Within the Institute for Global Food Security (IGFS) at Queen's University Belfast, (bio)analytical chemistry utilising mass spectrometry (MS), housed within the ASSET Technology Centre, is a fundamental enabling technology underpinning our food, environmental, and health research. Our capabilities extend across screening (spectroscopic platforms and ambient ionisation MS), targeted (liquid and gas chromatography single and triple quadrupole (TQ)-MS), and profiling methodologies from small molecules to proteins (quadrupole time-of-flight MS). One of the mainstay platforms used across our portfolio of work is TQ-MS coupled to liquid chromatography. This is used in, for example, targeted analysis for food contaminants, detection of antibiotics and their residues in food and environmental samples, and quantification of metabolites linked to health status. Much of this work is reliant on the sensitivity of instrumentation to detect and accurately quantify low abundant but biologically important metabolites, and to meet ever-increasingly demanding limits of detection and quantification for food and environmental regulations. Furthermore, the Xevo TQ Absolute shows excellent performance capabilities in direct infusion analysis which offers opportunities to remove the need for chromatographic separation in particular applications and therefore, decrease the environmental impact of each sample analysis. Furthermore, the use of direct infusion reduces the sample extraction and processing requirements, and the associated single-use plastic consumables and their environmental impact. The concept of 'green' (bio)analytical chemistry is becoming central to the sustainability of analytical methods. This involves both a movement away from fossil-fuel based consumables and solvents and increasing the analytical throughput of workflows to reduce the environmental impact of each sample analysis. As such, we propose to pair the Waters Xevo TQ Absolute instrument with the UPC2 supercritical fluid chromatography (SFC) and Acquity Premier system with a capability for two-dimensional liquid chromatography (2D-LC) from Waters Corporation. This will uniquely equip the ASSET Technology Centre at QUB with cutting-edge instrumentation to position itself at the forefront of the essential transition of (bio)analytical chemistry using mass spectrometry to improve sustainability. This dedicated combination of analytical equipment would be the first in the UK and internationally, and we believe would act as a focal point for collaborative research across public and private research organisations. Reflecting the breadth and strength of QUB research, this proposal has assembled an Investigatory team that will exploit the increased analytical capabilities enabled by this proposal in research that has already received substantial BBSRC funding (Cameron, Huws, and Elliott) and which fits into BBSRC Strategic Priorities including Animal Health (Huws, Hyland, and Elliott), Combatting AMR (Huws, Hyland, McGrath, and Gilpin), Data Driven Biology (Cameron, Huws, Hyland, Elliott, Connelly, McGrath, Gilpin, and Green), Food, Nutrition, and Health (Cameron, Elliott, Connelly, and Green), Healthy Ageing (Green), Integrative Microbiome Research (Cameron, Huws, Hyland, McGrath, Gilpin), Reducing Food Chain Waste (Huws, Hyland, and Elliott), Research to Inform Public Policy (Cameron, Huws, Hyland, Elliott, Connelly, McGrath, and Gilpin), Sustainably Enhancing Agriculture (Huws, Hyland, and Elliott), Systems Approaches (Cameron, Connelly, McGrath, Gilpin, and Green), and Welfare of Managed Animals (Huws, Hyland, and Elliott).

We propose to take a proteomics approach to the study of protein secretion and turnover in two yeasts: the microbial cell factory, Pichia pastoris, and the model eukaryote, Saccharomyces cerevisiae. We shall use a proteomics based approach to look, in both species, at the dynamics of transport of a heterologous protein from the site of its synthesis to the cell exterior. In both hosts we shall use recombinant human lysozyme (HuLy) as the test object. Previous work has demonstrated, using a series of lysozyme mutants, that the degree of unfolding of HuLY is a major factor in determining its secreted yield (1). Highly unfolded variants show poor secretion yield and trigger the unfolded protein response (UPR). We shall use a global proteomics approach to determine where in the secretion pathway from the ER to the cell exterior lysozyme and its variants accumulate. Highly unfolded proteins are known to induce both ER stress and the unfolded protein response (UPR). Our rationale for taking a global approach to dissect the secretion pathways of heterologous proteins in yeasts, is based on the need to determine not only the subcellular locations at which such proteins accumulate, but also their binding partners within each specific location. Standard methodologies to capture protein complexes using immunopreciptation of a bait do not yield information about compartmentalisation of intermediates and the dynamic nature of binding partners within compartments. We will thus make use of state-of-the-art technologies developed in the Lilley laboratory which allow accurate assignment of proteins to subcellular locations using distribution patterns of subcellular compartments on density gradients as determined by quantitation proteomics methods coupled with sophisticated statistical tools (2). In this project we will work with Jim Langridge who is Director of Proteomics at Waters to further develop this method employing up-to-date label-free proteomics methodologies to determine the distribution of thousands of proteins simultaneously. This label-free approach has been pioneered by Waters and has many advantages over the methods that the Lilley lab. has used to date, namely isobaric stable isotope in vitro labels. Recent work by the Lilley lab. has shown that these labels, such as iTRAQ, have significant problems regarding both their precision and accuracy (3). Robust label-free approaches have been shown not to suffer from the same shortcomings as the iTRAQ tags (4,5) and their use in determining the distribution patterns of organelle proteins within density gradients are more likely to lead to accurate measurement of such patterns and thus better resolution of the patterns associated with different sub cellular structures. Moreover, the label-free method to be employed, MSE, also estimates the absolute amount of proteins within different fractions, enabling measurement of stoichiometeries of proteins in complexes, as absolute distributions of protein species in terms of molecules of protein per compartment. Having further developed label free quantitative proteomics approaches to determine methods accurate subcellular locations of proteins and their binding partners, we will focus on examining the compartmentalisation of the recombinant protein. We will carry out global analysis of its association with other proteins including the unfolded-protein chaperone, Kar2p (a BiP ortholog) and the proteasome. We shall be particularly interested in the amyloidogenic version of HuLy (I156T) and will validate our results using this variant by expressing the Alzheimer's protein Abeta, both in its native form and as Abeta42 -GFP fusions. 1. Kumita, JR et al (2006) FEBS J273(4):711-20 2. Dunkley, T et al, (2006)Proc. Natl. Acad. Sci 103(17):6518-23 3. Karp, NA et al (2010) Mol. Cell Prot. in press 4. Silva, JC et al (2005), Anal Chem. 1;77(7):2187-200 5. Stapel, M et al (2010) Sci Signal.2;3(111)

