Mass spectrometry of protein complexes - from networks to structures

质谱法在蛋白质组学和代谢组学领域已经建立。它在破译蛋白质复合物的整体结构方面的作用还不太确定。使用程序，我们从完整的组装开始，我们已经表明，我们可以将组装分解成最简单的构建块，看看它们的整体形状，然后将它们集成到整体复杂的3D模型中。为了做到这一点，我们首先必须使用已知结构的配合物来验证该方法。我们已经与世界各地的许多团队合作，以获得具有各种不同拓扑安排和结构的配合物。通过对这些复合物的系统调查，我们希望确定这种方法是否适合用于确定未知蛋白质复合物的整体结构。我们的进一步研究涉及使用改进的分离方案在内源性表达水平分离复合物，其中单个亚基被标记，并用亲和方法纯化复合物。我们建议继续并巩固我们已经获得的涉及RNA翻译和剪接的复合物的非常令人兴奋的初步数据。这一系列的第一个配合物由延伸起始因子组成。通过比较和对比人类和酵母复合物，我们希望对它们的亚基相互作用图和整体结构获得新的见解。类似地，对于参与剪接的复合物，酵母和人类复合物都将进行研究，要么与Olga和E Makarov合作，要么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.