Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping.
In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.
Li
Chunyan
C
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Takazaki
Shinya
S
Jin
Xiuri
X
Kang
Dongchon
D
Abe
Yoshito
Y
Hamasaki
Naotaka
N
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism; D002700:Chloramines / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017136:Ion Transport; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D014105:Tosyl Compounds / Q000737:chemistry
D001457:Anion Exchange Protein 1, Erythrocyte; D002700:Chloramines; D014105:Tosyl Compounds; C016300:chloramine-T; D008715:Methionine