Sun Gang, Anderson Vernon E
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
Electrophoresis. 2004 Apr;25(7-8):959-65. doi: 10.1002/elps.200305800.
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, cytochrome c was 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, cytochrome c isolated 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.
在蛋白质组学研究中，由于常规使用凝胶电泳来分离多种蛋白质，因此防止十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳过程中发生的人为蛋白质氧化对于鉴定与人类疾病相关的生理蛋白质氧化至关重要。为了开发一种能完全防止SDS聚丙烯酰胺凝胶电泳中人为蛋白质氧化的方法，将细胞色素c在聚丙烯酰胺凝胶上进行电泳，然后进行胰蛋白酶胶内消化，接着通过质谱对胰蛋白酶肽段进行分析。研究发现，在凝胶聚合前对丙烯酰胺溶液进行脱气以去除分子氧，是保护电泳蛋白质免受电泳过程中产生的活性氧影响的关键步骤。然而，仍存在显著的人为蛋白质氧化现象，只有当蛋白质在以黄素作为光引发剂、阴极缓冲液中包含硫代乙醇酸盐作为活性氧清除剂进行光聚合的SDS凝胶上进行电泳时，才能完全消除这种现象。使用这种方法组合，从成年大鼠心脏线粒体中分离的细胞色素c被纯化并消化，随后进行质谱分析，证明了聚丙烯酰胺凝胶所需的高分辨率以及人为氧化的完全消除。
