Measurement of Isoketal Protein Adducts by Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry

  • Sean S. Davies
  • Cynthia J. Brame
  • Olivier Boutaud
  • Nathalie Bernoud-Hubac
  • L. Jackson RobertsII
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Oxidative stress has been increasingly implicated in the pathogenesis of a wide variety of diverse human diseases. Free radical damage to lipids, proteins, and DNA may all contribute to the pathogenesis of disease. We have recently discovered a series of highly reactive γ-ketoaldehydes that are formed by rearrangement of bicyclic endperoxide intermediates in the isoprostane (IsoP) pathway of free radical-mediated peroxidation of arachidonic acid (1), which we now term isoketals (IsoKs) (2) (Fig. 1). IsoKs rapidly react with the ε-amine of lysyl residues on proteins to form Schiff base, lactam, and hydroxylactam adducts (1,3,4) (Fig. 2). The rapidity with which IsoKs adduct to proteins exceeds that of other known reactive products of lipid peroxidation, e.g., 4-hydroxynonenal, by orders of magnitude (1). Adduction of proteins frequently leads to altered protein function (5, 6, 7, 8). This in turn can lead to cellular dysfunction, which may be causally linked to the pathogenesis of disease processes.
Fig. 1.

Formation of isoketals. Oxidation of arachidonic acid generates a series of bicyclic endoperoxide intermediates that undergo rearrangement to form a series of γ-ketoaldehyde stereo- and regio-isomers termed isoketals.

Fig. 2.

Formation of lysyl-isoketal adducts. Isoketals rapidly react with the ε-amine of lysine and lysyl residues on proteins to form Schiff base adducts, lactam adducts, and crosslinks.


Schiff Base Potassium Hydroxide Solution Form Schiff Base Resuspend Pellet Butylate Hydroxyl Toluene 
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Copyright information

© Humana Press Inc.,Totowa, NJ 2003

Authors and Affiliations

  • Sean S. Davies
    • 1
  • Cynthia J. Brame
    • 1
  • Olivier Boutaud
    • 1
  • Nathalie Bernoud-Hubac
    • 1
  • L. Jackson RobertsII
    • 1
  1. 1.Departments of Pharmacology and MedicineVanderbilt UniversityNashville

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