The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins toform covalent adducts, and this damage has been implicated in pathologies associated with oxidativestress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that mayserve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry(LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE withHSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues.The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNEMichael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selectedreaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constantsranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 >H247
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K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity insubdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kineticstogether with HSA structure and target residue p
Ka values suggest that location in the hydrophobic bindingcavity and low predicted p
Ka of H242 account for its high reactivity toward HNE. H242 adducts may bepreferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers foroxidative stress.