My research focuses on studying a type of proteins called intrinsically disordered proteins to find new drug targets. Unlike most proteins that exist in a defined 3-dimensional structure, IDPs switch quickly between multiple shapes. This renders them notoriously difficult to study. IDPs are involved in diseases such as cancer and neurodegenerative diseases, but their dynamic behaviour makes them challenging to study using traditional methods. I use a technique called ion mobility-mass spectrometry (IMMS), which is effective for analysing IDPs. This method helps me understand the different shapes these proteins can take, even the temporary ones. It's also useful for figuring out how proteins interact with each other or with drugs. In the initial three years of my Fellowship, I demonstrated that IMMS is effective for characterising IDPs based on their range of shapes, and for understanding how they change when a drug is involved. I've also showed that IMMS can help us understand how IDPs change when they undergo a process called liquid-liquid phase separation (LLPS), which is important in both normal cell function and disease. For the next phase of the Fellowship, the main goals are as follows: 1. To develop faster IMMS methods to quickly screen large sets of molecules for potential new drugs that could target an IDP of interest 2. Use newly developed methods to investigate how the shapes of IDPs change during LLPS, especially focusing on a protein related to prostate cancer. To achieve the first goal, I will continue to collaborate with Waters, an IMMS vendor that is dedicated to advancing instrumentation. We'll use a novel device to quickly test a variety of compounds to see which ones interact with the IDP and change its shape. The specific protein we're looking at is important in advanced stages of prostate cancer. For the second goal, we aim to further understand how IDPs change shape during LLPS, particularly in the context of prostate cancer. We'll investigate how a molecule that is known to disrupt this process affect the shape distribution of the androgen receptor, which is the key protein involved in prostate cancer. The ultimate aim is to develop strategies to interfere with this process and potentially treat advanced prostate cancer. In summary, my research aims to develop novel techniques to study IDPs and find new ways to target them for medical purposes.

Ion mobility-high resolution mass spectrometry (IM-HRMS) is the next-generation analytical platform in research and industry. Unlocking its full potential across applications as varied as biotherapeutics, environment and food safety requires not only pushing back the frontiers of instrumentation, fundamental understanding and applications - but harmonisation is essential. To achieve this, current shortcomings in data collection, analysis and reporting across instrument types, laboratories and research areas need to be scrutinised and overcome. The MobiliTraIN Doctoral Network will form 10 Doctoral Candidates (DCs) who will bring a new fundamental understanding of IM- HRMS, provide reference materials and guidelines for standardisation, develop state-of-the-art methods for the application of IM- HRMS in biopharmaceutical development, biological 'omics studies, and nontarget screening of contaminants, and lay the foundations for IM-HRMS adoption in industry. Through an interdisciplinary research programme, including an open science approach and training in technical, business and transferable skills, the MobiliTraIN DCs will become leading experts in ion mobility with a unique skill set to successfully advance their careers while supporting Europe's innovation capacity. Building on existing collaborations and research excellence covering the entire innovation chain of IM-HRMS development and application, MobiliTraIN unites 8 academic institutions, 3 leading instrumentation companies, 1 regulatory agency, 1 pharma industry leader and 5 SMEs from 8 countries. With complementary expertise, know-how and mentoring experience, our consortium is ideally suited to unveil the potential of IM-HRMS as a key technology for safer therapeutics, better understanding of complex disease progression and improved monitoring of food, water and public health safety.

The project is aimed at the development of new tools for the identifications of various microorganisms including bacteria causing a wide range of diseases from common cold to bloodstream infections. Knowing exactly which type of bacterium is involved in a disease is very important, since the choice of appropriate medication largely depends on it. Likewise, in case of public health or food safety, the correct classification of bacterial contaminations helps with the identification of their source and elimination of the contamination. Currently, samples containing bacterial cells are collected and sent to laboratories. Microbiologists grow the bacteria in Petri-dishes containing special nutrients. Based on the types of nutrients the bacteria can use and the results of multiple chemical tests, the bacterium is tentatively identified. If proper classification is necessary, nucleic acids are extracted from the bacterial cells, and their base-pair sequence is determined, which helps in the unambiguous identification. All of these processes are time consuming, which considerably delays the efficient intervention both in case of infectious diseases and in case of a waterborne disease outbreak. The purpose of the proposed research is to develop an alternative, much faster technique for the identification of bacteria. Mass spectrometry is an analytical technique capable of the measurement of the weight of molecules, and also the selective detection of hundreds of different molecules at the same time. We plan to use this well-established technique for looking at some special building blocks of bacterial cells. One novel aspect of the research is that mass spectrometers are not used in the traditional way, including a lengthy preparation of bacterial cells prior to analysis, but the cells are simply heated up, and electrically charged molecules formed on the boiling of cells are analysed using mass spectrometry. The idea of rapid mass spectrometric analysis by using simply heat has already been applied in case of surgery, where cancer tissue is identified in a similar way. In course of the proposed project we plan the adopt this technology (Rapid Evaporative Ionization Mass spectrometry; REIMS) for the analysis of bacteria grown in the laboratory and also for the direct analysis of liquid samples (ranging from pond water to blood) containing bacterial agents. We plan to build a large library of the spectroscopic fingerprints of the bacteria, which will be used as a training set for computer based search algorithms. The method, the database and the algorithm together will enable the unambiguous identification of bacteria in considerably shorter timeframe than the current routine. Furthermore, the proposed research can potentially lead to an approach, where bacteria are directly identified in their natural environment (e.g. in urine for a urinary infection) without growing them in the laboratory for several hours or days.

