突触蛋白质组学分析中双向电泳技术的优化

背景：双向电泳是突触蛋白质组学分析中最流行最通用的蛋白质分离方法之一.但文献中突触蛋白双向电泳对线性固相pH梯度（immobilized pH gradients,IPG）胶条和十二烷基磺酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electroph-oresis,SDS-PAGE）浓度的选择很多,尚未见统一标准.目的：对突触蛋白双向电泳的IPG胶条和SDS-PAGE凝胶浓度进行优化,以获得高质量的突触蛋白双向电泳图谱.方法：以大鼠海马突触蛋白为试材,比较pH 5.0-8.0与pH 3.0-10.0线性IPG胶条,线性与非线性pH 3.0-10.0IPG胶条,以及单一浓度10％与12％ SDS-PAGE对双向电泳的影响.此外,还使用计算机检索了1989至2013年中国知网及PubMed数据库中关于突触蛋白双向电泳的文献,对文献中选择的IPG胶条、SDS-PAGE凝胶浓度进行了统计和评价,并总结了突触蛋白在不同pH值IPG胶条和不同浓度SDS-PAGE凝胶的双向电泳图谱上的分布特征.结果与结论：结果表明,使用pH 3.0-10.0的非线性胶条及单一浓度10％ SDS-PAGE凝胶进行突触蛋白双向电泳较为适宜,电泳图谱质量好,实验操作便利;同时还推荐合并使用pH 4.0-7.0和pH 6.0-11.0的IPG胶条,以及线性梯度浓度9％-16％ SDS-PAGE凝胶,也适用于突触蛋白双向电泳分析.

Optimization of two-dimensional gel electrophoresis for synaptic proteomics

BACKGROUND： Two-dimensional electrophoresis （2-DE） is one of the most popular methods ot protein separation in synaptic proteomic analysis. However, there was no agreed standard on selection of immobilized pH gradients （IPG） strips and concentration of sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） in the research literature on 2-DE of synaptic proteins. OBJECTIVE： To optimize IPG strips and concentration of SDS-PAGE in 2-DE for synaptic proteomic analysis, and to get high-quality 2-DE maps of synaptic proteins. METHODS： Rat hippocampal synapses were adopted in this experiment to compare the effect of 2-DE, which were involved with IPG strips with the linear range of pH 5.0-8.0 and pH 3.0-10.0, IPG strips with the linear and nonlinear range of pH 3.0-10.0, as well as single 10% and 12% SDS-PAGE. Meanwhile, a computer-based online search of PubMed （1989/2013） and CNKI （1989/2013） was performed for the articles about 2-DE of synaptic proteins. The selection of IPG strips and concentration of SDS-PAGE in the research literature weresummarized and evaluated, and the distribution characteristics of the synaptic proteins in 2-DE maps of IPG strips with t . various pH range and SDS-PAGE gels with different concentration were summanzed. RESULTS AND CONCLUSION： The results demonstrated that the synaptic proteins were well separated conveniently and obtained high quality 2-DE maps by nonlinear IPG strips range of pH 3.0-10.0 and single 10% SDS-PAGE gels. At the same time, it was also recommended that combination use of IPG strips range of pH 4.0-7.0 and pH 6.0-11.0, as well as linear gradient 9-16% SDS-PAGE gels. They were all suitable for 2-DE of synaptic proteins.