Mass spectrometry imaging for biology and biotechnology

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

Mass spectrometry is a 'broadband' technology - everything has a mass. Mass spectrometers have developed over the past 15 years to attack more and more problems in biology, addressing the issue of complexity of the sample and the subtleties of the biomolecular domain. However, it is only recently that there have been significant developments in the ability to acquire a mass spectrum from a biological material in a spatially defined fashion - 'MS imaging'. The instruments designed for this application can generate an image in which the measured signal is not simply a colour (as in optical imaging) but a mass spectrum.

We propose to establish an MS imaging platform at the University of Liverpool that is accessible to other scientists within and outside the University. The system will couple a high resolution, high sensitivity mass spectrometer with two different imaging modalities - a matrix assisted laser desorption ionisation method (MALDI) and a laser activated electrospray ionisation method (LAESI). In MALDI, the sample is coated with a matrix molecule that absorbs the ultraviolet laser energy and which directs that energy to activation and charge addition to generate ions from the tissue or sample. In LAESI, an infrared laser is used to effect localised heating and volatilisation of the sample in close proximity to a probe that generates the ions for analysis. MALDI is preferred for small biomolecules, whereas LAESI is preferred for biological macromolecules. The two different sources permit the entire range of biological molecules, from low molecular weight metabolites to macromolecules such as proteins to be analysed.

The mass spectrometer we propose to install has several advantages. First, it is a high resolution instrument and the exquisite sensitivity is enhanced by a new design within the ion optics that steers informative ions into the mass analyser and away from contamination and background - this greatly increases sensitivity. Secondly, the instrument includes an ion mobility device that can further resolve ions according to their cross sectional area. This is particularly advantageous in imaging applications, where the complexity of the sample can compromise signal to noise and thus, sensitivity.

The main application for this platform has traditionally been in tissue visualisation - the exemplar project here is in the spatial mapping of damage to joints, or particular relevance to the biology of ageing. Whilst tissue imaging will be a major demand on instrument time, we will also explore new applications for one or the other ionisation methods. These include interrogation of proteins or carbohydrates that have been captured on arrays, rapid screening of metabolites in microorganisms that are engineered to generate new chemicals, for rapid phenotyping of proteins in biological fluids and for the direct visualisation of scent marks deposited in the environment. Thus, the new capacity will have broad applicability to a large set of BBSRC-funded projects.

To our knowledge, there is no system of comparable sophistication and scope that is in the UK and openly available to UK researchers. By establishing this platform at Liverpool, it will be under the management of the University of Liverpool Technology Directorate. The Technology Directorate has the mission of providing access to the very best research facilities for the maximal number of users, both inside and outside the University. It has the mechanisms and structures to ensure full time operation and to ensure access, as well as providing financial support for properly open facilities such as this, and by awarding access grants to allow academics, and particularly early career staff, to be able to use such facilities in advance of winning substantive funding.

Technical Summary

We propose to install a comprehensive mass spectrometry system that will allow the acquisition of 'mass images' from a range of samples, including tissues (plant and animal), protein and carbohydrate arrays, liquid samples for high throughput applications and naturally deposited samples such as those present in competitive scent marks. A comprehensive system should be capable of handling the full range of biological molecules, from low molecular weight metabolites (e.g lipids, including pheromonal steroid sulphates, microbial metabolites, carbohydrates and proteins). The diversity of the analytes requires that different ionisation methods be used. We have selected MALDI/LDI for low molecular weight analytes, and LAESI for both low molecular weight metabolites and high molecular weight macromolecules - LAESI is able to generate multiply-charged protein molecules to bring them into the accessible m/z range of the instrument. Both sources will be coupled to the same mass spectrometer, a Synapt G2si, that has a resolution of 50,000 FWHM, 1ppm mass accuracy, high sensitivity (in part from the use of a 'Stepwave' device to increase signal:noise ratio) and a travelling wave ion mobility cell to enhance peak capacity, specificity and sensitivity of analyses. The spatial resolution of the MALDI source is 15um, and for LAESI, 200um. The exemplar projects for which the platform will be used include:

= Spatial imaging of rodent scent marks
= Imaging of tendons to define functional performance and damage
= Rapid phenotyping by intact mass protein profiling
= Imaging and mass-based analysis of protein arrays
= Sequencing of enriched glycans on lectin arrays assembled onto gold
= High throughput microbial metabolite identification during rapid evolution of new enzyme catalysts

The range of projects is designed to indicate the breadth and broad applicability of these new ionisation and imaging approaches in analytical biological mass spectrometry.