This project addresses the application of high resolution and high sensitivity mass spectrometry to characterize the protein components in rodent scent marks. Recent research in rodent semiochemistry, in substantial part from the academic applicants laboratories, has revealed a depth and complexity to rodent chemical communication previously unanticipated. We are building a detailed picture of the receptor repertoire for these signals, and of the higher level processing that collates these signals into behavioural responses, but our understanding of the molecular composition of the scent marks is some way behind. Chemical communication is capable of conveying an incredibly subtle stream of information, especially between conspecifics. Scent marks, predominantly urinary, contain an astonishing array of information that transmits variable indicators of the state of the scent owner (e.g. health status, pregnancy, recent food ingested and time since deposition). This status information is primarily associated with proteins (lipocalins and ESPs) that provide information on genetically invariant parameters such as sex and individual identity. In this project, the student will bring to bear advanced mass spectrometric methodologies in the characterization of these proteins. The samples will be recovered from wild-caught rodents, and are sometimes vanishingly small (such as in tear secretions). The challenges in the study of these scent mark proteins are two fold. First, there is a pressing need for accurate quantification of the proteins in the scent mark. Second, it is clear that the highly polymorphic gene cluster that encodes these proteins is genetically unstable, leading to each wild animal being able to express a unique pattern of these proteins. Thus the challenges are both quantitative and qualitative. The student will address these challenges in collaboration with Waters, using a combination of intact mass profiling, making use of the high resolution QToF instruments and new algorithmic approaches to spectral deconvolution developed at Waters, label-free quantification using the Waters-developed MS^E analytical workflow, coupled with Hi3 peptide quantification, and through the use of selected reaction monitoring, using an artificial QconCAT concatenated standard peptide assembly (technology invented and patented by the academic partner). Finally, discovery of new polymorphic variants will be based on intact mass survey, followed by electron transfer dissociation (ETD, using a new front-end source designed at Waters but not yet widely available) to isolate and characterise the amino acid sequences of the variant proteins. The plan of the programme will follow the outline below, although we expect that from year 2 onwards, the student will take some responsibility for the direction of the research. Year 1: Induction, Design of QconCAT proteins, expression and validation, experience of wild rodent sample collection and diversity. Training in intact mass profiling, bioinformatics tools and peptide level label-fre and label-mediated quantification. Year 2: Development of ion mobility methodologies to improve resolution of complex mixtures of isoforms, and the use of gas phase cross-sectional area to assess the conformational stability and consequent degree of protonation on electrospray ionsation behaviour. Expression of recombinant lipocalins for model studies, appropriately engineered to alter electrostatic potential for charge state manipulation. Instruction on ETD fragmentation. Year 3: Application of ETD to discover amino acid sequence variation in new isoform variants, leading to quantification by surrogate peptides. Includes an exploration of the effect of sequence variation on ESI signal intensity and charge state for quantification. Year 4: Completion of thesis and papers, exploration of new areas of investigation.

Life sciences research is increasingly being performed using high-tech instrumentation, producing vast quantities of data. In turn, the science as a whole is being transformed from a knowledge-based discipline, into a Big Data discipline. Some of these techniques are called "omics" - from genomics (studying genes on a large scale), proteomics (proteins) and metabolomics (metabolites). Proteins are the functional molecules in cells, and by studying the levels at which different proteins are present in cells (for example comparing healthy versus diseased cells), we can understand how the system as a whole is behaving (or going wrong) and we can begin to understand the function played by the individual proteins. The pervasive technique used for proteomics is mass spectrometry (MS), which is capable of measuring many thousands of proteins from a single sample. One of the biggest challenges in omics research is the interplay between often complex and noisy data produced by the instrument and the requirement for bespoke software for data analysis. Both areas are under active development in academic research groups and by industrial organisations (commercial instrument and software manufacturers) - and it is indeed a major challenge ensuring that academic research and development has maximum impact on industry. In proteomics, there are various software packages, both commercial and free/open source, capable of analysing the raw data collected from MS to give a list of proteins identified, along with an abundance value in or between samples of interest. One popular package is called Progenesis QI - marketed internationally by instrument manufacturer Waters. Waters are a large global corporation, with headquarters near Manchester, UK. However, in proteomics, there is currently a shortage of good software for taking the quantified protein list, and performing downstream data analysis to arrive at a real understanding of the biological system, as required by scientists that make use of proteomics techniques in their research. These downstream analyses include visualising the large data set to check the data quality, and starting to understand which groups of proteins may be changing in the system of interest. It is also necessary to perform specialised statistical analyses to ensure that only significant results are taken for further study and published. In this project, an academic biologist and data analyst (Dr Dean Hammond) will take part in an industry interchange, to work directly with Waters, to develop new software for proteomics data analysis, developing a package called ProteoAnalytics. The package will be able to take input from Progenesis QI (and other suitable upstream packages), enabling biologists easy access to cutting-edge methods for data interpretation (such as mapping proteins to biological pathways), performing specialised statistical analyses, creating unique and powerful visualisations of large data sets, and assisting users to prepare high-quality figures and charts for scientific publications. The software will enable Waters to understand very rapidly the features that work for users, and will assist proteome scientists to perform large scale data analyses. The interchange will allow Dr Hammond to take his knowledge of academic proteomics data analysis to Waters, and gain experience of the industry perspective in software development. The interchange also enables the creation of a collaborative partnership between the principal investigator (Dr Andy Jones, who leads an academic software development group) with the commercial software development team at Waters.