Mass Spectrometry and Its Contribution To Hybrid Structure Determination

Abstract

Standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organization of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. We have combined two existing methodologies to tackle these challenges: affinity purification and nanoflow ESI-MS. We use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. Structural models will be presented for oligomeric protein complexes with different degrees of structural information including the human U1snRNP and eIF3 complexes. These models will then be examined within the context of their function.Recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. Using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections.Very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase. This enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. I will demonstrate some of the advantages of this approach by presenting recent insights into the structures of intact V-type ATP synthases.