Angiotensin II modification by decomposition products of linoleic acid-derived lipid hydroperoxide

•EDE and HPNE preferentially modified the N-terminus and His6 of angiotensin (Ang) II.•Mixture of [12C18]- and [13C18]-13-HPODE was incubated with Ang II and ascorbic acid.•Doublet MS peak and mass difference aided the identification of modified Ang II.•Major modifications derived from linoleic acid peroxidation were determined.•Identified modifications as potential biomarkers of lipid-mediated oxidative damage.

Polyunsaturated fatty acids are highly susceptible to oxidation induced by reactive oxygen species and enzymes, leading to the formation of lipid hydroperoxides. The linoleic acid (LA)-derived hydroperoxide, 13-hydroperoxyoctadecadienoic acid (HPODE) undergoes homolytic decomposition to reactive aldehydes, 4-oxo-2(E)-nonenal (ONE), 4-hydroxy-2(E)-nonenal, trans-4,5-epoxy-2(E)-decenal (EDE), and 4-hydroperoxy-2(E)-nonenal (HPNE), which can covalently modify peptides and proteins. ONE and HNE have been shown to react with angiotensin (Ang) II (DRVYIHPF) and modify the N-terminus, Arg2, and His6. ONE-derived pyruvamide-Ang II (Ang P) alters the biological activities of Ang II considerably. The present study revealed that EDE and HPNE preferentially modified the N-terminus and His6 of Ang II. In addition to the N-substituted pyrrole of [N-C4H2]-Ang II and Michael addition products of [His6(EDE)]-Ang II, hydrated forms were detected as major products, suggesting considerable involvement of the vicinal dihydrodiol (formed by epoxide hydration) in EDE-derived protein modification in vivo. Substantial amounts of [N-(EDE − H2O)]-Ang II isomers were also formed and their synthetic pathway might involve the tautomerization of a carbinolamine intermediate, followed by intramolecular cyclization and dehydration. The main HPNE-derived products were [His6(HPNE)]-Ang II and [N-(HPNE − H2O)]-Ang II. However, ONE, HNE, and malondialdehyde-derived modifications were dominant, because HPNE is a precursor of these aldehydes. A mixture of 13-HPODE and [13C18]-13-HPODE (1:1) was then used to determine the major modifications derived from LA peroxidation. The characteristic doublet (1:1) observed in the mass spectrum and the mass difference of the [M + H]+ doublet aided the identification of Ang P (N-terminal α-ketoamide), [N-ONE]-Ang II (4-ketoamide), [Arg2(ONE − H2O)]-Ang II, [His6(HNE)]-Ang II (Michael addition product), [N-C4H2]-Ang II (EDE-derived N-substituted pyrrole), [His6(HPNE)]-Ang II, [N-(9,12-dioxo-10(E)-dodecenoic acid)]-Ang II, and [His6(9-hydroxy-12-oxo-10(E)-decenoic acid)]-Ang II as the predominant LA-derived modifications. These modifications could represent the majority of lipid-derived modifications to peptides and proteins in biological systems.