Kim Helen, Eliuk Shannon, Deshane Jessy, Meleth Sreelatha, Sanderson Todd, Pinner Anita, Robinson Gloria, Wilson Landon, Kirk Marion, Barnes Stephen
Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USA.
Methods Mol Biol. 2007;371:349-91. doi: 10.1007/978-1-59745-361-5_24.
This chapter describes protocols for two-dimensional (2D) gel electrophoresis (isoelectric focusing [IEF] followed by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis [PAGE]), staining of gels with the fluorescent dye Sypro Ruby, 2D gel image analysis, peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS), liquid chromatography (LC)-tandem mass spectrometry (MS/MS), Western blot analysis of protein oxidations, and mass spectrometric mapping of sites of protein oxidations. Many of these methods were used to identify proteins affected in rat brain following ingestion of grape seed extract (GSE), a dietary supplement touted for anti-oxidant activity. Although beneficial actions in cell and animal models of chronic disease have been described for GSE, it has not been shown whether specific proteins were affected, or the nature of the effects. Applying 2D gel proteomics technology allowed discovery of proteins targeted by GSE without a priori knowledge of which one(s) might be affected. The newer 2D blue native (BN) electrophoresis methodology, which resolves protein complexes in a nondenaturing first dimension and then the components of these complexes in a denaturing second dimension, is discussed as a complementary approach. Analysis of protein oxidations and protein-protein interactions have special relevance to aging-related research, since oxidative stress and altered protein interactions may be at the heart of aging-related diseases. Finally, quality control issues related to implementation of high throughput technologies are addressed, to underscore the importance of minimizing bias and randomizing human and technical error in generating large datasets that are expensive and time-consuming to repeat.
本章介绍了二维（2D）凝胶电泳（等电聚焦[IEF]，随后进行十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳[PAGE]）的实验方案、用荧光染料Sypro Ruby对凝胶进行染色、2D凝胶图像分析、使用基质辅助激光解吸电离（MALDI）-飞行时间（TOF）质谱（MS）进行肽质量指纹（PMF）分析、液相色谱（LC）-串联质谱（MS/MS）、蛋白质氧化的蛋白质印迹分析以及蛋白质氧化位点的质谱定位。这些方法中有许多被用于鉴定摄入葡萄籽提取物（GSE）后大鼠脑中受影响的蛋白质，GSE是一种因具有抗氧化活性而备受吹捧的膳食补充剂。尽管在慢性疾病的细胞和动物模型中已描述了GSE的有益作用，但尚未表明是否有特定蛋白质受到影响，以及影响的性质如何。应用二维凝胶蛋白质组学技术能够发现GSE靶向的蛋白质，而无需事先了解哪些蛋白质可能会受到影响。还讨论了更新的二维蓝色天然（BN）电泳方法，该方法在非变性的第一维中分离蛋白质复合物，然后在变性的第二维中分离这些复合物的组分，作为一种补充方法。蛋白质氧化和蛋白质-蛋白质相互作用的分析与衰老相关研究特别相关，因为氧化应激和改变的蛋白质相互作用可能是衰老相关疾病的核心。最后，讨论了与高通量技术实施相关的质量控制问题，以强调在生成昂贵且耗时的大型数据集时尽量减少偏差并随机化人为和技术误差的重要性，这些数据集很难重复获取。
