Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway

Prostaglandin H-2 has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E-2 and D-2. As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E-2 and prostaglandin H-2-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E-2 and prostaglandin H-2-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.