Conformer selected Top Down Sequencing - A Novel Approach to Structural Proteomics

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

The development of 'soft ionisation' techniques have positioned mass spectrometry as the central go-to technique for proteomic investigations. Electrospray ionisation (ESI)-MS is used extensively in this post-genomic era to determine the primary structure of proteins and has really become the essential tool in so called 'bottom-up' proteomic analysis. Extensive effort and resource has gone into mass spectrometry based proteomics, the majority of which relies on so called 'bottom-up' characterization where proteins are enzymatically cleaved into peptides for MS analysis followed by database correlation to identify (and quantify) the proteins under study. This approach has many analytical advantages; critically it is high throughput and sensitive and clearly has succeeded in many studies however it has some drawbacks. Perhaps the most obvious is that "bottom up" approaches cannot provide direct information on the active fold and interactions of the proteins being analysed, this limits the functional data that could be obtained from these studies.
The analytical advantages of mass spectrometry also apply to its use to examine intact proteins and complexes and there is an emerging research field that identifies proteins this way - so called "top-down" methodologies where proteins are sequenced in the mass spectrometer. Most top down approaches destroy the functional form of the protein prior to sequencing, to enable higher throughput and to facilitate more productive analysis from higher charged parent ions. This is not quite the route we will take, rather we will probe intact proteins and complexes , with careful use of nano-electrospray ionisation (nESI) to retain solution structures.
This proposal will develop so called "top down" methods to examine conformations and dynamics of proteins and protein complexes. The research program will enhance the gamut of predominantly solution-phase based techniques which evaluate protein structure and interactions. Methodologies will assess conformational stability, and dynamics of proteins both in solution and in a solvent free environment. Funds and research time are requested to construct novel instrumentation with which to measure conformations, and unfolding and refolding dynamics over timescales ranging from microseconds to minutes. The proposed instrument will also be able to photo-dissociate mass and conformer selected ions, and detect the product ions. The program of work following technology development will focus on two areas:
1. Top down structural proteomics; combining photo-dissociation and ion mobility mass spectrometry (IM-MS) to map protein structure and interactions.
2. Performing FRET combined with IM-MS, to determine protein unfolding pathways and the effect of fluorescent makers on protein structure.
The equipment will comprise a novel duel ion guide mobility mass spectrometer, wherein IM-MS will be used to determine collision cross sections, and optical methods will interrogate structure and stability via FRET and/or photo dissociation. This combined IM-MS photo activation approach will be termed photo-IM-MS.
Ions will be externally generated via ESI and transferred into a customised ion mobility mass spectrometer. Once in the duel drift region of the IM-MS, the drift time of ions (under the influence of a weak electric field) is related to their rotationally averaged collision cross section with the buffer gas. This mobility measurement can be made, or alternatively the ions will be 'stepped' into a parallel stacked ring ion guide drift region (2SRIG), which has a laser beam passing through it as well as optical detection. Ions in this region will interact with light and either be optically detected, or be pushed back into the first mobility cell. On exiting the cell ions will be transferred to a time-of-flight mass spectrometer and thus will be detected as a function of both mass, charge state, and cross section and potentially following optical interaction

Technical Summary

This research program will design, develop and implement an analytical platform which will allow us to determine collision cross sections on mass selected ions and also to characterise conformer selected ions using optical methods. We will be able to perform FRET (Fluorescence Resonant Energy Transfer) as well as ultraviolet and infra-red action spectroscopy and perhaps most widely applicable photo-dissociation. Our proposed approach is based on the technique termed ion mobility mass spectrometry (IM-MS) which determines the rotationally averaged collision cross sections of mass selected ions. The novel aspect will be the design of a parallel stacked ring ion guide mobility cell which will provide an off axis region to further optical interaction. We will develop "top down" sequencing/characterisation methods for intact and native proteins and protein complexes using photo-dissociation with ion mobility. Solution based methods will complement solvent free experiments. Preliminary work will use small peptides, model protein and protein complexes to demonstrate the technology. In later studies we will apply this instrumentation to study dynamic and more disordered systems.
The work will develop the commercially available mass spectrometer known as a Synapt HDS MS. Initial work will take place at our project partners Waters at the Waters Mass Spectrometry Technology Center in Manchester and subsequent development and implementation will take place in Edinburgh.
Molecular mechanics calculations will generate structures to compare with those determined experimentally and will be trained via experimental data. Studies will be supported by comparison to data obtained from conventional techniques, including, solution phase FRET, NMR, and AUC. We will also employ pulse labeling techniques with the mass spectrometer as the detector. This will allow us to evaluate the effects of solvation on protein fold and on conformation.

