title: Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins.
abstract: BACKGROUND
  Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative.
  
  
  RESULTS
  We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity.
  
  
  CONCLUSIONS
  Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation.
authors.affiliation: Department of Biochemistry, University of Nebraska, Lincoln, 68588, USA.
authors.forename: Xinwen
authors.initials: X
authors.lastname: Liang
authors.forename: Alaattin
authors.initials: A
authors.lastname: Kaya
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Dung Tien
authors.initials: DT
authors.lastname: Le
authors.forename: Deame
authors.initials: D
authors.lastname: Hua
authors.forename: Vadim N
authors.initials: VN
authors.lastname: Gladyshev
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology; D000818:Animals; D019295:Computational Biology; D003545:Cysteine / Q000378:metabolism; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006861:Hydrogen Peroxide / Q000737:chemistry; D008715:Methionine / Q000031:analogs & derivatives* / Q000737:chemistry*; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003313:Zea mays / Q000201:enzymology
chemical_list: D011994:Recombinant Proteins; D008715:Methionine; D006861:Hydrogen Peroxide; D051150:Methionine Sulfoxide Reductases; D003545:Cysteine; C013111:methionine sulfoxide