Mass spectrometry underpinning synthetic biology, industrial biotechnology and world class bioscience

Lead Research Organisation: University of Sheffield
Department Name: Chemical & Biological Engineering

Abstract

The application of biological mass spectrometry, in particular in the field of proteomics has revolutionised the ability of study a wide range of biological systems and answer important questions. Biological mass spectrometry is of central importance and currently underpins a diverse range of studies, across a wide range of biological systems. Within the Department of Chemical and Biological Engineering (CBE) we have significant expertise in the application of mass spectrometry approaches and have used this technology to answer important questions including; 1) how bacteria defend themselves from viruses 2) protein organisation in photosynthesis 3) how can we optimise cells to produce important products.

The Q Exactive Plus instrument is new to market (released Sept 2013) and provides the opportunity for the University of Sheffield and it's research partners to make a step-change in the scope of research activities. The instruments unique features will accelerate our academic and industrial research.

Underpinning our strong industry connections, the University of Sheffield has recently established an Advanced Biomanufacturing Centre (ABC). Arising from the Dept of Chemical and Biological Engineering, the aim is to develop interdisciplinary industrially-relevant research for biomanufacturing and bioprocessing within the UK bioindustry. The proposed instrument will be a critical asset; underpinning research associated with biomanufacturing and bioprocess development and will underpin an extensive portfolio of current BBSRC funded research, demonstrating the huge value of the equipment.

Technical Summary

Within the emerging areas of synthetic biology and industrial biotechnology, the development and application of high-throughput, quantitative biological mass spectrometry (MS) approaches will underpin established and potential research in these critically important areas. However, the demand for increased throughput, unmatched sensitivity and quantitative applications of biological MS will require new instrumentation that is not currently available at the University of Sheffield.

The Q Exactive Plus instrument is new to market (released Sept 2013) and provides the opportunity for the University of Sheffield and it's research partners to make a step-change in the scope of research activities. The instruments unique features, allow for targeted quantification of peptides, small molecules and nucleic acids and delivers high throughput, targeted quantitation combined with high-resolution accurate-mass data, that will accelerate our academic and industrial research. Fast polarity switching, parallel reaction monitoring (PRM), high-resolution accurate-mass selected ion monitoring (HR/AM-SIM) are just some of the techniques we will employ to our systems biology research with enhanced sensitivity and selectivity.

The proposed instrument will underpin an extensive portfolio of current BBSRC funded research, demonstrating the huge value of the equipment. Moreover, this will facilitate new and novel research opportunities and new impacts within industrial biotechnology, synthetic biology, and world leading bioscience. Furthermore, the proposed instrument will be a critical asset in the recently established Advanced Biomanufacturing Centre; underpinning research associated with biomanufacturing and bioprocess development. This will enable a step change in the biological MS workflows and analytical capability for users (academic and industrial), beyond current through-put and quantitation limitations, as a complement to existing assets.

Planned Impact

The aim of this proposal is to purchase a new mass spectrometer. The primary impact of the proposal will result from the research that we propose to carry out in the immediate future. Furthermore, it is expected that further impact will result from new and novel research opportunities and new impacts within industrial biotechnology, synthetic biology, and world leading bioscience areas.
The projects that focus on the areas of Industrial Biotechnology/ Synthetic Biology are of direct relevance to UK bioindustry and a wide range of the proposed research will be of fundamental importance to pharmaceutical / biopharmaceutical industries, with more immediate impact than those addressing more fundamental science. The proposed equipment will support projects aimed at developing biological medicines including protein therapeutics produced by E. coli and mammalian cells in culture. It will also support projects related to the identification and characterisation of oligonucleotide therapeutics. As part of the proposed research, methods will be generated that can be implemented immediately to reduce time and costs spent in the creation of biomanufacturing systems - this represents a clear economic benefit and increased capability and competitiveness for UK bioindustry.

In addition, the equipment will underpin a number of projects relating to more fundamental science that are likely to have more longer term impact, however will benefit those pharmaceutical companies including those developing novel antifungal agents and Health professionals preventing and managing C. albicans infection. In order to generate suitable therapeutics for treatment of disorders involving mRNA export it will be essential to understand the specific roles of target proteins in this process. This study will greatly expand our knowledge in this area and identify new potential targets for future therapeutic interventions involving mRNA export diseases. The understanding of photosynthesis is of fundamental importance as the basis of life on Earth, but also has the potential to impact upon the search for a sustainable energy future.

