyrosine—ortho-tyrosine and meta-tyrosine—appear after hydroxyl radical modifies protein-bound phenylalanine residues (2, 3, 4). In contrast, o,o′-dityrosine forms when hydroxyl radical crosslinks tyrosine residues. o,o′-Dityrosine also appears when free or protein-bound tyrosine is attacked by tyrosyl radical (5), which is produced from tyrosine and H2O2 by the heme enzyme myeloperoxidase (6,7). Tyrosyl radical does not generate ortho-tyrosine and meta-tyrosine, however (2, 3, 4, 5). Another oxidant, hypochlorous acid (HOCl), produces 3-chlorotyrosine when it reacts with tyrosine (8,9). HOCl is generated only by myeloperoxidase, which requires H2O2 and Cl to perform the reaction. Thus, determining relative levels of ortho-tyrosine, meta-tyrosine, o,o′-dityrosine, and 3-chlorotyrosine can indicate which pathway might have inflicted protein damage in vivo in a particular tissue. These amino acid products are useful markers because they are stable to acid hydrolysis, an essential analytical step.


Tyrosyl Radical Oxidize Amino Acid Phenyl Methyl Silicone Amino Acid Hydrolysate Dityrosine Form 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Shigenaga, M. K., Hagen, T. M., and Ames B. N. (1994) Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA 91, 10,771–10,778.PubMedCrossRefGoogle Scholar
  2. 2.
    Pennathur, S., Wagner, J. D., Leeuwenburgh, C., Litwak, K. N., and Heinecke, J. W. (2001) A hydroxyl radical-like species oxidizes cynomolgus monkey artery wall proteins in early diabetic vascular disease. J. Clin. Invest. 107, 853–860.PubMedCrossRefGoogle Scholar
  3. 3.
    Leeuwenburgh, C., Rasmussen, J. E., Hsu, F. F., Mueller, D. M., Pennathur, S., and Heinecke, J. W. (1997) Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J. Biol. Chem. 272, 3520–3526.PubMedCrossRefGoogle Scholar
  4. 4.
    Pennathur, S., Jackson-Lewis, V., Przedborski, S., and Heinecke, J. W. (1999) Mass spectrometric quantification of 3-nitrotyrosine, orthortho-tyrosine, and o,o′-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson’s disease. J. Biol. Chem. 274, 34,621–34,628.PubMedCrossRefGoogle Scholar
  5. 5.
    Heinecke, J. W., Li, W., Francis, G. A., and Goldstein, J. A. (1993) Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative cross-linking of proteins. J. Clin. Invest. 91, 2866–2872.PubMedCrossRefGoogle Scholar
  6. 6.
    Heinecke, J. W., Li, W., Daehnke, H. L. 3rd, and Goldstein, J. A. (1993) Dityrosine, a specific marker of oxidation, is synthesized by the myelo-peroxidase-hydrogen peroxide system of human neutrophils and macrophages. J. Biol. Chem. 268, 4069–4077.PubMedGoogle Scholar
  7. 7.
    Jacob, J. S., Cistola, D. P., Hsu, F. F., Muzaffar, S., Mueller, D. M., Hazen, S. L., and Heinecke, J. W. (1996) Human phagocytes employ the myeloperoxidase-hydrogen peroxide system to synthesize dityrosine, trityrosine, pulcherosine, and isodityrosine by a tyrosyl radical-dependent pathway. J. Biol. Chem. 271, 19,950–19,956.PubMedCrossRefGoogle Scholar
  8. 8.
    Hazen, S. L., Hsu, F. F., Mueller, D. M., Crowley, J. R., and Heinecke, J. W. (1996) Human neutrophils employ chlorine gas as an oxidant during phagocytosis. J. Clin. Invest. 98, 1283–1289.PubMedCrossRefGoogle Scholar
  9. 9.
    Hazen, S. L. and Heinecke, J. W. (1997) 3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is arkedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 99, 2075–2081.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc.,Totowa, NJ 2003

Authors and Affiliations

  • Jay W. Heinecke
    • 1
  1. 1.Department of MedicineWashington University School of MedicineSt. Louis

Personalised recommendations