Korolainen Minna A, Goldsteins Gundars, Alafuzoff Irina, Koistinaho Jari, Pirttilä Tuula
Department of Neuroscience and Neurology, A.I Virtanen Institute for Molecular Sciences, University of Kuopio, Finland.
Electrophoresis. 2002 Sep;23(19):3428-33. doi: 10.1002/1522-2683(200210)23:19<3428::AID-ELPS3428>3.0.CO;2-5.
There is a growing body of evidence that oxidative stress plays a major role in Alzheimer's disease (AD) pathogenesis. Identification of oxidatively altered proteins in AD is important for understanding the relationship between protein oxidation, protein aggregation and neurodegeneration. In this communication, we report a method that can be applied to study oxidative changes of individual proteins in brain. In order to analyze protein oxidation by detection of protein-bound carbonyls, cytosolic protein extracts were derivatized with 2,4-dinitrophenylhydrazine (DNPH) and then separated by two-dimensional (2-D) gel electrophoresis. After electrotransfer to polyvinylidene difluoride (PVDF) membranes, proteins were first stained with Sypro Ruby protein stain, and then the oxidized proteins were detected with anti-dinitrophenyl (DNP) antibody. About 150 proteins and more than 100 oxidized proteins were detected and quantified in both AD and control cases by 2-D image analysis. The amount of protein-bound carbonyls was decreased for six and increased for one protein in AD. The amount of protein was increased for three proteins in AD. Furthermore, the degree of oxidation was calculated as the ratio of protein-bound carbonyls to the total amount of an individual protein. Two proteins showed a significant decrease in the degree of oxidation in AD. Our results suggest that the balance of protein oxidation and degradation is altered in AD.
越来越多的证据表明，氧化应激在阿尔茨海默病（AD）发病机制中起主要作用。鉴定AD中发生氧化改变的蛋白质对于理解蛋白质氧化、蛋白质聚集与神经退行性变之间的关系很重要。在本通讯中，我们报告了一种可用于研究大脑中单个蛋白质氧化变化的方法。为了通过检测蛋白质结合的羰基来分析蛋白质氧化，将胞质蛋白提取物用2,4-二硝基苯肼（DNPH）衍生化，然后通过二维（2-D）凝胶电泳分离。电转移到聚偏二氟乙烯（PVDF）膜上后，蛋白质首先用Sypro Ruby蛋白染色剂染色，然后用抗二硝基苯基（DNP）抗体检测氧化的蛋白质。通过二维图像分析，在AD和对照病例中均检测并定量了约150种蛋白质和100多种氧化蛋白质。AD中有6种蛋白质结合羰基的量减少，1种蛋白质增加。AD中有3种蛋白质的量增加。此外，氧化程度计算为蛋白质结合羰基与单个蛋白质总量的比率。两种蛋白质在AD中的氧化程度显著降低。我们的结果表明，AD中蛋白质氧化和降解的平衡发生了改变。
