Development of a peptide mapping procedure to identify and quantify methionine oxidation in recombinant human alpha1-antitrypsin.
A peptide mapping procedure was developed to identify and quantify methionine oxidation in recombinant human alpha1-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.
Griffiths
Steven W
SW
Massachusetts Institute of Technology, Department of Chemical Engineering, Cambridge 02139, USA. swgriffi@mit.edu
Cooney
Charles L
CL
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D006801:Humans; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry*; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
D017931:DNA Primers; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D008715:Methionine