Enzyme catalysis is being industrialised at a phenomenal rate, offering routes to chemical transformations that avoid expensive heavy metal catalysts, high temperatures and pressures, and providing impressive enantio-, regio- and chemo-selectivities. In short, biocatalysts are a cornerstone of the bioeconomy: they are required individually, or as cascades, in live cells or cell-free preparations to manufacture every day chemicals, materials, healthcare products, fuels and pharmaceuticals; and they are integral to many diagnostic and industrial sensing applications. They are central components of technologies underpinning the circular economy and offer engineering biology routes to realising global challenges, including net zero, clean growth and the bioeconomy. An ability to exploit and tailor biocatalyst activities both rapidly and predictably is essential to realising the contemporary global challenges and the UK Government's Innovation Strategy. Despite their central importance, the vast majority of natural and engineered enzymes are thermally-activated. This dependence on thermally-activated catalysis: i) limits biocatalysis to those reaction types found naturally in biology; ii) places a high dependence on expensive and unstable cofactors / coenzymes; and iii) places a sizeable demand on the provision of energy source (biochemical / artificial reductants), 'bioreactor' designs (e.g. within cell-free formats, nanoscale devices or microbial cell factories); and iv) restricts approaches to regulating biocatalyst / bioprocess activity. The use of light to drive enzyme catalysis would bypass many of these hurdles. However, with only three known exceptions, nature does not make use of enzymatic photocatalysis. Therefore, biology cannot access a broad range of 'difficult-to-achieve' reactions that would be transformational in catalysis science, and applications of these reactions in the modern world. Light is freely available and non-invasive, yet the photochemical versatility of natural cofactors such as flavin is seldom used by enzymes. Therefore, securing generalised routes to predictive photobiocatalysis design is a fundamental biological challenge. If successful, identifying generalised routes to the engineering and design of photobiocatalysts would be transformative for catalysis science in the emerging bioeconomy. This project will address this urgent need by using the natural photochemistry of flavin to make possible photocatalysis by any flavin-containing protein. This programme (termed GENPENZ) is positioned at the frontier of biological photocatalysis and enzyme design and engineering. It will generalise the concept of photo-biocatalyst design and engineering using existing (top down) and man-made (bottom up) protein scaffolds to biologically encode new photo-biocatalysts with wide reaction scope, or to assemble de novo protein frameworks from synthetic peptides. It will unite time-resolved 1D / 2D spectroscopy in the visible / infra-red spectral regions, across 12 decades of time (fs - s), with emerging capabilities in photo mass spectrometry (ion mobility; hydrogen-deuterium exchange), EPR spectroscopy, and photo-biocatalyst design engineering. High-level computational chemistry will underpin all protein-design/engineering work, spectroscopy, and structure elucidation. GENPENZ is based on breakthroughs in discovery science relating to mechanisms of enzyme photocatalysis. Realisation of a generalised platform for photo-biocatalyst design will open up new high-energy reaction pathways, enrich catalysis outcomes, and sidestep many of the scientific / economic constraints of working with thermally-activated biocatalysis in the emerging bioeconomy.

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.

Good farm animal health is paramount for food safety and security. It also reduces the burden on the environment and public health by a reduction in disease treatments using antimicrobials or other drugs. Greater knowledge and earlier detection of agriculturally important diseases will result in better farm disease management and welfare of farm animals as well as improved food produce yield and safety. In addition, recent food adulteration scandals have highlighted the need for faster and more detailed knowledge of our food and its production line to determine and guarantee food authenticity. Information gathering for all of the above is still limited by the analytical methods available. Some of these are not sufficiently cost-effective and/or too cumbersome and slow for rapid action. Others can be fast but are often very targeted, allowing the detection of only one condition, and limited in their practical use, particularly in combination with other tests. Fortunately, advances in modern mass spectrometry now allow highly sensitive recordings of the molecular profiles of biological samples such as milk. In combination with advanced bioinformatics these recordings can be used to teach computer algorithms for future classification and thus detection of agriculturally important conditions such as mastitis. As modern mass spectrometry is one of the most sensitive molecular detection and characterisation techniques, allowing the analysis of many molecular biomarkers simultaneously, there is now a great opportunity to make a step change in the early detection of such markers for many diseases in a cost-effective and fast way. However, cost-effectiveness and speed of analysis can still be limited due to the sample preparation necessary for analysis by mass spectrometry. Our lab has recently demonstrated that biological samples such as milk can be effectively analysed by mass spectrometry with no or minimal sample preparation by using liquid MALDI mass spectrometry. In collaboration with the University of Reading's Department of Agriculture and Centre of Dairy Research (CEDAR) with its large research herd of around 600 cows, we now aim to explore and fully develop a rapid analysis workflow for biological fluids from farm animals based on modern liquid MALDI mass spectrometry. This workflow should allow for early and specific detection of agriculturally important diseases and conditions, exploiting to the fullest mass spectrometry's great potential in highly sensitive, fast and inexpensive characterisation of the health of our farm animals and their produce.