Developing new mass spectrometry methodologies for the determination of structures of heterogeneous protein complexes

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Mass spectrometry, a technique that was initially applied to individual atoms and small molecules, is increasingly being used to look at much larger molecules such as proteins, not only individually, but also as large assemblies. These assemblies are often important molecular machines that control many different reactions in living cells. One of the problems with studying these assemblies is that they rarely exist in a single form, but rather are often found as mixtures of different numbers of proteins molecules that have been modified in some way. Most methods of analysis are therefore unable to study them since the results are effectively blurred by averaging across the different assemblies. Mass spectrometry of these large assemblies, however, is capable of separating the different components according to their mass to charge ratio, thereby allowing us to characterise by mass all the components within a mixed assembly. Until now mass spectrometry has not been able to tell us much about the shape of these assemblies. In order to do this we need to couple mass spectrometry to other methods to get three-dimensional images of these assemblies. This proposal therefore sets out to do this: to combine mass spectrometry with an established imaging technique known as electron microscopy. In addition, we have recently shown that we can measure the cross section of mixtures of large protein assemblies within a mass spectrometer. This has allowed us to deduce that their shape is close to what we would expect from analysis of crystals of the same assembly with X-rays, and we intend to develop this research further. We believe that together this combined has the potential to provide an incredibly powerful structural biology tool that will be able to tackle many protein assemblies that are currently intractable.