A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins.
A comparison of protein extraction methods suitable for gel-based proteomic studies of aphid proteins.
复制标题
DOI：
--
复制
发表时间：
2009-09
期刊：
影响因子：
--
通讯作者：
M. Cilia;T. Fish;X. Yang;M. Mclaughlin;T. Thannhauser;S. Gray
中科院分区：
文献类型：
--
作者：
M. Cilia;T. Fish;X. Yang;M. Mclaughlin;T. Thannhauser;S. Gray
蛋白质提取方法在再现性和总蛋白质组的代表性方面可以有很大的不同，但是比较蛋白质分离方法的数据有限。蛋白质分离方法的系统比较是蛋白质组学研究的第一个关键步骤。为了解决这个问题，我们比较了三种方法的分离，纯化和溶解的昆虫蛋白。蚜虫Schizaphis graminum是一种农业害虫，是昆虫组织的来源。使用TCA在丙酮（TCA-丙酮）、苯酚或多洗涤剂中在离液剂溶液中提取蛋白质。将提取的蛋白质溶解在多个离液剂溶液中，并使用1-D和2-D电泳进行检查，并使用2-D差异凝胶电泳（2-D DIGE）直接进行比较。使用质谱法鉴定每种浸提类型的蛋白质。我们无法将提取的蛋白质的差异归因于特定的物理特征，细胞位置或生物功能。TCA-丙酮提取法提取的蛋白质含量最高。每种提取方法分离出蚜虫蛋白质组的一个独特的子集。使用2-D DIGE进一步探索TCA-丙酮方法的定量可靠性。主成分分析表明，数据中的变化很少是技术问题造成的，从而证明三醋酸-丙酮提取是制备用于定量蛋白质组学实验的蚜虫蛋白的可靠方法。这些数据表明，虽然TCA-丙酮方法是一个合适的方法，定量蚜虫蛋白质组学，提取方法的组合，建议增加蛋白质组覆盖率时，使用基于凝胶的分离技术。
Protein extraction methods can vary widely in reproducibility and in representation of the total proteome, yet there are limited data comparing protein isolation methods. The methodical comparison of protein isolation methods is the first critical step for proteomic studies. To address this, we compared three methods for isolation, purification, and solubilization of insect proteins. The aphid Schizaphis graminum, an agricultural pest, was the source of insect tissue. Proteins were extracted using TCA in acetone (TCA-acetone), phenol, or multi-detergents in a chaotrope solution. Extracted proteins were solubilized in a multiple chaotrope solution and examined using 1-D and 2-D electrophoresis and compared directly using 2-D Difference Gel Electrophoresis (2-D DIGE). Mass spectrometry was used to identify proteins from each extraction type. We were unable to ascribe the differences in the proteins extracted to particular physical characteristics, cell location, or biological function. The TCA-acetone extraction yielded the greatest amount of protein from aphid tissues. Each extraction method isolated a unique subset of the aphid proteome. The TCA-acetone method was explored further for its quantitative reliability using 2-D DIGE. Principal component analysis showed that little of the variation in the data was a result of technical issues, thus demonstrating that the TCA-acetone extraction is a reliable method for preparing aphid proteins for a quantitative proteomics experiment. These data suggest that although the TCA-acetone method is a suitable method for quantitative aphid proteomics, a combination of extraction approaches is recommended for increasing proteome coverage when using gel-based separation techniques.