Ba. Bruenner et al., MAXIMUM-ENTROPY DECONVOLUTION OF HETEROGENEITY IN PROTEIN MODIFICATION - PROTEIN ADDUCTS OF 4-HYDROXY-2-NONENAL, Rapid communications in mass spectrometry, 8(7), 1994, pp. 509-512
To explore the chemistry of the reactions of the cytotoxic aldehyde tr
ans-4-hydroxy-2-nonenal (HNE) with proteins, we incubated this aldehyd
e in vitro with beta-lactoglobulin B, a model protein of molecular wei
ght 18 277 Da. Direct characterization of reaction products using elec
trospray ionization mass spectrometry yielded spectra whose complexity
suggested extensive product heterogeneity. Spectra were transformed t
o a true mass scale using both a conventional transform algorithm and
a maximum entropy algorithm. Both transformations demonstrated the for
mation of aldehyde-protein adducts containing from three to nine aldeh
yde molecules per molecule of protein. Maximum entropy deconvolution r
esolved Schiff base adducts and/or dehydration products, differing fro
m the Michael addition adducts by 18 Da. The dominant reaction pathway
, however, was Michael addition of the aldehyde to nucleophilic functi
onal groups on the protein. The large number of Michael adducts relati
ve to the one available cysteine requires that other amino acids, such
as histidine and lysine, also be modified. The data suggest that meth
ods for analysis of HNE that involve displacement of Schiff base group
s from proteins will only recover a small fraction of HNE.