title: Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis.
abstract: Methionine (Met) oxidation is a major modification of proteins, which converts Met to Met sulfoxide as the common product. It is challenging to determine the level of Met sulfoxide, because it can be generated during sample preparation and analysis as an artifact. To determine the level of Met sulfoxide in proteins accurately, an isotope labeling and LC-MS peptide mapping method was developed. Met residues in proteins were fully oxidized using hydrogen peroxide enriched with (18)O atoms before sample preparation. Therefore, it was impossible to generate Met sulfoxide as an artifact during sample preparation. The molecular weight difference of 2 Da between Met sulfoxide with the (16)O atom and Met sulfoxide with the (18)O atom was used to differentiate and calculate the level of Met sulfoxide in the sample originally. Using a recombinant monoclonal antibody as a model protein, much lower levels of Met sulfoxide were detected for the two susceptible Met residues with this new method compared to a typical peptide mapping procedure. The results demonstrated efficient elimination of the analytical artifact during LC-MS peptide mapping for the measurement of Met sulfoxide. This method can thus be used when accurate determination of the level of Met sulfoxide is critical.
authors.affiliation: Protein Characterization, Alexion Pharmaceuticals, Inc. , 352 Knotter Drive, Cheshire, Connecticut 06410, United States.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Liu
authors.forename: Gomathinayagam
authors.initials: G
authors.lastname: Ponniah
authors.forename: Alyssa
authors.initials: A
authors.lastname: Neill
authors.forename: Rekha
authors.initials: R
authors.lastname: Patel
authors.forename: Bruce
authors.initials: B
authors.lastname: Andrien
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D002853:Chromatography, Liquid / Q000379:methods*; D003412:Cricetulus; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012451:Safrole / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011506:Proteins; D008715:Methionine; D012451:Safrole; C005746:sulfoxide