Hydrogen/Deuterium Exchange and Ion Mobility Mass Spectrometry to Underpin Research on Protein Interactions

This proposal will fund a mass spectrometer and associated inlet system for the analysis of protein structure, dynamics and interactions. The instrument resource will be used by research groups from the Faculties of Engineering and Physical Sciences, Life Sciences, and Medicine and Human Sciences at the University of Manchester, in collaboration with academic partners from 5 other Universities, along with two research council Institutes. It also will be available to our industrial partners in industrial biotechnology, biomedicine and agri-food sectors. The requested instrument is an HDX-MS platform recently developed by Waters Corporation, the only commercially-available complete system for HDX-MS studies. Innovations in LC/MS and automation coupled with software position HDMX as a robust tool to characterise protein structure and interactions. This will be the first automated HDXMS ion mobility capable instrument in a UK Higher Education Institute.
The procedure we will follow for intact protein analysis will first perform ion mobility mass spectrometry (IM-MS) from native conditions on proteins and protein complexes. Conformational change due to inherent protein dynamics, ligand binding, or protein interaction will be measurable. IM-MS records the time it takes mass selected ions to traverse a cell under the influence of a weak electric field filled with an inert buffer gas. Collisions with the buffer gas impede the progress of the ion, and this coupled with the charge of the ion, causes the ion to drift with a velocity that is proportional to their size. By measuring the time it takes ions to cross the cell, it is possible to obtain the rotationally averaged cross section, a coarse structural parameter that will tell us the size of any given molecule. This readout will inform on conformational dynamics for enzymes, and on the change in shape of proteins as they interact with small 'druglike' molecules and with other proteins as they aggregate.
Differential hydrogen/deuterium exchange coupled with mass spectrometry has emerged as a sensitive technique to characterise changes in protein conformation. The marrying of these two techniques is extremely fortuitous, since the favorable exchange of a hydrogen atom for a deuterium atom results in a mass increase of one. The second part of our screen for protein conformation will use the mass increase measured by HDX-MS to 'snapshot' a given protein in a particular state, which when coupled with data from enzymatic digestion provides details of conformational changes at the residue level, an approach which is analogous to NMR, but uses far less material. Based on the pioneering work of Englander, it is possible to predict intrinsic amide hydrogen exchange rates for amino acid in a polypeptide. This exchange rate is influenced by hydrogen bonding; measuring this property for a given amide hydrogen is an excellent way to probe protein structure and dynamics. Exchange rates are influenced by pH and temperature, and these must be precisely controlled to gain useful information. In a typical HDXMS workflow, a target protein is incubated for a set of predetermined time points, and over a range of pHs or with a potential binding partner, the HDX reaction is quenched at the end of each of these reactions by rapidly lowering the pH and temperature, and the protein is then digested, and introduced via an UPLC interface to the mass spectrometer. For each digested polypeptide, prior knowledge of sequence allows the percentage deuterium uptake to be plotted as a function of time. The use of ETC or ECD allows the precise amino acid that has exchanged to be located. Deuterium uptake information for each state of the protein can be mapped to the sequence or to the structure if available. This approach will also be applied to intact proteins, and is amenable to complex mixture analysis for example in food stuffs to determine the molecular basis for an allergic response.

Protein interactions with other proteins (PPIs), carbohydrates, lipids and small molecules can alter the conformation of the substrate thus modulating its function. Understanding dynamic conformational changes is a mainstay of biology and medicine and in turn of great importance to biotechnology and the pharmaceutical industries. Proteins are both targets for small molecules, and also can function directly as drugs, for example monoclonal antibodies. In all cases, knowledge of the interplay between structure and dynamics of protein interactions is key to understanding function and to exploiting it. Methods which can characterise PPIs are of substantial interest. The use of X-ray crystallography to provide atomic structures has had a profound impact on biology and its application; despite this, static atomic structures do not easily provide information on the dynamics of the protein, nor on long-range allosteric interactions. In addition, some protein and protein states are resistant to crystallisation. Since pioneering work 20 years ago by Chait, the use of solution phase hydrogen deuterium exchange coupled with mass spectrometry (HDXMS) has grown to be a complementary technique to protein crystallography to determine both protein structure and dynamics. In this proposal we will use ion mobility and HDXMS to determine the conformation of proteins and how this changes in response to mutations, and to protein or ligand interactions.

A new and novel instrument platform for ion mobility hydrogen-deuterium exchange mass spectrometry is proposed which will provide a unique resource to the academic community in the UK. Hosted in the cross-faculty Manchester Institute of Biotechnology (MIB), the instrument will be used for research to investigate protein interactions and conformational changes using novel technology which has the capacity to characterise molecular structures in unprecedented detail, even in complex mixtures. We will realise its this capability in close collaboration with our academic partners spanning five Universities, and two research council Institutes and reaching out to industrial partners that have existing collaborations with commercial organisations that span the industrial biotechnology, biomedicine and agri-food sectors. Notable are the collaboration in the north of the UK and especially our near neighbours at the Universities of Liverpool and Lancaster, which will make the instrumentation more accessible to students and early stage researchers in the region.

We will deliver impact by focussing our efforts in selected strategic areas, explicitly linking developments in practical experimentation with data curation and analysis. The latter will be key to effective exploitation of the instrumental platform. Each of the strategic research areas has significant industrial collaboration linked to academic researchers through existing collaborative grants including BBSRC IPAs, TSB and other funding. Thus, industrial partnership is integral to the research we will do. Exploration of the new capability will be realised by our hosting students and early stage researchers at MIB, initially from our project partners [both academic and industrial], using a 'Research Hotel' approach. In this way we will grow a community able to disseminate the new ways of acquiring knowledge this platform will offer. They will form a user group, making use of on-line resources to promote exchange of ideas and best-practice. This will compliment conventional ways of dissemination to the wider academic community through publication in the peer-reviewed literature. To ensure the financial sustainability of this instrumental platform it will be run as a small equipment facility, thus ensuring its being well maintained and fit-for-purpose and remains accessible to acadmic and industrial researchers in the longer term. Beyond the specific partner organisations we will work with networks and stakeholder/community groups, including the Biosciences Knowledge Transfer Network, to show-case applications, develop wider training materials through Webinars, for example, with Waters Corporation, that have a global reach. Specific training courses, aimed at continuing professional development of industrial researchers will be developed with the University of Reading through the BBSRC Advanced Training Partnership for Food. This will be used as a model to roll-out training to other sectors. In this way we will grow the human capital and knowledge as to how to use this new generation of mass spectrometers and realise its potential in strategic priority areas such as industrial biotechnology and food security.