Analytical methods for investigation of reactivities of peptide functional groups toward xenobiotic chemicals

Important issues in the analysis of modified proteins and peptides were investigated: identification of sites of modification, factors governing reactivities of cysteine thiols, and identification of putative biomarkers of protein oxidation.; Low energy CID MS/MS using a triple quadrupole mass spectrometer identified sites of protein modification and determined fragmentation dynamics in protonated acrolein thioether conjugates. Because S-(3-oxopropyl)-N-acetyl-L-cysteine undergoes facile retro-Michael loss of acrolein under CID conditions, fragmentation studies were performed on a series of acrolein-peptides and cysteine derivatives to determine whether extensive loss of acrolein impedes structural characterization of acrolein-peptide adducts. Differences in fragmentation dynamics suggest protonation at the sulfur of S-(3-oxopropyl)-N-acetyl-L-cysteine facilitates retro-Michael elimination of acrolein with a low activation energy relative to other fragmentations. Retro-Michael fragmentation was insignificant for acrolein conjugates of glutathione derivatives, suggesting proton sequestration occurs at the multiple amide linkages of the peptide backbone. Peptide-bond cleavage was observed on the N-terminal side of S-(3-oxopropyl)cysteine residues which can be useful in locating cysteine residues in peptides.; Thiol ionization as a determinant of the rate of Michael addition of N-acetylcysteine to 1,4-naphthoquinone was investigated. Rate constants for the Michael addition of N-acetylcysteine to 1,4-naphthoquinone were determined over the range of pH 3-8 using KPLC and diode array detection. The rapid reaction rate, under conditions where formation of thiolate anion was minimal suggests intramolecular general base catalysis involving the carboxylate group of N-acetylcysteine. General base catalysis may influence reactivities of protein thiols toward electrophilic xenobiotic substances at pH conditions where thiolate formation is minimal.; A protocol using gas chromatography-mass spectrometry for identification of putative biomarkers of protein oxidation is presented. Fenton oxidation of the model compound N-acetylphenylalanine produced a diverse mixture of products including acetylated o- and m-tyrosine--which were distinguishable based on relative abundances of peaks in their electron ionization mass spectra. Such oxidation products are promising indicators of in vivo formation of reactive oxygen species such as hydroxyl radical.