MAXIMUM-ENTROPY DECONVOLUTION OF HETEROGENEITY IN PROTEIN MODIFICATION - PROTEIN ADDUCTS OF 4-HYDROXY-2-NONENAL

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.