The new mass spectrometer will facilitate training in cutting edge mass spectrometry and inter-disciplinary science. As part of the Advanced Biomanufacturing Centre at the University of Sheffield, the aim is to develop interdisciplinary industrially-relevant research for biomanufacturing and bioprocessing within the UK bioindustry. The proposed instrument will be a critical asset underpinning research associated with biomanufacturing and bioprocess development. We will provide highly skilled PhD students and PDRAs capable of working in this inter-disciplinary environment throughout the UK. The new instrument will attract new users from other Universities and Industry who will benefit from the excellent training environment.

Publications

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Close ED (2016) Nucleic acid separations using superficially porous silica particles. in Journal of chromatography. A

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Hadfield TL (2016) AOAC SMPR(®) 2016.006. in Journal of AOAC International

 
Description This grant was used to fund a new mass spectrometer to underpin a broad range of BBSRC research and facilitate new research opportunities focussing on synthetic biology, industrial biotechnology and world leading bioscience.

The instrument has been successfully utilised to underpin a wide number of important research projects focussing on synthetic biology, industrial biotechnology and world leading bioscience. Research carried out on the instrument established new networks, collaborations and partnerships and increased our research capability. In addition data generated on this instrument has resulted in a number of publications.

The funding has also enabled collaborations to be established with Industry in the areas of Industrial Biotechnology/Synthetic Biology highlighting the importance to pharmaceutical / biopharmaceutical sector. Research on the instrument is essential for a number of collaborations including GSK, MedImmune, Syngenta, Lonza andPorton Biopharma.

We have developed and utilised a wide number of new analytical methods on the instrument. In addition we have trained a large number of researchers in the operation, maintenance and data analysis on the new instrument.
Exploitation Route Research performed on the instrument supported by this award is of direct and immediate relevance to bioindustrial partners in the Bioprocessing Research Industry Club (BRIC).

In addition data generated and methods developed utilising this instrument have published (see publications). Furthermore findings have been presented at important academic conferences in both the UK and USA.
Sectors Agriculture, Food and Drink,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Research supported by this grant for a new mass spectrometer was used underpin a broad range research including synthetic biology, industrial biotechnology and world leading bioscience. The one year award has only recently finished and much of the economic/societal impact is expected over the next few years relating to this award. The award has facilitated knowledge exchange, collaborations and potential translation of research outcomes and technology developed. In particular a number of research projects resulted in Industrial impact in collaboration with UK bioindustry including the pharmaceutical / biopharmaceutical sector. The instrument facilitated further engagement and the development of interdisciplinary industrially-relevant research for biomanufacturing and bioprocessing within the UK bioindustry.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Energy,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description BBSRC industrial CASE studentship
Amount £95,042 (GBP)
Funding ID BB/N504099/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2015 
End 09/2019
 
Description Development of a robust process analytical platform for the synthesis, purification and characterization of dsRNA
Amount £300,000 (GBP)
Organisation Syngenta International AG 
Sector Public
Country Global
Start 08/2014 
End 07/2017
 
Title Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry. 
Description Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact This research method has been adopted by Industrial collaborators Syngenta 
 
Title RNASwift 
Description RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. RNASwify does not use hazardous chemicals including phenol, chloroform and formamide which are often used in traditional RNA extraction methods. RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact The methods has been adopted by a wide number of users including industrial including Syngenta and Greenlight bioscience. This approach has economic benefits to the users and societal benefits. 
URL https://www.sciencedirect.com/science/article/pii/S0003269716302196?via%3Dihub
 
Title PRIDE PXD002638 
Description Mass spectrometry raw data deposited at PRIDE relating to the publication Hitchcock A, Jackson PJ, Chidgey JW, Dickman MJ, Hunter CN, Canniffe DP. Biosynthesis of Chlorophyll a in a Purple Bacterial Phototroph and Assembly into a Plant Chlorophyll-Protein Complex. ACS Synth Biol. 2016 May 24 PubMed: 27171912 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Mass spectrometry data used in the following publication Hitchcock A, Jackson PJ, Chidgey JW, Dickman MJ, Hunter CN, Canniffe DP. Biosynthesis of Chlorophyll a in a Purple Bacterial Phototroph and Assembly into a Plant Chlorophyll-Protein Complex. ACS Synth Biol. 2016 May 24 PubMed: 27171912 
URL https://www.ebi.ac.uk/pride/archive/projects/PXD002638
 
