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

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

We propose to install a state-of-the-art timsTOF Pro mass spectrometer with associated liquid chromatography system in the internationally-recognised Centre for Proteome Research at the University of Liverpool, enhancing capabilities for single-cell quantitative proteomics and the differentiation of isomeric covalently modified analytes (peptides/glycans). Specifically, the unique dual TIMS configuration of the timsTOF Pro system make it ideally suited for the prosecution of largely underexplored proteomics problems, due to an ability to trap and separate ions based on their mobility (conformation) prior to tandem MS, which permits:
i) parallel accumulation and serial fragmentation (PASEF) of ions, enhancing proteome coverage, as well as instrument duty cycle (and thus sensitivity) of analysis;
ii) separation of analytes based on their conformation in the gas phase. Peptides and/or glycans that contain the same type of covalent modification attached at different sites (or near isobaric modifications) that differentially effect their structure will be orthogonally separated prior to site localisation by classical MS/MS.

The possible gains in sensitivity (limits of detection), combined with the ion mobility separation are unparalleled, and thus unique with respect to the instrumentation, both within the CPR, and across the N8.
This advanced instrumentation is essential if we are to maintain UK proteomics research at the forefront of this rapidly developing field, which underpins mainstream biological and biomedical research. Our exemplar projects demonstrate the breadth and broad applicability of how this advanced MS instrumentation will expand the breadth and detail of information that can be elucidated from biological samples. Specifically, we will establish the first platform in the UK for single-cell proteomics, and improve the robust identification of sites and types of multiple types of covalent modifications.

In addition to the applicants, their collaborators and users of the CPR Research Hotel, a significant number of the primary beneficiaries of this proposal will be indirect, i.e. those scientists who are able to exploit the knowledge garnered from timsTOF-based investigation of single-cell heterogeneity and confident identification/discrimination of covalent modifications (see above). The number of potential applications and the broad impact that could thus arise for the UK bioscience community is vast, and our tried-and-tested facility business model is already in place to serve the anticipated needs of the community. The exemplar projects must be seen as a small subset of the entire user base, but even across this small group, the number of potential beneficiaries in BBSRC priority areas, are clear.

For example, several BBSRC-funded projects (BB/N021703/1, BB/M012557/1, BB/M027791/1, BB/L009501/1, BB/H007113/1; >£2M awarded) will benefit substantially from understanding the combinatorial effects of modification on protein complexes, most notably the competitive 'coding' that can take place on tyrosine residues, which can be modified by nitration, phosphorylation and sulfation, and the expanded landscape of mammalian protein phosphorylation through 6 non-Ser/Thr/Tyr amino acids. Such knowledge is critical in defining the multifactorial regulation PTM-based signalling in response to diverse types and modes of signal input, and will impact the BBSRC strategic priority areas of 'healthy ageing' and 'immune system function', and also 'basic bioscience underpinning health'.

In a similar vein, the ability to define heterogeneity of therapeutic glycoproteins at an early stage of product development will assist in getting low-cost, effective protein-based drugs to market in a timely manner, and is critical given the effects of variable glycosylation on efficacy and product stability. It is predicted that the rapidly growing biologics market will reach ~US $500 billion globally by 2020, with ~70% of new drug approvals by 2025 being biologics. However, development of significantly improved, robust characterization platforms which can determine product similarity, particularly with respect to low-cost biosimilars designed against 'off-patent' biotherapeutics, which this project seeks to address, is essential.

The technology development implicit in this application (we are not just associated with technology exploitation, but also method and software development) aligns with the BBSRC strategic priority 'Enabling New Ways of Working/Technology Development'. It follows therefore that access to cutting-edge equipment, currently inaccessible to UK scientists, and the methodology that will be developed, will have great impact in both basic & applied bioscience, ultimately benefitting the general populous. In the short term, this will be due to increasing availability of more/ cheaper biologics, and latterly as our basic understanding of the generic cell signalling mechanisms involved in immunity & infection, apoptosis etc. is translated into novel therapeutics or improved biomarkers.

By ensuring that UK researchers remain at the cutting edge of analytical science, we optimise progress towards realisable goals. Some of the outputs from this research may be realised within a five-year timeframe. A number of these areas have the potential to generate IP that in turn engenders new products and manufacturing processes. UK industry thus stands to gain directly from access to this enabling technology, and the applicants have an excellent track record of industrial collaboration and in the promulgation of their research.

Finally, the 'Research Hotel' will train large numbers of research scientists and support use of this technology for their own ends, significantly impacting the number of trained bioanalytical researchers that can exploit high-end MS techniques in the future. This training element alone will have significant impact.