Hydrogen/Deuterium Exchange and Ion Mobility Mass Spectrometry to Underpin Research on Protein Interactions
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
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.
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.
Technical Summary
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.
Planned Impact
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.
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.
Publications
Aitken JF
(2017)
Rutin suppresses human-amylin/hIAPP misfolding and oligomer formation in-vitro, and ameliorates diabetes and its impacts in human-amylin/hIAPP transgenic mice.
in Biochemical and biophysical research communications
Bellina B
(2014)
UV photodissociation of trapped ions following ion mobility separation in a Q-ToF mass spectrometer.
in The Analyst
Beveridge R
(2019)
Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclin A
in Angewandte Chemie
Beveridge R
(2015)
Relating gas phase to solution conformations: Lessons from disordered proteins.
in Proteomics
Beveridge R
(2019)
Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins.
in Journal of the American Chemical Society
Beveridge R
(2016)
Mass spectrometry locates local and allosteric conformational changes that occur on cofactor binding.
in Nature communications
Beveridge R
(2019)
Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclin A.
in Angewandte Chemie (International ed. in English)
Beveridge R
(2014)
A mass-spectrometry-based framework to define the extent of disorder in proteins.
in Analytical chemistry
Description | The grant is still on going and we have already found out 1. some differences between disordered and structured proteins 2. Mapped sties of allosteric inhibtion 3. Deviations from crystal structures in three heteromeric systems 4. new methods for operating Synapt mass spectrometers 5. The role of cofactors in protein structure 6. a method for enhancing the structural information we gain when performing top down sequencing |
Exploitation Route | This is an ongoing collaborative grant. Weare applying for follow on funding |
Sectors | Chemicals Healthcare Pharmaceuticals and Medical Biotechnology |
URL | http://www.mbc.manchester.ac.uk/ |
Description | This award is in collaboration with Waters. They are already using some of the technology we have co-developed to help synchronize event in the analysis of molecules. |
First Year Of Impact | 2015 |
Sector | Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | Antibiotic K16: Elucidation and Engineering Pathways to New Anti-infective Agents. |
Amount | £486,550 (GBP) |
Funding ID | BB/V008552/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2021 |
End | 04/2025 |
Description | Generalised Photocatalysis by Enzymes (GENPENZ) |
Amount | £3,178,051 (GBP) |
Funding ID | BB/X003027/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 02/2023 |
End | 01/2028 |
Description | MRC Case |
Amount | £96,000 (GBP) |
Funding ID | MRC_CASE |
Organisation | UCB Pharma |
Sector | Private |
Country | United Kingdom |
Start | 08/2010 |
End | 08/2014 |
Description | Methods for bioengineering NRPS/PKS assembly lines delivering peptide natural products with electrophilic warheads. |
Amount | £470,912 (GBP) |
Funding ID | BB/V016083/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2022 |
End | 08/2025 |
Description | Prospecting for pH sensors in host and pathogen systems |
Amount | £435,094 (GBP) |
Funding ID | BB/V006592/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2021 |
End | 09/2024 |
Description | Use of MS to understand the supramolecular assemblies |
Organisation | University of Edinburgh |
Department | Centre for Clinical Brain Sciences (CCBS) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | we make measurements with ion mobility mass spectrometry and UVPD the partners are: Professor Lee Cronin (University of Glasgow) Professor Kari Reisen and Dr. Elina Kalenius (University of Jyväskylä Finland) Professor David Leigh (University of Manchester) Professor Richard Winpenny (University of Manchester) Dr. Paul Lusby (University of Edinburgh) Professor KAte Jolliffe University of Sydney |
