Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate-gel electrophoresis.
Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate-gel electrophoresis.
复制标题
防止十二烷基硫酸钠凝胶电泳过程中产生的人为蛋白质氧化。
DOI：
10.1002/elps.200305800
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Anderson,VernonE
中科院分区：
文献类型：
--
作者：
Sun,Gang;Anderson,VernonE
十二烷基硫酸钠（SDS）丙烯酰胺凝胶电泳过程中发生的人为蛋白质氧化的预防是至关重要的，由于常规使用凝胶电泳分离蛋白质组学研究中的多种蛋白质，确定与人类疾病有关的生理蛋白质氧化。为了开发一种完全防止SDS丙烯酰胺凝胶电泳中人为蛋白质氧化的方法，将细胞色素固定在聚丙烯酰胺凝胶上，并进行胰蛋白酶凝胶内消化，然后通过质谱法进行胰蛋白酶肽分析。有人发现，脱气的丙烯酰胺溶液，以除去分子氧凝胶聚合之前是一个关键的过程，以保护电泳过程中产生的活性氧物种的蛋白质。然而，显著的人为蛋白质氧化仍然存在，只能完全消除，如果蛋白质在用黄素作为光引发剂和巯基乙酸盐作为活性氧清除剂包括在阴极缓冲液中的SDS凝胶上进行光聚合。使用这种组合的方法，cytochromecisolated从成年大鼠心脏线粒体进行纯化和消化，然后通过质谱分析，证明了所需的高分辨率的聚丙烯酰胺凝胶和人工氧化的整个消除。
Prevention of artifactual protein oxidation occurring during sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis is critical for identifying physiological protein oxidation implicated in human diseases due to the routine use of gel electrophoresis to separate the multiple proteins in proteomic studies. To develop a methodology that completely prevents artifactual protein oxidation in SDS acrylamide gel electrophoresis, cytochromecwas electrophoresed on polyacrylamide gels and subjected to trypsin in‐gel digestion followed by tryptic peptide analysis by mass spectrometry. It was found that degassing the acrylamide solution to remove molecular oxygen prior to gel polymerization is a crucial process to protect the electrophoresed protein from reactive oxygen species generated during electrophoresis. However, significant artifactual protein oxidation remains that can only be eliminated entirely, if proteins are electrophoresed on an SDS gel photopolymerized with flavin as the photoinitiator and thioglycolate included in the cathode buffer as a reactive oxygen species scavenger. Using this combination of methodologies, cytochromecisolated from adult rat heart mitochondria was purified and digested followed by mass spectrometric analysis, demonstrating the requisite high resolution of the polyacrylamide gel and the entire elimination of artifactual oxidation.