Mass Spectrometric Characterization of Protein Modification by the Products of Nonenzymatic Oxidation of Linoleic Acid

Autoxidation of linoleic acid (LA) enhanced by Fe(II)/ascorbate generates unsaturated hydroperoxides which undergo further oxidative evolution resulting in a mixture of electrophiles, including epoxyketooctadecenoic acid and dienones with intact C-18 chains as well as oxidative cleavage products such as 4-hydroxy-2(E)-nonenal (HNE), 4-oxo-2(E)-nonenal (ONE), 2(E)-octenal, 9-hydroxy-12-oxo-10(E)-dodecenoic acid, 9,12-dioxo-10(E)-dodecenoic acid, and 11-oxoundec-9(E)-enoic acid. Mass spectrometric (MALDI-TOF-MS and LC-ESI-MS/MS) studies have been performed following incubation of the model protein beta-lactoglobulin with LA, Fe(II), and ascorbate, which identified adducts of these electrophiles with three different protein nucleophiles. Deuterium labeled linoleic acid 17,17,18,18,18-d(5)-(9Z,12Z)-octadeca-9,12-dienoic acid (d(5)-LA) was synthesized to facilitate the detection and characterization of the protein modifications by mass spectrometry. Reduction by NaBH4 served to trap reversible adducts and to quantify the number of reducible functional groups in each adduct. This study, which mimics the distribution of reactive lipid peroxidation products generated by a continuous low level flux of reactive oxygen species present in vivo under conditions of oxidative stress, confirms that many irreversibly formed adducts previously identified following exposure of model proteins to pure electrophilic modifiers such as HNE and ONE are also generated during in situ oxidation of LA. These adducts include HNE-His Michael adducts (MA), ONE-Lys 4-ketoamide, ONE-Lys pyrrolinone, and a Cys/His-ONE-Lys pyrrole cross-link. However, reversibly formed adducts, Such as the HNE-Lys Schiff base, are not present at detectable levels. The isotopic labeling allowed less commonly identified mirror-image adducts derived from the carboxy terminus of LA to be identified. A novel 2-octenoic acid-His MA was discovered.