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

通过增强英国自上而下蛋白质组学的能力来了解翻译后修饰的复杂性

• 批准号: BB/R000182/1
• 负责人: Claire Eyers
• 金额: $ 77.82万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR000182%2F1
• 关键词: Understanding; complexity; post; translation; modifications

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.

质谱（MS）是一种基础和快速发展的技术，广泛应用于生物研究的所有领域。它可以通过测量带电粒子（称为离子）的质荷比（m/z）来准确确定分子（如蛋白质）的质量。这种测量很重要，因为它们使我们能够识别生物分子，并确定它们在不同条件下的组成和/或数量如何变化，这是理解生命的关键。这项提案的重点是通过MS分析完整的蛋白质，为新的仪器寻求资金，使我们能够更准确地测量和分析蛋白质。特别是，将测量通过连接化学基团（如磷酸盐）而发生的蛋白质的生物调节。许多蛋白质的生物学功能是由特定化学基团的可逆添加来调节的。有了我们的新工具，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，科学家们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）活性可以使用药物或通过与蛋白质伴侣的相互作用来改变，而且至关重要的是，这些因子的结合通常是特定于不同蛋白质形式的，就像PIN号码是特定个人帐户中特定银行卡的特定号码一样。我们的最终目标是了解特定修饰蛋白（“蛋白质”）的生物多样性，以及它们如何与药物和/或蛋白质结合。MS仪器的重大发展意味着现在可以从相对少量的材料中以复杂的深度来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、质量准确度和分辨率的提高将允许以更有针对性的方式测量和操作特定的蛋白质离子，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（UoL）的蛋白质组研究中心建立这个先进的MS平台。这些设备将提供给当地和英国各地的科学家，他们目前正在努力获得具有这种能力的高端仪器。该系统将广泛适用于英国广播公司研究中心资助的许多研究领域;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等领域的研究，这些研究要么在提案中作为示例项目提出，要么在申请所附的支持信中涵盖。据我们所知，BBSRC的研究人员没有可比较的系统。我们与ThermoScientific和英国唯一一个购买了该系统的英国学术团体（Lilley，剑桥，惠康信托基金资助，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，在那里它将被集成到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护和发展我们的“开放”和“透明”设施，为英国各地最大数量的用户提供最好的研究设施。它还授予“访问补助金”，允许学者，特别是ECR，在赢得实质性资金之前使用这些设施来支持新的研究。利用这种研究设施（在其他地方通常是临时性的）也加强了与工业界的合作，因为公司能够利用专业管理的最新技术和专门知识外包分析工作。

项目成果

Biochemical Analysis of AKAP-Anchored PKA Signaling Complexes.

• DOI: 10.1007/978-1-0716-2245-2_19
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)

Temporal modulation of the NF-?B RelA network in response to different types of DNA damage

NF-κB RelA 网络响应不同类型 DNA 损伤的时间调节

• DOI: 10.1101/2020.08.11.246504
• 发表时间: 2020
• 作者: Campbell A

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics

使用 Polo 样激酶 4 (PLK4) 抑制剂 centrinone 通过基于 SILAC 的磷酸蛋白质组学研究细胞内信号网络

• DOI: 10.1101/2020.05.22.110767
• 发表时间: 2020
• 作者: Byrne D