Title PRIDE PXD007825 
Description Mass spectrometry raw deposited in PRIDE for the following publication Kaneva IN, Longworth J, Sudbery PE, Dickman MJ.Quantitative Proteomic Analysis in Candida albicans Using SILAC-Based Mass Spectrometry.DOI: 10.1002/pmic.201700278 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Mass spectrometry raw data used in the following publication Kaneva IN, Longworth J, Sudbery PE, Dickman MJ.Quantitative Proteomic Analysis in Candida albicans Using SILAC-Based Mass Spectrometry.DOI: 10.1002/pmic.201700278 
 
Title PRIDE Project PXD003139 
Description Mass spectrometry raw data sets deposited at PRIDE for data used in Chen GE, Hitchcock A, Jackson PJ, Chaudhuri RR, Dickman MJ, Hunter CN, Canniffe DP. Two unrelated 8-vinyl reductases ensure production of mature chlorophylls in Acaryochloris marina. J Bacteriol. 2016 Feb 22 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact N/A 
URL https://www.ebi.ac.uk/pride/archive/projects/PXD003139
 
Title PRIDE Project PXD003149 
Description Mass spectrometry raw data deposited at PRIDE relating to Hollingshead S, Kopecná J, Armstrong DR, Bucinská L, Jackson PJ, Chen GE, Dickman MJ, Williamson MP, Sobotka R, Hunter CN. Synthesis of Chlorophyll-Binding Proteins in a Fully Segregated ?ycf54 Strain of the Cyanobacterium Synechocystis PCC 6803. Front Plant Sci. 2016 Mar 17;7:292. eCollection 2016 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Mass spectrometry data was used in the following publication- Hollingshead S, Kopecná J, Armstrong DR, Bucinská L, Jackson PJ, Chen GE, Dickman MJ, Williamson MP, Sobotka R, Hunter CN. Synthesis of Chlorophyll-Binding Proteins in a Fully Segregated ?ycf54 Strain of the Cyanobacterium Synechocystis PCC 6803. Front Plant Sci. 2016 Mar 17;7:292. eCollection 2016 
URL https://www.ebi.ac.uk/pride/archive/projects/PXD003149
 
Description Collaboration with Greenlight Bioscience, Boston USA 
Organisation GreenLight Biosciences, Inc.
PI Contribution Provided technical expertise, research direction and consultancy. Contributed to transfer of technology and methods developed in my laboratory to the industrial partner
Collaborator Contribution Provided experimental samples/systems for study at the University of Sheffield
Impact Multidisciplinary encompassing biological engineering, biomanufacturing, analytical chemistry and agritechnology. Outcomes included transfer of technology developed in my laboratory to the industrial partner. The company is now using the methods/technology we developed for their applications enabling solutions, increasing throughput and economic benefits to the company.
Start Year 2017
 
Description Collaboration with Medimmune 
Organisation MedImmune Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have initiated a collaboration with Medimmune, providing expertise and advice for future research plans within Medimmune. We have discussed potential research opportunites using the technology we have developed and applications to analyse current materials being developed at Medimmune.
Collaborator Contribution The industrial partners are providing materials to be sent to Sheffield to be analysed
Impact Multi-disciplinary project across the physical-life science interface. Disciplines included manufacturing, analytical chemistry and life science. Production of RNA therapeutics/medicines and characterisation using bioanalytical tools.
Start Year 2018
 
