Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China.
Mol Cell Proteomics. 2012 Sep;11(9):786-99. doi: 10.1074/mcp.M111.012450. Epub 2012 Feb 27.
The major challenge of "protein complexomics" is to separate intact protein complexes or interactional proteins without dissociation or denaturation from complex biological samples and to characterize structural subunits of protein complexes. To address these issues, we developed a novel approach termed "broad-spectrum four-dimensional orthogonal electrophoresis (BS4-DE) system," which is composed of a nondenaturing part I and denaturing part II. Here we developed a mild acidic-native-PAGE to constitute part I, together with native-thin-layer-IEF and basic-native-PAGE, widening the range of BS4-DE system application for extremely basic proteins with the range of pI from about 8 to 11 (there are obviously 1000 kinds of proteins in this interval), and also speculated on the mechanism of separating. We first proposed ammonium hydroxide-ultrasonic protein extractive strategy as a seamless connection between part I and part II, and also speculated on the extractive mechanism. More than 4000 protein complexes could be theoretically solved by this system. Using this approach, we focus on blood rich in protein complexes which make it challenging to sera/plasma proteome study. Our results indicated that the BS4-DE system could be applied to blood protein complexomics investigation, providing a comprehensively feasible approach for disease proteomics.
“蛋白质复合物组学”的主要挑战是从复杂的生物样品中分离完整的蛋白质复合物或相互作用的蛋白质，而不使其解离或变性，并对蛋白质复合物的结构亚基进行鉴定。为了解决这些问题，我们开发了一种新的方法，称为“广谱四维度正交电泳（BS4-DE）系统”，它由非变性部分 I 和变性部分 II 组成。在这里，我们开发了一种温和的酸性天然-PAGE 来构成部分 I，与天然薄层等电聚焦和碱性天然-PAGE 一起，拓宽了 BS4-DE 系统的应用范围，用于等电点（pI）约为 8 到 11 的极碱性蛋白质（这一区间内显然有 1000 种蛋白质），并对分离机制进行了推测。我们首先提出了氨超声蛋白提取策略，作为部分 I 和部分 II 的无缝连接，并对提取机制进行了推测。该系统理论上可以解决 4000 多种蛋白质复合物。通过这种方法，我们专注于富含蛋白质复合物的血液，这使得血清/血浆蛋白质组学研究具有挑战性。我们的研究结果表明，BS4-DE 系统可应用于血液蛋白质复合物组学研究，为疾病蛋白质组学提供了一种全面可行的方法。
