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

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.

We propose to install a state-of-the-art Orbitrap Lumos Tribrid mass spectrometer (MS) with associated liquid chromatography system in the internationally-recognised Centre for Proteome Research at the University of Liverpool, enhancing capabilities for top-down proteomics and the analysis of complex combinatorial post-translational modifications. Unique features of the Lumos system that will be essential for undertaking complex analysis of high mass, low abundant proteoforms include:
i) ultra-high resolution, accurate mass measurement essential for analysis of large intact protein ions;
ii) high capacity transfer tube with significantly enhanced transmission of both intact protein ions and their fragments following dissociation;
iii) multiple modes of ion dissociation including collision-induced dissociation (CID), higher-energy collision dissociation (HCD) and high capacity electron transfer dissociation (ETD-HD), which can be used alone or in combination for MSn, an absolute requirement for defining the composition of proteins and post-translationally modified polypeptides;
iv) multi-notch isolation (also known as synchronous precursor selection) to selectively isolate multiple specific ions in the ion trap, thereby increasing the signal quality for subsequent MSn.

This advanced instrumentation is essential if we are to maintain UK proteomics research at the forefront of this rapidly developing field, which itself underpins mainstream biological and biomedical research. Our exemplar projects will expand the scope of information that can be elucidated from biological samples, from understanding the roles of combinatorial protein modification in cell signalling and ligand binding through to deciphering glycan heterogeneity of biopharmaceuticals.

The range of projects included in this application ably demonstrates the breadth and broad applicability of this advanced instrumentation in analytical biological mass spectrometry.

In addition to the applicants, their collaborators and users of the CPR Research Hotel, a significant number of the primary beneficiaries will be indirect, i.e. those scientists who are able to exploit knowledge garnered from TDP on PTM-induced regulation of proteins to further enhance biological understanding, including a more-informed use of small molecule compounds in fundamental research. 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 for the anticipated needs of the bioscience community. The exemplar projects should be seen as a small subset of the entire possible user-base, but even across this small group, the number of potential beneficiaries in BBSRC priority areas are very clear.
For example, the BBSRC-funded grants 'Systems Biology of biological timers and inflammation' (Manchester, Liverpool & Warwick; sLOLA £3.8M) and 'DNA damage-induced phosphorylation and regulation of NF-kB' (Liverpool & Newcastle; £509k) will both benefit from understanding the stimulation-induced dynamics of NF-kB (RelA) modification and the combinatorial effects of modification on protein complexes and transcriptional activity. Such knowledge is critical in defining the multifactorial regulation of NF-kB signalling in response to diverse signal input, which will have impact in the BBSRC strategic priority areas of 'healthy ageing' and 'immune system function', as well as 'basic bioscience underpinning health'.
Similarly, the ability to define heterogeneity of therapeutic glycoproteins during early stage 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. Development of significantly improved, robust characterization platforms that can assess product similarity, particularly with respect to low-cost biosimilars designed against 'off-patent' biotherapeutics, which this project seeks to address, is thus essential.
The technology development implicit in this application (NB: we are not just technology 'users', but also method and software 'developers') aligns closely with the BBSRC strategic priority 'Enabling New Ways of Working/Technology Development'. It follows therefore that access to cutting-edge equipment and associated methodologies, especially those that are currently inaccessible to UK-based scientists, will have major impact in both basic & applied bioscience. It will also benefit the general populous: In the short term, impact will likely be realised due to increasing availability of better and/or cheaper biologics, and as our basic understanding of PTM-regulated cell signalling mechanisms involved in immunity & infection and apoptosis are uncovered, through translation into therapeutics and improved biomarkers.
By ensuring that UK remains at the cutting edge of analytical science, progress towards realisable goals is afforded. We believe that several research outputs may be realised within a five-year timeframe. A number of these have the potential to generate IP that will in turn engender new products and manufacturing processes. UK industry thus stands to gain directly from access to such enabling technology, especially given the excellent track record of industrial collaboration amongst the research applicants.
Finally, because the 'Research Hotel' will train research scientists and support their exploitation of this advanced technology for their own ends, our proposal will significantly increase the number of trained bioanalytical researchers that can exploit high-end MS techniques. This training element alone will have significant impact on the UK science infrastructure.