Description International collaboration with Wageningen University, The Netherlands 
Organisation University of Wageningen
Country Netherlands 
Sector Academic/University 
PI Contribution During the course of this study and the work developed facilitated the collaboration with international partners at the Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands. This work enabled the collaboration on the CRISPR/Cas system using the developed methodology of the funded research. The collaboration with the Wageningen group facilitated the application and development of novel approaches for the direct purification of RNA from RNA/protein complexes prior to MS analysis. The collaboration enabled the development of novel analytical techniques (in Sheffield) to study the relevant biological system (Wageningen). This resulted in a number of publications from the collaborative work.
Collaborator Contribution Collaborators provided materials for analysis using our analytical methods.
Impact Publications doi: 10.1042/BJ20131474 doi: 10.1093/nar/gkx1264; doi:10.1073/pnas.1111854108; doi: 10.1038/nsmb.2019; doi: 10.1126/science.1159689.
Start Year 2008
 
Description Research Collaboration with GSK 
Organisation GlaxoSmithKline (GSK)
Department Research and Development GSK
Country United Kingdom 
Sector Private 
PI Contribution Development of 2DLC methods for the analysis of therapeutic oligonucleotides.
Collaborator Contribution Industrial partner provides research supervision and direction, In addition industrial partner provides samples for use in the research projects
Impact PhD CASE studentship award (BBSRC) (BB/K501086/1) Nucleic acid separations using superficially porous silica particles. Close ED, Nwokeoji AO, Milton D, Cook K, Hindocha DM, Hook EC, Wood H, Dickman MJ. J Chromatogr A. 2016 Apr 1;1440:135-44. doi: 10.1016/j.chroma.2016.02.057. Multidisciplinary collaboration across life science/physical science interface
Start Year 2012
 
Description Research collaboration with Australian National Laboratory 
Organisation Australian National University (ANU)
Department College of Medicine, Biology & Environment (CMBE)
Country Australia 
Sector Academic/University 
PI Contribution Development and application of novel bioseparations and mass spectrometry analysis. Applications to a number of different biological systems including identification and quantification of histone PTMs and RNA modifications
Collaborator Contribution Collaborators provided samples for LC MS analysis.
Impact Extensive histone post-translational modification in honey bees Dickman MJ, Kucharski R, Maleszka R, Hurd PJ. Insect Biochem Mol Biol. 2013 Feb;43(2):125-37. doi: 10.1016/j.ibmb.2012.11.003. Epub 2012 Nov 20. Multidisciplinary collaboration across the physical sciences (bioanalytical) and life sciences (honeybee biology and environment)
Start Year 2010
 
Description Research collaboration with Gurdon Institute, University of Cambridge 
Organisation University of Cambridge
Department Gurdon Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution As part of this collaboration we performed mass spectrometry analysis on wide range of samples, including quantitative proteomic analysis using SILAC. The collaboration utilised the MS equipment on the grant and additional data and bioinformatic analysis.
Collaborator Contribution Research partners generated the samples using affinity pulldowns using designed nucleic acids probes with specific nucleic acid modifications.
Impact This is a multidisciplinary collaboration across the physical science-life science interface.
Start Year 2014
 
Description Research collaboration with Porton Biopharma LTD 
Organisation Porton Biopharma LTD
Country United Kingdom 
Sector Private 
PI Contribution Provided analytical support including HPLC and mass spectrometry analysis.
Collaborator Contribution Provided appropriated samples and research discussions.
Impact Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry. Gervais D, King D, Kanda P, Foote N, Elliott L, Brown P, Lee NO, Thalassinos K, Pizzey C, Rambo R, Minshull TC, Dickman MJ, Smith S. Pharm Res. 2015 Nov;32(11):3636-48. doi: 10.1007/s11095-015-1722-2. Epub 2015 Jun 4.
Start Year 2015
 
Description Research collaboration with Roman Sobotka (University of South Bohemia, Czech Republic) 
Organisation University of South Bohemia
Country Czech Republic 
Sector Academic/University 
PI Contribution Mass spectrometry has been used to underpin a number of important investigations in collaboration with Roman Sobotka (University of South Bohemia, Czech Republic). WE have used both qualitative and quantitative mass spectrometry studies on the QExactive HF instrument to provide further Investigation of protein profiles across a light-dark time-course in Synechocystis.In addition we have used MS to determine the stoichiometry of interaction partners in FLAG-ChlG, FLAG-Ycf39 and His-ChlG pulldown experiments using QconCAT-based quantification.
Collaborator Contribution The collaborators provided samples for MS analysis.
Impact This is multidisciplinary collaboration across the life science and physical science interface.
Start Year 2016
 
