Development and application of wide-range gradient gel electrophoresis to proteome analysis
Development and application of wide-range gradient gel electrophoresis to proteome analysis
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DOI：
10.1039/c5ay01072a
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发表时间：
2015-09
影响因子：
3.1
通讯作者：
Hai-Hong Yu;Hui Liu;Yizhong Yan;Zhipan Dai;Shuai Yan;Jianglin Li;Z. Duan;Zhen Liu;Xianchun Wang
中科院分区：
文献类型：
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作者：
Hai-Hong Yu;Hui Liu;Yizhong Yan;Zhipan Dai;Shuai Yan;Jianglin Li;Z. Duan;Zhen Liu;Xianchun Wang
SDS广泛用于处理难以溶解和消化的蛋白质，并改善SDS-PAGE中的蛋白质分离。然而，SDS会干扰后续分析，需要在消化和LC-MS/MS分析之前去除，而常规SDS-PAGE缺乏有效去除SDS并保留低分子量蛋白质和肽的能力。在本工作中，我们建立了一个宽范围梯度凝胶电泳（WGGE）系统中的垂直板凝胶电泳池，它主要由一个4-20%的连续梯度聚丙烯酰胺凝胶分离层和两个拦截层，甚至更高的浓度（30%和50%，分别）。该系统的主要优点是同时净化SDS溶解的样品，分离蛋白质并拦截低分子量蛋白质和肽，从而简化了实验操作，提高了蛋白质回收率，提高了蛋白质组分析的总效率。利用该系统，样品和凝胶中约87.25%的SDS被去除，并有效地拦截了分子量为3.75 kDa的肽。结合CapLC-MS/MS，将WGGE系统应用于大鼠肝膜富集蛋白样品的分析，结果表明，基于WGGE的策略适用于分子量、pI、疏水性等不同的蛋白质的鉴定，这表明在各种蛋白质组的全局和比较分析中的潜在应用。
SDS is widely used to treat proteins that are difficult to solubilize and digest and improve protein separation in SDS-PAGE. However, SDS interferes with subsequent analyses and needs to be removed prior to digestion and LC-MS/MS analysis, whereas the conventional SDS-PAGE lacks the ability to efficiently remove SDS and retain low-molecular-weight proteins and peptides. In the present work, we developed a wide-range gradient gel electrophoresis (WGGE) system in a vertical slab gel electrophoresis cell, which was primarily composed of a 4–20% continuous gradient polyacrylamide gel separation layer and two interception layers with even higher concentrations (30% and 50%, respectively). The main advantages of the system are simultaneously cleaning up SDS-solubilized samples, separating proteins and intercepting low-molecular-weight proteins and peptides, thereby simplifying experimental operation, improving protein recovery and enhancing the total efficiency of proteome analysis. Using this system, about 87.25% of SDS in the sample and gel was electrophoretically removed and a peptide with a molecular weight of 3.75 kDa was efficiently intercepted. Combined with CapLC-MS/MS, the WGGE system was applied to the analysis of rat liver membrane-enriched protein samples and the results indicated that the WGGE-based strategy is suitable for the identification of proteins varying in molecular weight, pI, hydrophobicity, etc., suggesting potential applications in global and comparative analyses of various proteomes.