Direct characterization of protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal using electrospray mass spectrometry.

Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that > 99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2-nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins.