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Description

Reactive aldehydes generated during lipid peroxidation contribute to cell and tissue damage associated with oxidative stress. Alpha,beta-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily interact with nucleophilic groups on proteins, leading to intramolecular or intermolecular protein crosslinking. Two types of HNE modifications exist, the first type is formed by a 1:1 Michael adduct reaction with lysine amine, histine imidazole, or cysteine sulfhydryl, while the second type is produced by one HNE reacting simultaneously with two free amine groups from two N-alpha-acetyllysines (NALs), so-called Michael-Schiff base adduct. In the latter case, the initial nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base further undergoes oxidative cyclization to form a fluorescent 2-hydroxy-3-imino-1,2-dihydropyrrol derivative commonly referred to as HNE fluorophore. Reversibly formed HNE-Lys Michael adduct is not stable and reduction by sodium borohydride (NaBH4) is often employed to preserve Lys HNE mofidication in protein samples. Reductive stabilization by NaBH4 treatment is also used to prevent 4-HNE loss from 4-HNE-Cys Michael adduct by retro-Michael addition reaction (RMA) during collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS.