Mass SpectrometryCharacterization of Thiol ConjugatesLinked to Polyoxygenated Polyunsaturated Fatty Acid Species

Radical mediated oxidation of polyunsaturated fatty acids (PUFA) are known to generate a series of poly-oxygenated cyclic products (PUFA-On, n≥3). Here we describe the characterization of glutathione (GSH)-conjugates bound to poly-oxygenated docosahexaenoic (DHA-On, n=3-9), arachidonic (ARA-On, n=3-7), α-linolenic (ALA-O3) and linoleic (LA-O3) acid species. Similar conjugates were also characterized for N-acetylcysteine (NAC) and Cu,Zn-superoxide dismutase (SOD1). Extensive LC-MS/MS characterization using a synthetic α-linolenic hydroxy-endoperoxide (ALA-O3) derivative revealed at least two types of mechanisms leading to thiol adduction. A mechanism involving the nucleophilic attack by thiolate anion on 1,2-dioxolane to form a sulfenate ester-bonded conjugate and a mechanism involving cleavage of the dioxolane to form a α,β-unsaturated carbonyl followed by Michael addition reaction. Finally, we detected a GSH conjugate with hydroxy-endoperoxide derived from linoleic acid (LA-O3) in mice liver. In summary,...