Subunit architecture of non-covalent complexes isolated directly from the cells.

以前的质谱方法虽然成功地提出了许多复合物，但依赖于分离所有蛋白质并从中产生肽来鉴定组分蛋白质。虽然这种方法在提供蛋白质目录方面非常出色，但它无法揭示来自稳定复合物的蛋白质是否可以完整提取。我们正在开发的方法依赖于蛋白质的组装，这样它们就可以作为一个完整的复合物被提取出来。使用我们的方法提取的蛋白质组装体也带来了与核酸和辅因子的关联，并揭示了所有蛋白质是否一直存在。我们选择了一组参与RNA加工的重要复合物来发展这些想法，并希望从这项研究中发现新的相互作用。我们还计划使用质谱法来揭示蛋白质是如何通过使用各种方法来产生更小的亚复合物而在组装结构中组织的。由于有些蛋白质会出现在不止一个亚复合体中，这将使我们能够将它们拼凑在一起，以发现整个复合体的整体结构。一旦我们有了这些信息，一个主要的问题是这些亚单位是以什么方向或形状组织的？为了解决这个问题，我们正在开发可以直接在质谱仪中测量离子截面的方法。该方法依赖于离子通过一个装置的运动，该装置测量它们的速度作为它们的横截面积的函数。这将使我们能够区分基本形状。我们想进一步扩展这一点，以区分形状上更细微的差异。将亚基的数量、可能的排列和整体形状等信息综合起来，我们希望能够开始预测一些大分子机器的分子结构及其相互作用，这些结构迄今为止在细胞中仍然难以捉摸。

Previous mass spectrometry approaches although successful in proposing numerous complexes have relied on separating all the proteins and generating peptides from them to identify the component proteins. While that approach is excellent at providing a catalogue of proteins it is unable to reveal whether or not the proteins from stable complexes that can be extracted intact. The approaches that we are developing rely upon the proteins being assembled, so that they can be extracted as an intact complex. The protein assemblies extracted using our methods also bring with them associations with nucleic acids and cofactors and reveal whether or not all of the proteins are present all of the time. We have chosen an important set of complexes involved in processing RNA to develop these ideas and from this research hope to discover new interactions. We also plan to use mass spectrometry to uncover how the proteins are organised within the structure of the assembly by using various methods to generate smaller subcomplexes. Since there will be some proteins that occur in more than one subcomplex this will allow us to piece them together to discover the overall architecture of the whole complex. Once we have this information a major question is in what orientation or shape are these subunits organised? To address this question we are developing methods that can measure the cross section of ions directly in the mass spectrometer. The method relies upon the movement of ions through a device that measures their speed as a function of their cross sectional area. This will enable us to distinguish basic shapes. We would like to extend this further to distinguish more subtle differences in shape. Taken together information about the number of subunits, their possible arrangements and overall shape we hope that we will be able to start to predict molecular structures of some of the macromolecular machines and their interactions that have so far remained elusive in cells.