Long-lived 4-oxo-2-enal-derived apparent lysine Michael adducts are actually the isomeric 4-ketoamides

There has been significant recent interest in the protein reactivity and biological activity of 4-oxo-2-nonenal (ONE), the 4-keto cousin of HNE. Despite the ability of HNE to form at least metastable Michael adducts with protein Lys residues, mass spectrometric evidence for the existence of apparent ONE-Lys Michael adducts is not supported by preparative solution studies. We herein show that the m+154 adducts on Lys residues that persist upon incubating with ONE represent the isomeric 4-ketoamides. The same type of 4-ketoamide represents the apparent Michael adduct on Lys residues formed by the carboxy-terminating ONE-like 4-oxo-2-enal arising along with ONE from the oxidation of linoleic acid.