Advanced MS instrumentation for enhanced proteomics capabilities

Mass spectrometry (MS) is a rapidly advancing fundamental technology employed in many areas of 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 extremely important since they allow us to structurally characterise chemical entities and determine how these molecules change (in composition and/or amount) under different conditions. This proposal focusses on the analysis of proteins, with a view to determining how they change, either by the attachment of chemical groups such as phosphate, or by their binding to other regulatory molecules, all of which result in a mass shift that can be measured.
The biological function of proteins can be regulated by the reversible addition of specific chemical entities. Hence, by defining when and where these occur, scientists can firstly understand how they regulate proteins and ultimately their physiological relevance. Moreover, the function and/or enzyme activity of proteins can be manipulated using small molecule inhibitors and protein binding partners. Remarkably, the binding of these factors is often specific to distinct protein forms, rather like a PIN number is specific for a particular bank card.

Significant developments in MS instrumentation means that it is now possible to characterise proteins and their modifications in depth, a feat which has previously been hampered by an inability to distinguish ions with very similar m/z values. The improved ability to manipulate ions within the instrument, permits related ions to be accumulated, and significantly increases the sensitivity; detection of a protein 'needle in a haystack' can become a reality. This increased sensitivity is particularly important when analysing biological samples that are of low abundance, as is typical when looking at the different modified protein forms found in cells. Moreover, this instrument can also be used to control ion fragmentation in new ways, meaning that significantly more structural information can be elucidated. We are therefore in a position to start characterising, and thus understanding, the mechanisms of action of compounds that interact with different sites on a protein and how modification affects protein function, stability and subcellular localisation in complex biological samples.
We propose to establish such an advanced MS platform at the University of Liverpool (UoL). The platform will be available to scientists both locally and from across the UK, who currently struggle to access such high-end instrumentation. The system will thus have broad applicability to a large number of BBSRC-funded research areas, impacting ageing, industrial biotechnology, animal health, food security & allergy, many of 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 that is openly available to UK researchers. Our excellent working links with the two academic groups in the UK (Lamond, Dundee & Lehner, Cambridge) who have such systems installed (Wellcome Trust funded) will help us establish our platform, which will be managed by UoL's Technology Directorate (TD) and be integrated into the Proteomics Shared Research Facility. The TD has the mission of providing access to the very best research facilities for the maximal number of users, both inside and outside the University, and provides financial support for 'open' and 'transparent' facilities such as ours. It also awards specific 'access grants', which permit academics, in particular early career research staff, to use these 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.

We propose to install a state-of-the-art mass spectrometry (MS) system capable of
i) acquiring high resolution, accurate mass MSn data essential for the analysis of large intact protein ions,
ii) multiple modes of ion dissociation including collision-induced dissociation (CID), higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), an absolute requirement for both the structural interrogation of proteins and post-translationally modified polypeptides,
iii) multi-notch isolation (also known as synchronous precursor selection) to enhance the abundance of multiple specific ions in the ion trap, thereby increasing the signal quality for subsequent MSn.

These advanced instrumental features are essential if we are to routinely exploit the power of MS for aspects of proteomics that are more advanced than, say, the quantitative characterisation of changes in protein levels. Our exemplar projects all expand the scope of information that can be elucidated from biological samples, including understanding the roles of combinatorial protein modification in cell signalling and ligand binding, advanced quantitative MS to elucidate the roles of post-translational modification in sub-cellular trafficking, and deciphering glycan heterogeneity of biopharmaceuticals.

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

The main beneficiaries of this proposal will be indirect, i.e. those scientists who are able to utilise the knowledge gained by exploiting the enhanced analytical capability of a high-end ultra-high resolution MS system. The number of potential applications and the impact that could thus arise is vast, and our tried-and-tested facility business model is already in place for anticipated needs of the community. The exemplar projects must be seen as a small subset of the entire constituency of users, but even within this small group, the number of beneficiaries undertaking projects in BBSRC priority areas that will be enhanced significantly, are clear.

For example, the BBSRC-funded grants studying '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 complex formation and transcriptional activity. Such knowledge is critical in defining the multifactorial regulation of NF-kB signalling in response to diverse types and modes of signal input, which will have impact in the BBSRC strategic priority areas of 'healthy ageing' and 'immune system function', not to mention 'basic bioscience underpinning health'.

Similarly, 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 expected that >50% of new drug approvals by 2015 will be biologics, reaching ~70% by 2025, and this rapidly growing market is projected to reach ~US$500 billion globally by 2020. 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 technology users, but will drive development into new research spheres) also aligns with the BBSRC strategic priority 'Enabling New Ways of Working/Technology Development'. It follows therefore that access to such cutting-edge equipment, currently largely inaccessible to UK scientists, and the methodology that will be developed, will have great impact in both basic & applied bioscience, ultimately benefiting the general populous. In the short term, this will be due to increasing availability of more and cheaper biologics, and latterly as our basic understanding of the mechanisms involved in healthy ageing, immunity & infection, food-based allergies etc. is translated into novel therapeutics or improved biomarkers and diagnostic platforms.

By ensuring that researchers are maintained 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. Most of these areas have the potential to generate IP that in turn engenders new products and manufacturing processes. The applicants have an excellent track record of industrial collaboration and in the promulgation of their research. Thus, UK industry stands to gain directly from access to this enabling technology (as demonstrated by the letters of support and previously awarded funding from Industrial collaborators, including those actively supported by TSB applications i.e. Discuva).
Finally, by establishing a 'Fusion Hotel' where scientists will be trained and supported to use this technology for their own ends there will be significant impact in the number of able bioanalytical research scientists that can exploit high-end MS techniques in the future.