Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping
Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping
复制标题
DOI：
10.1021/bi060627f
复制
发表时间：
2006-10-03
期刊：
影响因子：
2.9
通讯作者：
Hamasaki, Naotaka
中科院分区：
文献类型：
--
作者：
Li, Chunyan;Takazaki, Shinya;Hamasaki, Naotaka
本研究采用肽图结合液相色谱/电喷雾质谱（LC/ESI MS）技术，对红细胞膜蛋白带3的甲硫氨酸氧化进行了研究。最初，我们确定了由氯胺T（N-氯-对甲苯磺酰胺），一种亲水性试剂氧化的蛋氨酸位点。有三个氧化蛋氨酸（蛋氨酸559，蛋氨酸741，和蛋氨酸909）在带3，这些蛋氨酸位于亲水性区域由以前的拓扑研究带3。此外，我们发现，C12 E8，聚氧乙烯去污剂，导致氧化的蛋氨酸在带3的跨膜段，这种氧化发生在C12 E8预孵育时间依赖性的方式。在先前的研究中，发现过氧化物在聚氧乙烯洗涤剂中积累。因此，我们的方法能够直接和定量检测由于洗涤剂过氧化物引起的蛋白质损伤。此外，我们还研究了在4，4 '-二硝基二苯乙烯-2，2'-二磺酸（DNDS）或焦碳酸二乙酯（DEPC）存在下蛋氨酸的氧化，这分别诱导带3中的外向或内向构象。我们的结果表明，Met 741的位置与带3介导的阴离子转运诱导的带3构象有关。总之，我们发现蛋氨酸氧化可用于检查膜蛋白结构如下：（1）膜蛋白的拓扑学研究，（2）用于评估去污剂溶解研究中蛋白质的质量，和（3）用于检测膜蛋白的构象变化。
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.