Modified TCA/acetone precipitation of plant proteins for proteomic analysis
Modified TCA/acetone precipitation of plant proteins for proteomic analysis
复制标题
DOI：
10.1371/journal.pone.0202238
复制
发表时间：
2018-12-17
期刊：
影响因子：
3.7
通讯作者：
Wang, Wei
中科院分区：
文献类型：
--
作者：
Nie, Liangjie;Zhang, Hang;Wang, Wei
在蛋白质组学分析中，从细胞或组织中获得的蛋白质提取物通常需要去除干扰物质以制备高质量的蛋白质样品。许多蛋白质提取方法已应用于各种生物样品。TCA/丙酮沉淀和苯酚提取是蛋白质提取的常用方法，被认为可以最大限度地减少蛋白质降解和蛋白酶活性，并减少盐和多酚等污染物。然而，TCA/丙酮沉淀法依赖于组织粉末的完全粉碎和反复冲洗以去除干扰物质，这是费力且耗时的。此外，通过在TCA/丙酮中长时间孵育，沉淀的蛋白质更难以再溶解。本文介绍了一种改进的TCA/丙酮沉淀植物蛋白的方法。使用含SDS的缓冲液提取细胞或组织的蛋白质，用等体积的20%TCA/丙酮沉淀，并用丙酮洗涤。与经典的TCA/丙酮沉淀和简单的丙酮沉淀相比，该方案产生了相当的产率、斑点数量和蛋白质组分析，但需要更少的时间（约10分钟）。45分钟），从而避免在TCA/丙酮或丙酮中延长时间孵育后过量的蛋白质修饰和降解。改良TCA/丙酮沉淀法简便、快速，适用于植物各种组织的蛋白质组学分析。
Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.