Structural studies of proteins and protein complexes by mass spectrometry methods
My research is aimed at development and applications of mass spectrometry-based methods to understand cellular functions at the protein level. We are particularly interested in analyzing biomolecular complexes which are at the nodes of the intricate network of protein interactions in the cell. Current projects include the investigation of assembly and disassembly of non-covalent complexes in response to conformational and post-translational modification states, and their link with structure and function.
We are employing new, cutting-edge technologies such as ion mobility in conjunction with high-resolution tandem mass spectrometry of non-covalent complexes under non-denaturing conditions to obtain information on subunit architecture and stoichiometry as well as size and shape of assemblies.
We are also developing advanced fragmentation techniques for the sequencing of proteins and mapping of post-translational modification sites, in collaboration with instrument manufacturers.Current research is directed at structures and conformational transitions of various kinases in response to phosphorylation states, and the interactions of kinases with elements of the molecular HSP90 chaperone machinery. We are also investigating key proteins involved in the hypoxia response and ubiquitination system.
More recently we have begun to characterize ion channels and other membrane-bound proteins in a non-denaturing environment using proteomics approaches.
- Sobott F, Robinson CV. Understanding protein interactions and their representation in the gas phase of the mass spectrometer. Chapter in: Principles of mass spectrometry applied to biomolecules (Eds: C Lifshitz, J Laskin), Wiley, 2006. ISBN: 0-471-72184-0
- Sobott F, McCammon MG, Hernandez H, Robinson CV. The flight of macromolecular complexes in a mass spectrometer. Philos Transact A Math Phys Eng Sci. 2005; 363(1827):379-91
- Sobott F, Robinson CV. Characterising electrosprayed biomolecules using tandem-MS - the noncovalent GroEL chaperonin assembly. Int J Mass Spectrom 2004; 236:25-32
- Sobott F, Benesch JL, Vierling E, Robinson CV. Subunit exchange of multimeric protein complexes. Real-time monitoring of subunit exchange between small heat shock proteins by using electrospray mass spectrometry. J Biol Chem. 2002; 277(41):38921-9
- Sobott F, Hernandez H, McCammon MG, Tito MA, Robinson CV. A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal Chem. 2002; 74(6):1402-7
Figure 1: Non-denaturing mass spectrum of HSP90 in complex with the co-chaperone p50 (Cdc37) and cyclin-dependent kinase Cdk4 as a client protein shows a 2:1:1 stoichiometry. The inset on the right shows a 3D reconstruction from single-particle electron microscopy (negative stain) of the HSP90 ternary complex (Vaughan CK et al. Mol Cell 23 (2006) 697)
Figure 2: Non-denaturing mass spectrum (y axis: m/z) plotted against ion mobility (x axis: drift time) showing three distinct conformers for the enzyme CarC (carbapenem synthase; manuscript in preparation). The inset shows an x-ray structure of the enzyme which crystallises as a hexamer, confirming three different conformations (Clifton IJ et al. J Biol Chem 278 (2003) 20843). Work performed in collaboration with Prof. Chris Schofield and Dr James McCullagh
Graduate Student and Postdoctoral Positions: Enquiries with CV welcome