Understanding complexity of post-translation modifications by enhancing UK capability for top-down proteomics

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Mass spectrometry (MS) is a fundamental and rapidly advancing technology, widely used in all areas of biological research. It can accurately determine the mass of molecules, such as proteins, by measuring the mass-to-charge ratio (m/z) of charged particles, called ions. Such measurements are important because they allow us to characterise biological molecules and determine how they change in composition and/or amount under different conditions that are key to understanding life. This proposal focusses on the analysis of intact proteins by MS, seeking funds for new instrumentation that will permit us to measure and characterise proteins more accurately. In particular, the biological regulation of proteins that occurs through the attachment of chemical groups, such as phosphate, will be measured. The biological function of many proteins is regulated by the reversible addition of specific chemical groups. With our new instrumentation, we will be able to define when and where these modifications occur, and in what order and combination. With this information, scientists can better understand how these modification regulate protein function in biological systems.Moreover, the function and/or catalytic (enzyme) activity of many proteins can be altered using drugs and through interactions with protein partners, and crucially, the binding of these factors is often specific to distinct protein forms, rather like a PIN number is specific for a particular bank card from the account of a given individual. Our ultimate goal is to understand the biological diversity of specific modified proteins ('proteoforms') and how they bind differentially to drugs and/or proteins. Significant developments in MS instrumentation mean that it is now feasible to characterise proteins and their modifications in intricate depth from relatively small amounts of material, which has previously been impossible due to technological limitations. The improved sensitivity, mass accuracy and resolution of the Thermo Scientific Orbitrap Lumos mass spectrometer will permit specific protein ions to be measured and manipulated in a more targeted manner, so that discovery of a protein 'needle in a haystack' can become a reality. We propose to establish this advanced MS platform in the Centre for Proteome Research at the University of Liverpool (UoL). The equipment will be available to scientists locally and across the UK, who currently struggle to access high-end instrumentation with this capability. The system will have broad applicability to a large number of BBSRC-funded research areas; we have specifically focused on research impacting the areas of infection, immunity, fundamental cell growth mechanisms, the response to hypoxia and biotherapeutics, which are either presented as example projects in the proposal or covered in the letters of support included with the application.To our knowledge, there is no comparable system available to BBSRC researchers. Our excellent working links with ThermoScientific and the single UK-based academic group in the UK that has purchased this system (Lilley, Cambridge, Wellcome Trust funded, although not for TDP) will allow us to rapidly establish a working platform in Liverpool, where it will be integrated into the UoL Proteomics Shared Research Facility and supported by UoL's Technology Directorate (TD). The TD provides access to the very best research facilities for the maximum number of users, across the UK, by providing financial support to maintain and develop our 'open' and 'transparent' facility. It also awards 'access grants', permitting academics, in particular ECRs, to use these facilities to support new research in advance of winning substantive funding. Access to such research facilities, which is typically available on an ad hoc basis elsewhere, also enhances collaboration with industry, since companies are able to outsource analysis using professionally-managed state-of-the-art technology and expertise.