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

以前的质谱分析方法虽然成功地提出了许多复合物，但它们依赖于分离所有的蛋白质并从中产生肽来识别组成蛋白质。虽然这种方法在提供蛋白质目录方面非常出色，但它无法揭示稳定复合物中的蛋白质是否可以完整地提取出来。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体被提取出来。用我们的方法提取的蛋白质组合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通过使用各种方法生成更小的亚复合物来揭示蛋白质在组装结构内的组织方式。由于会有一些蛋白质出现在多个亚复合体中，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按什么方向或形状组织的？为了解决这个问题，我们正在开发可以在质谱仪中直接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。综合亚基的数量、它们可能的排列和整体形状的信息，我们希望我们将能够开始预测一些大分子机器的分子结构以及它们在细胞中迄今仍难以捉摸的相互作用。

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.