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

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.

This proposal is aimed at coupling ion mobility and electron microscopy (EM) to nano-electrospray mass spectrometry (MS). Specifically, the technology is directed at applications in structural biology, especially those involving large, non-covalently associated, protein-protein complexes. Such complexes exhibit a large variety of topologies making the cross-sectional area measurement produced by ion mobility MS a powerful tool for structural characterization. Similarly, electron microscopy samples prepared by soft-landing mass-selected material can offer the ability to characterize heterogeneous materials that had previously only provided structures that are averaged over all the oligomeric states and stoichiometries of an assembly present in solution. Ion mobility has been used for over a decade to determine the structure peptides and small proteins. Recently, we applied ion mobility-mass spectrometry to protein complexes where large structural changes were expected as a function of ligand binding and gas-phase activation. In this proposal, we extend these studies to heterogeneous and aggregation-prone protein complexes.EM is an established structural probe for biological assemblies, typically of high-purity and inhabit in a single structure. MS separation followed by soft-landing represents an attractive alternative for the preparation of samples for EM. Mass-selection allows for the analysis of each individual oligomeric states without averaging over the entire population, thus leading to higher-resolution images of heterogeneous materials. Here, we propose the development of this technology in parallel with an ion mobility approach in an effort compare structures obtained for soft-landed protein complexes with the structures assigned to the same complex in the gas-phase. We also envision obtaining EM images of protein complexes for which, due to their polydisperse nature, a high-resolution structure has yet to be obtained