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 react ive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. T his aldehyde exhibits a variety of biological effects and has been rep orted as a marker of lipid peroxidation. The toxicity and atherogenici ty of 4-hydroxy-2-nonenal have been attributed to the formation of cov alent protein adducts. In the current study, two model proteins, beta- lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-none nal, and the protein adducts were characterized using electrospray ion ization mass spectrometry. Our findings provided clear and direct evid ence that >99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmatio n of this result was obtained via quantitative conversion of the modif ied proteins to oxime and pentafluorobenzyl oxime derivatives as demon strated by electrospray ionization mass spectrometry, spectrophotometr ic protein carbonyl assays, and gas chromatography/mass spectrometry d etermination of 4-hydroxy-2-nonenal released upon treatment with hydro xylamine. These results further demonstrate the availability of the pr otein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schi ff base adducts also suggests that most methods for determining 4-hydr oxy-2-nonenal in biological tissues or fluids are based on erroneous a ssumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonen al from proteins.