Advanced MS instrumentation for enhanced proteomics capabilities

质谱仪(MS)是一种应用于许多研究领域的快速发展的基础技术；它可以通过测量带电粒子(即离子)的质量/电荷比(或m/z)来精确确定分子的质量。这种测量非常重要，因为它们使我们能够从结构上表征化学实体，并确定这些分子在不同条件下如何变化(在组成和/或数量上)。这一建议侧重于对蛋白质的分析，以期确定它们是如何变化的，无论是通过与磷酸盐等化学基团的结合，还是通过它们与其他调节分子的结合，所有这些都会导致可以测量的质量移动。蛋白质的生物功能可以通过特定化学实体的可逆添加来调节。因此，通过确定它们发生的时间和地点，科学家可以首先了解它们是如何调节蛋白质的，并最终了解它们的生理相关性。此外，蛋白质的功能和/或酶活性可以使用小分子抑制剂和蛋白质结合伙伴来操纵。值得注意的是，这些因素的结合通常是特定于不同蛋白质形式的，就像PIN号码是特定于特定银行卡的一样。MS仪器的重大发展意味着现在有可能深入表征蛋白质及其修饰，这一壮举以前曾因无法区分具有非常相似m/z值的离子而受阻。改进了在仪器内操作离子的能力，允许相关离子积累，并显著提高了灵敏度；检测蛋白质的大海捞针可以成为现实。在分析低丰度的生物样品时，这种提高的灵敏度尤其重要，因为在观察细胞中发现的不同修饰蛋白质形式时，这是典型的。此外，这种仪器还可以用来以新的方式控制离子碎裂，这意味着可以阐明更多的结构信息。因此，我们能够开始表征并了解与蛋白质不同位点相互作用的化合物的作用机制，以及修饰如何影响复杂生物样本中的蛋白质功能、稳定性和亚细胞定位。我们建议在利物浦大学(UOL)建立这样一个先进的MS平台。该平台将向当地和来自英国各地的科学家开放，他们目前正努力获得这样的高端仪器。因此，该系统将广泛适用于BBSRC资助的大量研究领域，包括影响老龄化、工业生物技术、动物健康、食品安全和过敏，其中许多领域要么在提案中作为范例项目提出，要么在申请中包含在支持信中。据我们所知，英国研究人员还没有公开可用的类似系统。我们与英国的两个学术团体(Lamond，Dundee&Lehner，Cambridge)建立了良好的工作关系，他们安装了这样的系统(惠康信托基金资助)，这将帮助我们建立我们的平台，该平台将由UOL的技术局(TD)管理，并整合到蛋白质组学共享研究设施中。运输署的使命是为大学内外最多的使用者提供最好的研究设施，并为我们这类“开放”和“透明”的设施提供财政支援。它还授予特定的“准入补助金”，允许学者，特别是早期职业研究人员，在赢得更多实质性资金之前，利用这些设施支持新的研究想法。使用此类研究设施还可以加强与行业的合作，因为公司能够使用专业管理的技术外包一些分析，从而帮助推动成功。

Mass spectrometry (MS) is a rapidly advancing fundamental technology employed in many areas of research; it can accurately determine the mass of molecules by measuring the mass-to-charge ratio (or m/z) of charged particles, called ions. Such measurements are extremely important since they allow us to structurally characterise chemical entities and determine how these molecules change (in composition and/or amount) under different conditions. This proposal focusses on the analysis of proteins, with a view to determining how they change, either by the attachment of chemical groups such as phosphate, or by their binding to other regulatory molecules, all of which result in a mass shift that can be measured.The biological function of proteins can be regulated by the reversible addition of specific chemical entities. Hence, by defining when and where these occur, scientists can firstly understand how they regulate proteins and ultimately their physiological relevance. Moreover, the function and/or enzyme activity of proteins can be manipulated using small molecule inhibitors and protein binding partners. Remarkably, the binding of these factors is often specific to distinct protein forms, rather like a PIN number is specific for a particular bank card. Significant developments in MS instrumentation means that it is now possible to characterise proteins and their modifications in depth, a feat which has previously been hampered by an inability to distinguish ions with very similar m/z values. The improved ability to manipulate ions within the instrument, permits related ions to be accumulated, and significantly increases the sensitivity; detection of a protein 'needle in a haystack' can become a reality. This increased sensitivity is particularly important when analysing biological samples that are of low abundance, as is typical when looking at the different modified protein forms found in cells. Moreover, this instrument can also be used to control ion fragmentation in new ways, meaning that significantly more structural information can be elucidated. We are therefore in a position to start characterising, and thus understanding, the mechanisms of action of compounds that interact with different sites on a protein and how modification affects protein function, stability and subcellular localisation in complex biological samples.We propose to establish such an advanced MS platform at the University of Liverpool (UoL). The platform will be available to scientists both locally and from across the UK, who currently struggle to access such high-end instrumentation. The system will thus have broad applicability to a large number of BBSRC-funded research areas, impacting ageing, industrial biotechnology, animal health, food security & allergy, many of 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 that is openly available to UK researchers. Our excellent working links with the two academic groups in the UK (Lamond, Dundee & Lehner, Cambridge) who have such systems installed (Wellcome Trust funded) will help us establish our platform, which will be managed by UoL's Technology Directorate (TD) and be integrated into the Proteomics Shared Research Facility. The TD has the mission of providing access to the very best research facilities for the maximal number of users, both inside and outside the University, and provides financial support for 'open' and 'transparent' facilities such as ours. It also awards specific 'access grants', which permit academics, in particular early career research staff, to use these facilities to support new research ideas in advance of winning more substantive funding. Access to such research facilities also enhances collaboration with Industry, since companies are able to outsource some of their analysis using professionally-managed technology thus helping drive success.