Mass Spectrometry Based Lipidomics and Metabolomics to Drive Bioscience Discovery

Lipids are key components of biological system ranging from plants to mammals. Lipid molecules are critically involved in membrane structures of cells and also provide an energy store and act as signalling molecules within and between cells. Hence, understanding lipid metabolism is important for all areas of the BBSRC remit.
Mass spectrometry (MS) represents the primary technology in lipidomics and when combined with liquid chromatography (LC) is suitable for the unbiased identification and quantification of lipids in complex mixtures. Extra dimensions of separation are provided by high resolution MS, where isobaric ions (ions with the same nominal mass but different exact mass) are resolved, and by tandem mass spectrometry (MS/MS) or multi-stage fragmentation (MSn), where characteristic ions are separated from background. A more recent addition to MS-tool box is MS-imaging (MSI) adding further to the available bioimaging technologies.
The UK has a burgeoning lipidomic community but lacks a MSI resource. The current proposal is for a high resolution MS instrument with MS/MS and MSn capabilities combined with interchangeable interfaces for high spatial-resolution atmospheric pressure (AP)-MALDI and electrospray ionisation (ESI). The AP-MALDI source offers high spatial-resolution (5 - 10 microm), at the single cell level, while the ESI interface is compatible with other imaging modalities e.g. Liquid Extraction for Surface Analysis (LESA). The instrument will be a resource for BBRSC-remit research and for the UK lipidomic community both academic and industrial. The new instrument will underpin scientific research in several key strategic priority areas for BBSRC, including (1) Healthy ageing across the life course; (2) Sustainably enhancing agricultural production; and (3) The replacement, refinement and reduction (3Rs) in research using animals.

The main aim of this proposal is to establish a mass spectrometry imaging (MSI) resource for the BBSRC lipidomics community in the UK. This will be achieved through the siting in Swansea University of an MSI instrument capable of high spatial-resolution (5 - 10 microm), high mass-resolution (>200,000 (FWHM) at m/z 400) and capacity to perform tandem-MS (MS/MS) and multistage-fragmentation (MSn). The instrument will be equipped with a AP-MALDI source with 10 kHz Nd:YAG laser at 355 nm and spot size of <10 microm. An advantage of the AP-MALDI source is that it can be rapidly interchanged with an electrospray ionisation (ESI) source, minimising down-time between different applications. The ESI source can be utilised with other imaging modalities, e.g. Liquid Extraction for Surface Analysis (LESA). The mass spectrometer will have a resolution of >200,000 (FWHM) at m/z 400, allowing separation of lipid signals from isobaric (ions with the same nominal mass but different exact mass) chemical noise. This will be important for the imaging of low-abundance lipids. The instrument will be able to perform both MS/MS and MSn with high mass-resolution and high mass-accuracy measurement of fragment-ions. MS/MS will add an extra dimension to MSI allowing the exploitation of multiple-reaction monitoring methods commonly used for LC-MS applications to enhance signal to noise ratio. In addition, MSn is a particularly important fragmentation method for the structural identification of unknown lipids.

This application is to install a high spatial-resolution mass spectrometry imaging (MSI) instrument at Swansea University. The instrument will be a resource for BBRSC-remit research and the UK lipidomic community. As illustrated in the application work-packages, the equipment will drive bioscience discovery in a breadth of research areas ranging from food and agriculture to animal and human health and consequently have significant social and economic impact.
Impact on food and nutrition: Global consumption of edible oil has increased steadily at an annual rate of about 5% for the past 50 years. Innovation in oilseed technology is required to create a hybrid vegetable oil replacement for fish oil and for production of high value oils. High spatial-resolution MSI will elucidate the compartmentation of oil assembly in developing seeds, which is essential for the redesign of lipid biosynthesis in seeds in order to improve yield.
Impact on agriculture: A major limitation to plant growth is restricted access to nutrients in the soil. To improve nutrient acquisition, most land plants enter a beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. Incomplete understanding of the mechanism underlying this symbiosis makes it difficult to exploit for plant crop improvement. A recent discovery made at Rothamsted Research that plants supply lipids to arbuscular mycorrhiza was a paradigm shift. MSI will now provide a complete spatial understanding of lipid exchange and allow us to learn how to facilitate this process for agricultural benefit.
At Swansea University, a patented (WO2016198852A1) technology "symbiont-mediated RNA interference" was developed to eliminate insect pests. Lipidomics will be used to identify the RNAi target in insects to prevent transmission of a plant pathogenic virus. This virus has a huge worldwide impact on agricultural productivity. Current control strategies for agricultural insect pests are heavily reliant on chemical pesticides. However, there is no safe targeted chemical pesticide. This research aims to replace harmful non-targeted chemical pesticides with a smart, environmentally-friendly alternatives and so improve worldwide agricultural productivity. In addition, the technology could be translated to limit transmission of viruses by insects that cause human and animal disease.
Impact on animal and human health: Lipid metabolism underpins normal physiology and homeostatic control during early development and across the lifespan. One particular example is cholesterol metabolism. Cholesterol is implicated in various neurological disorders and much more research into the involvement of cholesterol metabolism in healthy brain function is still required. We will use the proposed instrument with Swansea University patented (US9851368B2) technology to generate the first 3D-attlas of the sterol content of brain, which will fill significant gaps in our understanding of brain biology across the life span and also generate considerable interest from academics, clinicians, the pharmaceutical industry and the public in general.
Impact with 3Rs: Through better characterisation of the metabolic consequences of toxin/carcinogen exposure to human 3D tissue models, we will validate these models as an alternative approach to animal testing. This will impact the safety assessment in chemical and pharmaceutical industries and offer the possibility of replacing animal use in future.
Impact on drug discovery: Improved methods and technologies to image drugs and their metabolites in tissue will impact the drug discovery process
Impact on training for the lipidomics community: The availability of a MSI lipidomic community resource, also available for young researchers to access, will greatly improve training options available in the UK.