Planned Impact

We do our research to make an impact. This goes beyond the fame or money rewards that many in other sectors might be content with. From taking good data, that we understand, we wish to develop new knowledge, and new ways of obtaining knowledge. We also want to share that knowledge and for it to be of wide use. In the Barran group it is a matter of great pride that 15 students have received PhD's in the past 5 years, and along with 4 postdoctoral researchers, have then gone on to work in many other areas, trained and educated by their experience. This is one of the most immediate impacts from our research portfolio, but there is more to what we do than that. We have an on-going aim to use evidence from the gas phase to understand biological function. As a consequence of this, we collaborate widely with life science researchers so that we can bring the accuracy and selectively of gas phase analysis to assist with their understanding of the complicated processes that go on in the messy environment of cells.

Optical methods are used widely by cell biologists and biophysical chemists and we will here use similar methods albeit in the reductionist environment of the gas phase, which will contrast and complement cell work. Here we will build a novel instrument capable of determining the structures and dynamics of protein and protein complexes, adding to the emerging field of top down protein characterization. This will allow for rapid characterisation of protein structure, using very small amounts of sample .

We will have impact in a number of areas with this technology development. An early outcome will be that we will enable spectroscopy to be performed on a high resolution mass spectrometer capable of analyzing large intact bioactive protein complexes. The fact that we can also examine conformer and mass selected species with optical methods will have relevance to the proteomics community and in particular to those that are interested in mapping protein structure.

Once constructed, we apply our new technology platform to examine disordered and dynamic proteins and their complexes where much is predicted but little has been experimentally verified. The competing technologies for understanding IDPs are NMR and ultra-analytical centrifugation, but both require more sample than electrospray and neither can access as short timescales as we will here. In addition the high throughput capabilities of mass spectrometry are well suited to providing a global understanding unfolding dynamics, an area that we will exploit once this technology is implemented.

The research will have the most immediate benefit to research groups interested in the chosen protein systems; this will of course be those working on transcription factors but also will be biophysics groups interested in the nature of the disordered state of proteins and in related amyloidgenic systems. We are making an instrument that could have wide application. The use of photo dissociation as applied to peptides and sugars is growing and the 2SRIG system could readily be applied to such species. There are other non-biological systems which may also be well interrogated with this technology and we will seek collaborations in synthetic chemistry to test this.


Waters are very keen to see the possibilities of this research, we will work closely with them and our business development executives to best exploit this technology; Waters are our preferred partner since the 2SRIG is a technology relation to existing Synapt instrumentation, and since we have had several years of successful collaboration with them.

Publications

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Camacho IS (2019) Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor. in Proceedings of the National Academy of Sciences of the United States of America

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Pacholarz KJ (2016) Molecular Insights into the Thermal Stability of mAbs with Variable-Temperature Ion-Mobility Mass Spectrometry. in Chembiochem : a European journal of chemical biology

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Surman AJ (2016) Sizing and Discovery of Nanosized Polyoxometalate Clusters by Mass Spectrometry. in Journal of the American Chemical Society

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Ujma J (2018) Initial Steps of Amyloidogenic Peptide Assembly Revealed by Cold-Ion Spectroscopy. in Angewandte Chemie (International ed. in English)

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Yan C (2017) Real-Time Screening of Biocatalysts in Live Bacterial Colonies. in Journal of the American Chemical Society

 
Description We have been able to separate ions by their shape and then break each ion up into smaller parts using a UV laser to find out what it is and also why it has different shapes.
This helps us to 'sequence' peptides and proteins in the mass spectrometer and to gain unique structural information.
Exploitation Route We are examining a range of proteins and protein complexes to rapidly gain structural information,
We are involved in a consortium with FELIX (Radbourg University NL) and Waters where we will install a second Photo IMS system there.
Ohio State University are also setting up a new system.
Sectors Chemicals,Digital/Communication/Information Technologies (including Software),Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.mbc.manchester.ac.uk/
 
Description Waters are going to make a product with this modification
First Year Of Impact 2017
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description MRC Case
Amount £96,000 (GBP)
Funding ID MRC_CASE 
Organisation UCB Pharma 
Sector Private
Country United Kingdom
Start 09/2010 
End 08/2014
 
Description Development of software to analyse MS data - Uo Manchester and Waters 
Organisation Waters Corporation
Department Micromass UK Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have built and designed 2 software packages. One with Waters and one reliant on their instrumentation but as a stand alone analytical tool
Collaborator Contribution Host of Lukasz Migas for PIP. Offer of Case top up funding to this award
Impact 1. Two papers in review 2. Software developed currently arranging licensing agreement
Start Year 2015
 
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
 
Company Name Bioshape ltd 
Description Bioanalysis 
Year Established 2015 
Impact so far 10 clients
Website http://www.bio-shape.com