Collaborator Contribution | they supply compounds and we discuss research questions |
Impact | Sizing and discovery of nanosized polyoxometalate clusters by mass spectrometry AJ Surman, PJ Robbins, J Ujma, Q Zheng, PE Barran, L Cronin Journal of the American Chemical Society 138 (11), 3824 2016 Making hybrid [n]-rotaxanes as supramolecular arrays of molecular electron spin qubits A Fernandez, J Ferrando-Soria, EM Pineda, F Tuna, IJ Vitorica-Yrezabal, ... Nature communications 7 2016 The potential of ion mobility mass spectrometry for tuning synthetic host guest systems: A case study using novel zinc (II) dipicolylamine anion sensors C Nortcliffe, LG Migas, X Liu, HT Ngo, KA Jolliffe, PE Barran International Journal of Mass Spectrometry 391, 62-70 |
Start Year | 2014 |
Description | Use of MS to understand the supramolecular assemblies |
Organisation | University of Glasgow |
Department | Institute of Cardiovascular and Medical Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | we make measurements with ion mobility mass spectrometry and UVPD the partners are: Professor Lee Cronin (University of Glasgow) Professor Kari Reisen and Dr. Elina Kalenius (University of Jyväskylä Finland) Professor David Leigh (University of Manchester) Professor Richard Winpenny (University of Manchester) Dr. Paul Lusby (University of Edinburgh) Professor KAte Jolliffe University of Sydney |
Collaborator Contribution | they supply compounds and we discuss research questions |
Impact | Sizing and discovery of nanosized polyoxometalate clusters by mass spectrometry AJ Surman, PJ Robbins, J Ujma, Q Zheng, PE Barran, L Cronin Journal of the American Chemical Society 138 (11), 3824 2016 Making hybrid [n]-rotaxanes as supramolecular arrays of molecular electron spin qubits A Fernandez, J Ferrando-Soria, EM Pineda, F Tuna, IJ Vitorica-Yrezabal, ... Nature communications 7 2016 The potential of ion mobility mass spectrometry for tuning synthetic host guest systems: A case study using novel zinc (II) dipicolylamine anion sensors C Nortcliffe, LG Migas, X Liu, HT Ngo, KA Jolliffe, PE Barran International Journal of Mass Spectrometry 391, 62-70 |
Start Year | 2014 |
Description | Use of MS to understand the supramolecular assemblies |
Organisation | University of Jyvaskyla |
Department | Department of Chemistry |
Country | Finland |
Sector | Academic/University |
PI Contribution | we make measurements with ion mobility mass spectrometry and UVPD the partners are: Professor Lee Cronin (University of Glasgow) Professor Kari Reisen and Dr. Elina Kalenius (University of Jyväskylä Finland) Professor David Leigh (University of Manchester) Professor Richard Winpenny (University of Manchester) Dr. Paul Lusby (University of Edinburgh) Professor KAte Jolliffe University of Sydney |
Collaborator Contribution | they supply compounds and we discuss research questions |
Impact | Sizing and discovery of nanosized polyoxometalate clusters by mass spectrometry AJ Surman, PJ Robbins, J Ujma, Q Zheng, PE Barran, L Cronin Journal of the American Chemical Society 138 (11), 3824 2016 Making hybrid [n]-rotaxanes as supramolecular arrays of molecular electron spin qubits A Fernandez, J Ferrando-Soria, EM Pineda, F Tuna, IJ Vitorica-Yrezabal, ... Nature communications 7 2016 The potential of ion mobility mass spectrometry for tuning synthetic host guest systems: A case study using novel zinc (II) dipicolylamine anion sensors C Nortcliffe, LG Migas, X Liu, HT Ngo, KA Jolliffe, PE Barran International Journal of Mass Spectrometry 391, 62-70 |
Start Year | 2014 |
Description | Use of MS to understand the supramolecular assemblies |
Organisation | University of Sydney |
Department | Medical Radiation Sciences |
Country | Australia |
Sector | Hospitals |
PI Contribution | we make measurements with ion mobility mass spectrometry and UVPD the partners are: Professor Lee Cronin (University of Glasgow) Professor Kari Reisen and Dr. Elina Kalenius (University of Jyväskylä Finland) Professor David Leigh (University of Manchester) Professor Richard Winpenny (University of Manchester) Dr. Paul Lusby (University of Edinburgh) Professor KAte Jolliffe University of Sydney |