Planned Impact

The main beneficiaries of this technology will be the indirect users of research driven by the academic users and thus, will be broad ranging. The exemplar projects must be seen as a small subset of the entire constituency of users, but even with this small group, the beneficiaries are clear.

For example, the large BBSRC-funded sLoLa programme on rodent scent communication (between Liverpool and Rothamsted) has the potential to radically change our approach to rodent pest control, reducing harm to non-target species and greatly decreasing losses to foodstuffs through pest damage. Similarly, the ability to screen evolved microorganisms quickly would greatly enhance our route to 'green chemistry' in the search for new chemicals such as antibiotics - this too is a BBSRC sLoLa programme. The programme on tissue imaging focuses on tendon structure and function, with particular reference to damage and age-related changes - of particular relevance to a rapidly ageing population. Lastly, the ability to develop mass spectrometry to image arrays offers a previously unrealised degree of sophistication in the analysis of biomarkers and diagnostics.

The technology development implicit in this application (we are not just users of this technology, we wish to develop it into new research areas) related to the BBSRC strategic priority of 'Enabling New Ways of Working(ENWW)/Technology Development". The sub-projects that are highlighted link directly to "Basic Bioscience Underpinning Health /Ageing Research (tendon structure/function relationships), Food Security/Global Food Security (new methods to control rodent pests), "Industrial Biotechnology/New Strategic Approaches" (green catalysts) and ENWW/Systems approaches (global proteome reduction and top-down analysis)

It follows that access to the type of technology, currently almost inaccessible in the UK, has the potential to enhance areas of research. The search for new antibiotics is approaching crisis point. We will soon reach the point where the planet cannot produce enough food to feed the expanding population. Further, we are an ageing population, and we need to understand the ageing process in greater detail. We need improved biomarkers and diagnostic platforms to help us maintain our health into old age.

Thus, the beneficiaries of this research are the general population, who will benefit from improved diagnostics, improved security of pre- and post-harvest crops and the means to extend their lifespan healthily and productively. By ensuring that researchers working in these areas are maintained at the cutting edge of analytical science, we ensure optimal progress towards realisable goals. Some of the outputs from this research may be realised within a five-year timeframe.

All of these areas (green chemistry, improved diagnostics, rodent pest control) have the potential to generate intellectual property that in turn engenders new products and manufacturing processes. The applicants and the protagonists of the exemplar projects have a good track record of industrial collaboration and in the promulgation of their research. Thus, UK industry stands to gain very directly from the availability of this enabling technology from new catalysts, new biomarker and biosentinel molecules to rodent control methods that do not rely on toxic anticoagulants. Each of these categories will generate intellectual property that can be commercialised and used to bring new products to market.

Publications

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Description To soon to evaluate. MS Imaging has not proven to be popular, but we continue to use the instrument as a MALDI analyser, on a virtually daily basis. The instrument is functioning well. The REIMS source is completely novel.We have also installed a REIMS source on one of the instruments, and this is yielding valuable new data, leading to a CASe studentship, and further collaborations.

We continue to develop and offer Imaging, and are actively seeking new collaborations. We have a full trained, University postdoctoral research fellow who is fully trained in the technology and an established imager, but at the present, her efforts are being directed to supporting the instrument in other ways, in order to make optimal use of her time, and of the instrument, ensuring a good return on investment for BBSRC.

The Centre for Proteome Research (CPR) was a beta test site for a new approach to analysing biological samples, called REIMS (rapid evaporative ionisation mass spectrometry). Essentially, this is based on the combustion of a sample using diathermy, and the smoke that is generated is then ionised at high temperature to generate complex patterns of ions that create diagnostic signatures. These signatures are information rich, and although the initial development of REIMS and the application was for identification of tumour margins during surgery, we have shown that this method of data acquisition has considerable added uses in the biological arena. In particular, we have demonstrated, and are in the process of writing up, a paper that has confirmed the ability of REIMS to provide accurate species identification from faecal samples produced by small rodents, at a few seconds per analysis.

This has confirmed the utility of REIMS in field biological applications, and CPR has been talking with colleagues in International Pheromone Systems (IPS) about the application of REIMS. IPS are pioneers in the manufacture of pheromones, lures and traps used to promote sustainable integrated pest management. We have discussed the potential of REIMS for identification of small insect crop pest species. Often, these are subtly different in appearance and require the skills of a trained entomologist.

