Zhou Jian, Li Jianglin, Li Jianjun, Chen Ping, Wang Xianchun, Liang Songping
The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, PR China.
Electrophoresis. 2010 Dec;31(23-24):3816-22. doi: 10.1002/elps.201000255.
Sample preparation holds an important place in MS-based proteome analysis. For effective proteolysis and MS analysis, it is essential to eliminate the interferences while extracting the analytes of interest from complex mixtures. To address this, herein we describe a new dried polyacrylamide gel absorption method. In this method, the protein sample prepared using high concentration of SDS was directly and completely absorbed by vacuum-dried polyacrylamide gel, and then the interfering substances including SDS and some other salts were efficiently removed by in-gel washing steps while retaining the denatured proteins in the gel, thus offering a clean environment amenable to downstream buffer exchange, proteolytic digestion and digest recovery, etc. In combination with in-gel digestion and LC-MS/MS, the newly developed method was applied to the proteome analyses of membrane-enriched fraction and whole tissue homogenate. It was demonstrated that the method is suitable for the analysis of a complex biological sample and can be widely used for sample cleanup in shotgun proteome analyses.
样品制备在基于质谱的蛋白质组分析中占有重要地位。为了实现有效的蛋白质水解和质谱分析，从复杂混合物中提取目标分析物时消除干扰至关重要。为解决这一问题，我们在此描述一种新的干燥聚丙烯酰胺凝胶吸附方法。在该方法中，用高浓度十二烷基硫酸钠（SDS）制备的蛋白质样品被真空干燥的聚丙烯酰胺凝胶直接且完全吸附，然后通过凝胶内洗涤步骤有效去除包括SDS和其他一些盐在内的干扰物质，同时将变性蛋白质保留在凝胶中，从而提供一个有利于下游缓冲液交换、蛋白水解消化和消化产物回收等的清洁环境。结合凝胶内消化和液相色谱-串联质谱（LC-MS/MS），将新开发的方法应用于富含膜的组分和全组织匀浆的蛋白质组分析。结果表明，该方法适用于复杂生物样品的分析，可广泛用于鸟枪法蛋白质组分析中的样品净化。
