Ba. Bruenner et al., DIRECT CHARACTERIZATION OF PROTEIN ADDUCTS OF THE LIPID-PEROXIDATION PRODUCT 4-HYDROXY-2-NONENAL USING ELECTROSPRAY MASS-SPECTROMETRY, Chemical research in toxicology, 8(4), 1995, pp. 552-559
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.