A mass spectrometry centre for the analysis of glycerolipids, glycerophospholipids and sphingolipids, and their lipid oxidation products

Lipids are the fatty molecules that make up the membranes that surround cells; without them life would not exist. However, this is only one of the important roles that lipids play in biology. For example, lipids are also involved in many forms of communication within and between cells, and the lipid composition of the cell membrane affects the activity of proteins embedded in it, such as those that transport molecules in and out of the cell. Damage to lipids by reaction with oxygen, in the same way that cooking oils go rancid, is related to many of their roles in disease. Hence, the analysis of lipids and understanding their roles in biology are very important areas of research. However, the comprehensive analysis of lipids is challenging as they are a very complex set of molecules, with over 100,000 different types in human cells, and many more when bacteria and other microorganisms are included. Many of them have very similar chemical structures, making it hard to tell them apart, but very diverse effects, so it is important to identify them correctly. The equipment we will buy with this grant has an extra dimension for the separation of molecules, based on their shape, which will greatly enhance the number of different lipids that we are able to distinguish and will enable a wide range of research to help understand their complex roles in biology.

There are many examples of how lipids are important in life. For example, they play a role in controlling cell growth to cell death, including processes particularly important in conditions such as inflammation. Lipids can also affect the activity of proteins in the cell, and particularly those in the cell membranes, many of which are targets for drugs such as morphine and insulin. Analysis of the lipids associated with membrane proteins, and the effects that changing of these lipids has on the activity of the proteins, is important in understanding these effects and how they may change with age or diet, or in other diverse areas, such as the production of biofuels or processes in bacterial replication that could be new targets for antimicrobials. Lipids contain many sites that can be attacked by reactive chemical species, and these damaged lipids can themselves have biological activity or react with other molecules impairing their function. An example is LDL, or bad cholesterol. Oxidative damage to the lipids in LDL is thought to be responsible for changes that lead to heart attacks and strokes. We need to be able to analyse the different lipids that are generated in these reactions, how they interact with or react with biological systems, and what effects this has on the biological system.

This grant proposal is provide instrumentation that will allow us to perform the complex analysis required to confidently identify and measure the amount of lipids that are present in complex biological samples. The main technique to be used is mass spectrometry, which measures the weight of molecules very accurately, as well as being able to break up the molecules to get information on their structure. However, the current methods are not always able to separate all the individual components in complex mixtures to allow their full analysis, especially of low abundance molecules that affect cell behaviour. The new instrument will provide extra capabilities through an additional dimension for separation of the molecules, called ion mobility, which is able to separate molecule based on their shape. The equipment will be the first available of a new design of instrument that allows much finer separation of molecules (it has a cyclic ion mobility cell providing much longer effective separation path lengths). This will allow us to do more accurate measurement of the lipids present and the way in which they are changed, leading to a much better understanding of biology in many important areas.

Separation and ionization methods coupled to mass spectrometry for lipid analysis are improving rapidly, but substantial further progress is needed to identify and quantify lipids and lipoxidation products effectively and efficiently in complex biological samples, and relate these to biological function.

This proposal is for the latest generation ion mobility mass spectrometer to create a resource for the analysis of glycero- and glycerophospholipids and sphingomyelins, their oxidation products and the reaction of these with proteins. Current methods struggle to provide a comprehensive identification and quantification of lipids in complex samples. There is huge congestion in lipid mass spectra due to the complexity of the lipidome (over 100,000 species, which increases exponentially when including lipids from microorganisms and oxidation products), the narrow mass range in which lipids occur, the high levels of isotopic peaks, and the many isomeric and isobaric species. Added to this is the complexity of analysis of the reaction products of oxidised lipids with proteins.

Ion mobility mass spectrometry is being increasingly utilized to provide an additional, orthogonal separation step within the mass spectrometer, and is becoming essential for global lipid analysis. This is particularly important for identification and quantification of low levels of lipids or lipids that ionize poorly in complex mixtures, and to speed up analyses using faster chromatography or shotgun approaches.

Exemplar areas of research that would be enabled by this additional capability include: i) bioenergy & industrial biotechnology, including development of organisms for biofuels, chemical & protein production ; ii) membrane protein-lipid interactions in combatting antimicrobial resistance; iii) healthy ageing across the lifecourse; iv) lipid oxidation & lipoxidation in signaling pathways; v) the role of lipid metabolites in biological effects of extracellular vesicles.

The new ion mobility mass spectrometry resource for the analysis of glycerolipids, glycerophospholipids and sphingomyelins, their oxidation products and reactions to give lipoxided proteins established by this grant will enable world class research and open up new areas of biology. It will be available to all interested parties, with a focus on the Midlands Innovation University group, and will be supported by technical and academic expertise. It will enable comprehensive identification and quantification of these molecules in complex and small samples. This will clearly benefit a wide range of scientists from diverse scientific communities.

Key beneficiaries will be:
i) The industrial partner on the project, UK-based Waters Scientific Mass Spectrometry, will benefit from the early application of their new technologies to complex, real-world samples in strategic research areas of lipid research, which instrument manufacturers often find challenging to do. The collaborative and co-operative approach will lead to exemplar data and facilitate increased competitiveness in the market, and ultimately benefit the UK economy.
ii) Biological scientists who have research that would benefit from the improved analysis of glycero(phospho)lipids and sphingomyelins, or their oxidation products, will have access to the resource, be able to utilize the new instrument and benefit from improved identification, resolution and quantification of their analytes. In the first instance this will be a fairly small community of researchers (~20) but longer term this will rapidly increase as we build further links with the community.
iii) The lipidomics / lipid oxidation / lipoxidation community as we develop new methods and demonstrate the benefits of the improved ion mobility resolution, and identify new lipids species, the data for which will be deposited in publicly accessible databases.
iv) The general public, in that the research enabled by the new resource will impact on health and wealth in areas such as green production methods, drug and drug target discovery, healthier ageing, and has the potential to lead to other benefits such as biomarker identification.

Direct benefits from the research projects described in the proposal will benefit industrial scientists in the biotechnology and bioproducts field; this will be beneficial both to UK and European companies. Looking ahead, there is also potential for impact on clinical diagnostics units and healthcare by improved understanding of inflammatory disease processes (e.g. in diabetes, cardiovascular disease) or identification of additional disease markers, although it has to be recognized that changes are difficult to implement in the health service and require extensive testing. Nevertheless, both these impacts have the potential to translate to societal value in the long term (10-15 years), and thus represent benefits to the public both in the UK and abroad. Additionally, through outreach events, the project aims to engage the public in aspects of the research such as "sticky lipids in health and disease", which we hope will increase awareness of underlying causes of obesity, diabetes and cardiovascular diseases, but also of the beneficial roles of lipids.