An ion-mobility mass spectrometry platform for single-cell proteomics and sensitive discrimination of isomeric biomolecules

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Mass spectrometry (MS) is a rapidly advancing fundamental technology used in all areas of biological research. It can accurately determine the mass of molecules by measuring the mass-to-charge ratio (or m/z) of charged particles called ions. Such measurements are very important, since they allow us to characterise and quantify proteins and carbohydrates, and determine how they change (in composition and/or amount) under different conditions. Through the acquisition of a state-of-the-art instrumentation, this proposal seeks to expand capability for protein analysis, building on the expertise and experience of the applicants, to focus on two key areas: i) Quantify the levels of different proteins in individual mammalian cells. Individual cells in a population often exhibit subtly different responses to e.g. in different parts of an organ, or following infection. However, unbiased investigation of protein changes at the level of an individual cell is extremely difficult due to the low amounts of material available for analysis. Recent methodological and instrumental developments mean that it is now feasible to start to understand the unique responses of individual cells in a heterogenous population of cells within an organ e.g. the liver or endometrium, or in cells following viral infection. ii) Characterise the exact patterns and sites of protein 'decoration' (covalent modification). Protein modification unlocks the regulatory potential of different proteins, rather like a PIN number controls access to a bank account. Consequently, defining what type of modification is present, and exactly where modification sites are positioned on a biomolecule, is essential to understanding their physiological relevance. However, this becomes particularly problematic as we consider multiple modifications that are close together on a protein, or where there are modifications that are (largely) indistinguishable by mass e.g. phosphate and sulfate. The sensitive separation of modified peptides based on conformation, as can be achieved with this new instrumentation, can go some way to addressing these issues in defining the precise type and location of these covalent modifications. We plan to bring this novel instrumentation, which combines extremely sensitive quantification with conformation-based separation of peptides, to the Centre for Proteome Research (CPR) at the University of Liverpool. Using a well-established cost recovery model to ensure long-term maintenance (supported by UoL's Technology Directorate (TD)), we will make it accessible to research scientists working across BBSRC strategic priority areas, both within Liverpool, and across the UK, with a focus initially on the research intensive 'Northern Powerhouse'. The TD provides access to the very best research facilities for the maximal number of users, both inside and outside Liverpool, providing financial support to maintain and develop 'open' and 'transparent' facilities such as ours. It also awards 'access grants', permitting academics, in particular ECRs, to use our facilities to support new research ideas in advance of winning more substantive funding. Access to such research facilities also enhances collaboration with Industry, since companies are able to outsource some of their analysis using professionally-managed technology thus helping drive success.This platform will be available to scientists both locally and nationally, who currently struggle to access such high-end instrumentation in the UK. The system will have broad applicability to numerous BBSRC-funded research areas; here we specifically focus on research impacting the areas of infection biology, fundamental cell signalling, and synthetic biology, which are either presented as example projects in the proposal or covered in the letters of support included with the application. Numerous other parties working in similar areas have expressed interest, although letters could not be included due to space cons