Description Research collaboration with ThermoFisher 
Organisation Thermo Fisher Scientific
Country United States 
Sector Private 
PI Contribution Development and application of novel HPLC methods for the analysis of nucleic acids.
Collaborator Contribution Industrial partner provides novel stationary phases and chromatography columns to evaluate and develop for nucleic acid separations
Impact Nucleic acid separations using superficially porous silica particles Elizabeth D. Close, Alison O. Nwokeoji, Dafydd Milton, Ken Cook, Darsha M. Hindocha, Elliot C. Hook, Helen Wood, Mark J. Dickman, In press. doi:10.1016/j.chroma.2016.02.057
Start Year 2010
 
Description Syngenta 
Organisation Syngenta International AG
Country Global 
Sector Public 
PI Contribution We have a partnership with Syngenta to develop and optimise analytical tools for the analysis of nucleic acids
Collaborator Contribution Syngenta provide research support, supervision, funding and materials for analysis
Impact doi: 10.1016/j.ab.2016.08.001; doi: 10.1016/j.chroma.2016.12.062; doi: 10.1002/rcm.8074; doi: 10.1021/acs.analchem.7b04000 Multi-disciplinary collaboration -chemistry/life science/engineering. Research focuses on the development of analytical methods, biomanufacturing and synthetic biology approaches. Direct Research funding from Syngenta £400,000 awarded to Prof M Dickman (3 grants) BBSRC Industrial CASE award (BB/N504099/1) Prof M Dickman £95,000; Industrial CASE Studentship from the EPSRC National Productivity Fund with Syngenta £95,00 Prof M Dickman EPSRC HEFCE Catalyst Studentship-Prof M Dickman
Start Year 2012
 
Description BRIC updates 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Research presentations at 6-monthly BRIC dissemination meetings (2015/2016) to demonstrate the methods developed and data generated on to a wide audience in the biopharmaceutical/biomanufacturing sector. 50-100 delagates were present from both academic/industrial and all BRIC partners.

Outcomes included demonstration of research capability and methods, networking and establishing industrial collaborations/partnerships.
Year(s) Of Engagement Activity 2015,2016
URL http://www.bbsrc.ac.uk/innovation/sharing-challenges/bric/
 
Description Invited seminar Queen Mary University of London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Approx 50 people attended including undergraduate, postgraduate students in addition of academic staff. Following seminar questions and discussion followed and future collaborative work was discussed with members of staff and the University.
Year(s) Of Engagement Activity 2016
 
Description Research seminar at Syngenta, Jealott's Hill, Berkshire 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Research presentation at Syngenta. Industry audience 11-50 members, including international attendees via weblink conference across the global company . Outcome/impacts include discussions for further funding and dissemination of research impact.
Year(s) Of Engagement Activity 2017
 
Description UCAS Schools visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Each year 50-200 UCAS school children visit the Dept. As part of the UCAS visit a short tour of the labs and an overview of my current research was provided and current research project taken by undergraduate students. The talk was followed by discussion and questions.

Enabled the Dept to build links with a number of schools for further outreach activities
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
 
Description Visit to Greenlight Bioscience 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Research presentation at Greenlight Bioscience in Boston, USA and expert panel discussions focussed on research activities and implementation of technology from my laboratory. Impact included the company now is using the technology/methods we developed.
Year(s) Of Engagement Activity 2017
 
Description • Invited Conference Seminar, KTN's Biopharmaceutical Analytical Group Symposium, Leeds, UK March 15 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Conference presentation at a Knowledge transfer network meeting- KTN's Biopharmaceutical Analytical Group Symposium
Outcomes-Dissemination of research and methods to a wide audience including industry and academia. Established new collaborations with both industrial and academic partners. Increased interest in the methods we have developed and future research opportunities.
Year(s) Of Engagement Activity 2015
 
Description • Working party/invited seminar with Syngenta LTD (UK and Ghent, Belgium) 2015 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Research presentation and research discussions as part of working group party established with Syngenta (both in the UK and Ghent, Belgium).

Outcomes-established/enhanced collaboration with industrial partner Syngenta. Outcomes dissemination of novel methods and techniques developed in our laboratory. Led to further funding from the industrial partner and future research plans.
Year(s) Of Engagement Activity 2015