Collaborator Contribution | they supply compounds and we discuss research questions |
Impact | Sizing and discovery of nanosized polyoxometalate clusters by mass spectrometry AJ Surman, PJ Robbins, J Ujma, Q Zheng, PE Barran, L Cronin Journal of the American Chemical Society 138 (11), 3824 2016 Making hybrid [n]-rotaxanes as supramolecular arrays of molecular electron spin qubits A Fernandez, J Ferrando-Soria, EM Pineda, F Tuna, IJ Vitorica-Yrezabal, ... Nature communications 7 2016 The potential of ion mobility mass spectrometry for tuning synthetic host guest systems: A case study using novel zinc (II) dipicolylamine anion sensors C Nortcliffe, LG Migas, X Liu, HT Ngo, KA Jolliffe, PE Barran International Journal of Mass Spectrometry 391, 62-70 |
Start Year | 2014 |
Title | BIOMARKERS AND USES THEREOF |
Description | The present invention relates to methods for detecting the presence or absence of Parkinson's disease (PD). |
IP Reference | WO2021156637 |
Protection | Patent / Patent application |
Year Protection Granted | 2021 |
Licensed | Yes |
Impact | We are seeking to translate this method and have also formed a spin out. |
Title | BIOMARKERS AND USES THEREOF |
Description | The present invention relates to methods of assessing whether an individual has Parkinson's Disease (PD) and/or is no longer responding to treatment, the method comprising the identification of one or more volatile compounds in the sebum of the individual. The present invention also relates to methods of extracting and detecting analytes from sebum. |
IP Reference | WO2020025967 |
Protection | Patent / Patent application |
Year Protection Granted | 2020 |
Licensed | Yes |
Impact | As before - spinout Sebomix Ltd. |
Title | DETECTION OF LIPID MARKERS |
Description | The present invention relates to methods for identifying high molecular mass lipids in samples. Such high molecular mass lipids may be useful as biomarkers for the identification of disease. |
IP Reference | WO2021156638 |
Protection | Patent / Patent application |
Year Protection Granted | 2021 |
Licensed | Yes |
Impact | Spin out |
Company Name | SebOMIX |
Description | SebOMIX develops a clinical diagnostic test for Parkinson's Disease, with the aim of commercialising its research. |
Year Established | 2021 |
Impact | The company has only just been spun out. It owns the IP of this technology. |
Company Name | Bio-Shape Ltd. |
Description | |
Year Established | 2015 |
Impact | so far 10 clients |
Website | http://www.bio-shape.com |
Description | Numerous Media Appearances on press release |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Due to the unusual origin of this project, it has already attracted a lot of attention. Joy Milne receives mail addressed to 'The woman that can smell Parkinson's, Perth, Scotland'. Barran and Milne have each given a TedX talk on this work (Barran PaloAlto 2019, Milne Manchester, 2023). They have appeared in four major documentaries/films (Paxman: Putting up with Parkinson's ITV 2022, The Woman who can Smell Parkinson's BBC 2017, Operation Ouch 2021, Natures Weirdest Events 2020) and numerous other media appearance have focused interest on this remarkable start to a scientific program. The PI and Joy receive on average 10 communications per week from people who thing they or someone they know has Parkinson's based on what they have heard from our work about the odour and also increased awareness of prodromal symptoms. We anticipate this public interest will continue and where appropriate will use it to inform people with PD about this study and its outputs. We will continue to use our connections to PD groups worldwide to engage with and communicate about, our research findings. These groups include national and regional patient and career research interest groups set up by Parkinson's UK, the Young Onset PD (YOPD) interest group, PD Avengers which addresses PPI and research required for People with PD, the Women's Group in PD - Joy Milne is founder member for the promotion of the 'Unmet Needs' of Women with Parkinson's Disease. |
Year(s) Of Engagement Activity | 2015,2016,2017,2018,2019,2020,2021,2022,2023 |