Understanding complexity of post-translation modifications by enhancing UK capability for top-down proteomics

质谱（MS）是一项基础和快速发展的技术，广泛应用于生物研究的各个领域。它可以通过测量带电粒子（称为离子）的质荷比（m/z）来精确地确定分子（如蛋白质）的质量。这些测量很重要，因为它们使我们能够表征生物分子，并确定它们在不同条件下的组成和/或数量是如何变化的，这是理解生命的关键。该提案的重点是用质谱分析完整的蛋白质，为新的仪器寻求资金，这将使我们能够更准确地测量和表征蛋白质。特别是，通过化学基团（如磷酸盐）的附着而发生的蛋白质的生物调节将被测量。许多蛋白质的生物学功能是通过可逆添加特定的化学基团来调节的。使用我们的新工具，我们将能够定义这些修改发生的时间和地点，以及以什么顺序和组合。有了这些信息，科学家可以更好地了解这些修饰如何调节生物系统中的蛋白质功能。此外，许多蛋白质的功能和/或催化（酶）活性可以通过药物和与蛋白质伴侣的相互作用而改变，至关重要的是，这些因素的结合通常是特定于不同的蛋白质形式的，而不是特定于特定个人账户的特定银行卡的PIN号码。我们的最终目标是了解特定修饰蛋白（“蛋白质形态”）的生物多样性，以及它们如何与药物和/或蛋白质结合。质谱仪器的重大发展意味着现在可以从相对少量的材料中深入表征蛋白质及其修饰，这在以前由于技术限制是不可能的。Thermo Scientific Orbitrap Lumos质谱仪的灵敏度、质量精度和分辨率的提高，将允许以更有针对性的方式测量和操纵特定的蛋白质离子，从而使“大海捞针”的蛋白质发现成为现实。我们建议在利物浦大学蛋白质组研究中心建立这个先进的质谱平台。这些设备将提供给当地和英国各地的科学家，他们目前正努力获得具有这种能力的高端仪器。该系统将广泛适用于大量bbsrc资助的研究领域；我们特别关注影响感染、免疫、基本细胞生长机制、缺氧反应和生物治疗领域的研究，这些研究要么在提案中作为示例项目提出，要么在申请中包含的支持信中涵盖。据我们所知，BBSRC的研究人员还没有可比较的系统。我们与ThermoScientific以及购买该系统的英国单一学术团体（Lilley, Cambridge， Wellcome Trust资助，尽管不是TDP）的良好工作联系将使我们能够在利物浦迅速建立一个工作平台，在那里它将被整合到UoL蛋白质组学共享研究设施中，并得到UoL技术理事会（TD）的支持。TD通过提供财政支持来维持和发展我们的“开放”和“透明”设施，为英国各地的最大数量的用户提供最好的研究设施。它还授予“获取资助”，允许学者，特别是ecr，在获得实质性资助之前使用这些设施来支持新的研究。这些研究设施通常是在其他地方临时提供的，由于公司能够使用专业管理的最先进技术和专业知识将分析外包，因此也加强了与行业的合作。

Mass spectrometry (MS) is a fundamental and rapidly advancing technology, widely used in all areas of biological research. It can accurately determine the mass of molecules, such as proteins, by measuring the mass-to-charge ratio (m/z) of charged particles, called ions. Such measurements are important because they allow us to characterise biological molecules and determine how they change in composition and/or amount under different conditions that are key to understanding life. This proposal focusses on the analysis of intact proteins by MS, seeking funds for new instrumentation that will permit us to measure and characterise proteins more accurately. In particular, the biological regulation of proteins that occurs through the attachment of chemical groups, such as phosphate, will be measured. The biological function of many proteins is regulated by the reversible addition of specific chemical groups. With our new instrumentation, we will be able to define when and where these modifications occur, and in what order and combination. With this information, scientists can better understand how these modification regulate protein function in biological systems.Moreover, the function and/or catalytic (enzyme) activity of many proteins can be altered using drugs and through interactions with protein partners, and crucially, the binding of these factors is often specific to distinct protein forms, rather like a PIN number is specific for a particular bank card from the account of a given individual. Our ultimate goal is to understand the biological diversity of specific modified proteins ('proteoforms') and how they bind differentially to drugs and/or proteins. Significant developments in MS instrumentation mean that it is now feasible to characterise proteins and their modifications in intricate depth from relatively small amounts of material, which has previously been impossible due to technological limitations. The improved sensitivity, mass accuracy and resolution of the Thermo Scientific Orbitrap Lumos mass spectrometer will permit specific protein ions to be measured and manipulated in a more targeted manner, so that discovery of a protein 'needle in a haystack' can become a reality. We propose to establish this advanced MS platform in the Centre for Proteome Research at the University of Liverpool (UoL). The equipment will be available to scientists locally and across the UK, who currently struggle to access high-end instrumentation with this capability. The system will have broad applicability to a large number of BBSRC-funded research areas; we have specifically focused on research impacting the areas of infection, immunity, fundamental cell growth mechanisms, the response to hypoxia and biotherapeutics, which are either presented as example projects in the proposal or covered in the letters of support included with the application.To our knowledge, there is no comparable system available to BBSRC researchers. Our excellent working links with ThermoScientific and the single UK-based academic group in the UK that has purchased this system (Lilley, Cambridge, Wellcome Trust funded, although not for TDP) will allow us to rapidly establish a working platform in Liverpool, where it will be integrated into the UoL Proteomics Shared Research Facility and supported by UoL's Technology Directorate (TD). The TD provides access to the very best research facilities for the maximum number of users, across the UK, by providing financial support to maintain and develop our 'open' and 'transparent' facility. It also awards 'access grants', permitting academics, in particular ECRs, to use these facilities to support new research in advance of winning substantive funding. Access to such research facilities, which is typically available on an ad hoc basis elsewhere, also enhances collaboration with industry, since companies are able to outsource analysis using professionally-managed state-of-the-art technology and expertise.