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 trans ‐4‐hydroxy‐2‐nonenal (HNE) with proteins, we incubated this aldehyde in vitro with β‐lactoglobulin B, a model protein of molecular weight 18 277 Da. Direct characterization of reaction products using electrospray ionization mass spectrometry yielded spectra whose complexity suggested extensive product heterogeneity. Spectra were transformed to a true mass scale using both a conventional transform algorithm and a maximum entropy algorithm. Both transformations demonstrated the formation of aldehyde‐protein adducts containing from three to nine aldehyde molecules per molecule of protein. Maximum entropy deconvolution resolved Schiff base adducts and/or dehydration products, differing from the Michael addition adducts by 18 Da. The dominant reaction pathway, however, was Michael addition of the aldehyde to nucleophilic functional groups on the protein. The large number of Michael adducts relative to the one available cysteine requires that other amino acids, such as histidine and lysine, also be modified. The data suggest that methods for analysis of HNE that involve displacement of Schiff base groups from proteins will only recover a small fraction of HNE.