Mass spectrometry imaging for biology and biotechnology

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.

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.

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.