Mass spectrometry of protein complexes - from networks to structures

质谱学在蛋白质组学和代谢组学领域得到了广泛的应用。它在破译蛋白质复合体整体结构方面的作用还远未得到证实。通过使用一个程序，我们从完整的组件开始，我们已经展示了我们可以将组件分解为最简单的构建块，查看它们的整体形状，然后将它们集成到整个综合体的3D模型中。为了做到这一点，我们首先必须使用已知结构的络合物来验证该方法。我们已经与世界各地的许多集团合作，获得了具有各种不同拓扑安排和结构的复合体。通过对这些复合体的系统研究，我们希望确定这种方法是否适用于确定未知蛋白质复合体的整体结构。我们的另一项研究涉及使用改进的分离方案在内源表达水平分离复合体，其中单个亚基被标记，并使用亲和方法纯化复合体。我们建议继续并巩固我们已经获得的关于RNA翻译和剪接的复合体的非常令人兴奋的初步数据。这一系列复合体中的第一个由延伸起始因子组成。通过比较和对比人类和酵母复合体，我们希望对它们的亚基相互作用图和整体结构有新的见解。同样，对于参与剪接的复合体，将研究酵母和人类复合体，要么与Olga和E Makarov合作，要么通过从内部培养的酵母菌株中分离出来。

Mass spectrometry is well established in the fields of proteomics and metabolomics. Much less established is its role in deciphering the overall architecture of protein complexes. Using a procedure whereby we begin with the intact assembly we have shown that we can decompose the assembly into its simplest building blocks, look at their overall shape and then integrate them into 3D models of the overall complex. In order to do this we first have to validate the method using complexes of known structure. We have collaborated with a number of groups worldwide to obtain complexes with a variety of different topological arrangements and structures. Through a systematic survey of these complexes we hope to determine whether or not this approach will be suitable for use in deciding the overall architecture of unknown protein complexes. A further stream of our research involves isolating complexes at endogenous expression levels using a modified isolation protocol in which a single subunit is tagged and complexes are purified using affinity methods. We propose to continue and consolidate very exciting preliminary data that we have obtained for complexes involved in translation and splicing of RNA. The first of this series of complexes consists of the elongation initiation factors. By comparing and contrasting both human and yeast complexes we hope to gain novel insight into their subunit interaction maps and overall architecture. Similarly for the complexes involved in splicing, both yeast and human complexes will be studied, either in collaboration with Olga and E Makarov or by isolation from yeast strains grown in-house.