We wished to explore the potential of REIMS as a rapid diagnostic tool. In brief, field collected insects will be killed by freezing and combusted (individually) using the REIMS source. There will need to be some optimisation of sample combustion protocols and data acquisition modes. Initial experiments will use different beetles (to define species separation) and more closely related species of ants, as these can be maintained in IPS facilities over the winter months. Subsequently, and outside the time frame of this project, we will deploy the same method for field-collected samples in the spring. At this time, all methods and equipment will be in place for a PhD student to develop this area further. It will form a major part of her thesis, and has the potential to greatly increase the company's ability to identify pest species.

This preliminary work was funded by a BBSRC internal grant. The University of Liverpool was awarded a BBSRC Agri-Food Technology Seeding Catalyst Award. The purpose of the award was to provide funding which enables academics to undertake a preliminary / early-stage translational project that may eventually enable the development of new agri-food technologies and processes that:

• increase productivity in the agri-food sector;
• provide healthy, safe, high quality and nutritious food for UK consumers and global markets, or
• ensure supply chain integrity and long-term environmental resilience.

In this instance, we have proven that REIMS can be used for a range of filed-compatible processes, including insect identification and characterisation of rodents through faecal analyses. The instruments have thus been used exhaustively in a broader range of applications. IN a paper being prepared we write: We describe a new approach to recovery of information from faecal samples, based on analysis of the molecular signature generated by rapid evaporative ionisation mass spectrometry (REIMS). Faecal pellets from five different rodent species were analysed by REIMS and complex mass spectra were rapidly acquired (typically a few seconds per sample). The uninterpreted mass spectra (signatures) were then used to seed linear discriminant analysis and classification models based on random forests. It was possible to classify each species of origin with a high rate of accuracy, whether faeces were from animals maintained under standard laboratory conditions or wild-caught. REIMS signatures were stable to prior storage of the faecal material under a range of different conditions and were not altered rapidly or radically by changes in diet. Further, within species, REIMS could discriminate faeces from adult versus juvenile mice, male versus female mice and those from three different laboratory strains. REIMS offers a completely novel method for the rapid analysis of faecal samples, extending faecal analysis (previously focused on DNA) to an assessment of phenotype, and has considerable potential as a new tool in the armamentarium of the field biologist.
Exploitation Route MS Imaging is relatively new technology. It is not yet clear how widely this can be deployed, and the information that can ensue. Ms Imaging is offered to external users through the Technology Directorate at Liverpool.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Instrumentation platform recently installed, training now in place. Too early to evaluate impacts. A major problem has been the lack of specialist skilled staff able to use the instrumentation. However, the University of Liverpool has in 2015/2016 allowed appointment of a specialist imaging scientists, and we expect this to greatly enhance usage. Update: the addition of a REIMs source has also given us new approaches to biological sample analysis and imaging.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology
 
Description Catalyst Fund
Amount £45,540 (GBP)
Funding ID 16-VUW-011-CSG 
Organisation Royal Society of New Zealand 
Sector Learned Society
Country New Zealand
Start 05/2017 
End 09/2018
 
Description Seeding Catalyst Award
Amount £6,000 (GBP)
Funding ID BB/SCA/Liverpool/17 
Organisation University of Liverpool 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 02/2018
 
Title REIMS for biological sample analysis 
Description A new approach to remote sensing of phenotype using REIMS analysis of faecal samples. Developed with a beta version of the ion source. Publication is pending exploration of IP 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact A completely new approach to environmental sampling 
 
Description Access to advanced MS Unilever 
Organisation Unilever
Department Unilever UK R&D Centre Port Sunlight
Country United Kingdom 
Sector Private 
PI Contribution Application of advanced proteomics to Unilever challenges
Collaborator Contribution Access to advanaed MS and expertise
Impact Report (confidential) to company New collaborations Potential new PhD students
Start Year 2015
 
Description MS collaboration (Waters) 
Organisation Waters Corporation
Department Micromass UK Ltd
Country United Kingdom 
Sector Private 
PI Contribution Development of new applications for ion sources Collaboration on publications
Collaborator Contribution Access to advanced technology Support for CASE studentship applicationd
Impact Joint publications CASE students
Start Year 2010
 
Description BSPR Nominated Lecturer 2016-2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact BSPR Lecture "The proteomics of sex" delivered to Duke University, CALTEH, Dundee, and the London Biol Sci Ms group
Year(s) Of Engagement Activity 2016,2017