Development of a peptide mapping procedure to identify and quantify methionine oxidation in recombinant human α1-antitrypsin

A peptide mapping procedure was developed to identify and quantify methionine oxidation in recombinant human α1-antitrypsin. Due to the protein’s complex structural biochemistry, chromatographic analysis of methionine containing digest peptides was a significant challenge. However, by using a combination of mass spectrometry, protein engineering, and high-temperature reversed-phase liquid chromatography, we were able to identify methionine residues that are susceptible to oxidation by hydrogen peroxide, and quantify their reactivity. Our results show that five of the protein’s 10 methionine residues are susceptible to oxidation at neutral pH, four of which are localized to the active site region.