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

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
–
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.