An ion-mobility mass spectrometry platform for single-cell proteomics and sensitive discrimination of isomeric biomolecules

质谱分析（MS）是一种快速发展的基础技术，用于生物研究的所有领域。它可以通过测量被称为离子的带电粒子的质荷比（或m/z）来精确地确定分子的质量。这种测量非常重要，因为它们使我们能够对蛋白质和碳水化合物进行定量和定量，并确定它们在不同条件下如何变化（成分和/或量）。通过购买最先进的仪器，该提案旨在扩大蛋白质分析能力，以申请人的专业知识和经验为基础，重点关注两个关键领域：i）量化单个哺乳动物细胞中不同蛋白质的水平。群体中的单个细胞通常表现出微妙的不同反应，例如在器官的不同部位，或感染后。然而，由于可用于分析的材料量少，在单个细胞水平上对蛋白质变化进行无偏调查是极其困难的。最近的方法学和仪器的发展意味着现在可以开始了解器官（例如肝脏或子宫内膜）内异源细胞群中单个细胞或病毒感染后细胞中单个细胞的独特反应。ii）表征蛋白质“修饰”（共价修饰）的确切模式和位点。蛋白质修饰释放了不同蛋白质的调节潜力，就像密码控制银行账户一样。因此，定义存在什么类型的修饰，以及修饰位点在生物分子上的确切位置，对于理解它们的生理相关性至关重要。然而，当我们考虑在蛋白质上靠近在一起的多个修饰时，或者当存在（很大程度上）无法通过质量区分的修饰时，这变得特别成问题，例如磷酸盐和硫酸盐。基于构象的修饰肽的灵敏分离，如可以用这种新仪器实现的，可以在某种程度上解决这些问题，定义这些共价修饰的精确类型和位置。我们计划将这种新型仪器带到利物浦大学的蛋白质组研究中心（CPR），该仪器将极其灵敏的定量与基于构象的肽分离相结合。使用完善的成本回收模型，以确保长期维护（由UoL的技术理事会（TD）的支持），我们将使它访问跨BBSRC战略优先领域的研究科学家工作，无论是在利物浦，并在英国各地，重点最初是研究密集型的“北方发电站”。TD为利物浦内外的最大数量的用户提供最好的研究设施，提供财政支持，以维护和发展“开放”和“透明”的设施，如我们。它还授予“访问补助金”，允许学者，特别是ECR，使用我们的设施，以支持新的研究思路之前赢得更多的实质性资金。使用这些研究设施也加强了与工业界的合作，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。这个平台将提供给当地和全国的科学家，他们目前在英国很难使用这种高端仪器。该系统将广泛适用于许多BBSRC资助的研究领域;在这里，我们特别关注影响感染生物学，基本细胞信号传导和合成生物学领域的研究，这些研究要么作为提案中的示例项目，要么包含在申请的支持信中。在类似领域工作的许多其他方面也表示了兴趣，但由于篇幅限制，无法将信件包括在内

Mass spectrometry (MS) is a rapidly advancing fundamental technology used in all areas of biological 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 very important, since they allow us to characterise and quantify proteins and carbohydrates, and determine how they change (in composition and/or amount) under different conditions. Through the acquisition of a state-of-the-art instrumentation, this proposal seeks to expand capability for protein analysis, building on the expertise and experience of the applicants, to focus on two key areas: i) Quantify the levels of different proteins in individual mammalian cells. Individual cells in a population often exhibit subtly different responses to e.g. in different parts of an organ, or following infection. However, unbiased investigation of protein changes at the level of an individual cell is extremely difficult due to the low amounts of material available for analysis. Recent methodological and instrumental developments mean that it is now feasible to start to understand the unique responses of individual cells in a heterogenous population of cells within an organ e.g. the liver or endometrium, or in cells following viral infection. ii) Characterise the exact patterns and sites of protein 'decoration' (covalent modification). Protein modification unlocks the regulatory potential of different proteins, rather like a PIN number controls access to a bank account. Consequently, defining what type of modification is present, and exactly where modification sites are positioned on a biomolecule, is essential to understanding their physiological relevance. However, this becomes particularly problematic as we consider multiple modifications that are close together on a protein, or where there are modifications that are (largely) indistinguishable by mass e.g. phosphate and sulfate. The sensitive separation of modified peptides based on conformation, as can be achieved with this new instrumentation, can go some way to addressing these issues in defining the precise type and location of these covalent modifications. We plan to bring this novel instrumentation, which combines extremely sensitive quantification with conformation-based separation of peptides, to the Centre for Proteome Research (CPR) at the University of Liverpool. Using a well-established cost recovery model to ensure long-term maintenance (supported by UoL's Technology Directorate (TD)), we will make it accessible to research scientists working across BBSRC strategic priority areas, both within Liverpool, and across the UK, with a focus initially on the research intensive 'Northern Powerhouse'. The TD provides access to the very best research facilities for the maximal number of users, both inside and outside Liverpool, providing financial support to maintain and develop 'open' and 'transparent' facilities such as ours. It also awards 'access grants', permitting academics, in particular ECRs, to use our 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.This platform will be available to scientists both locally and nationally, who currently struggle to access such high-end instrumentation in the UK. The system will have broad applicability to numerous BBSRC-funded research areas; here we specifically focus on research impacting the areas of infection biology, fundamental cell signalling, and synthetic biology, which are either presented as example projects in the proposal or covered in the letters of support included with the application. Numerous other parties working in similar areas have expressed interest, although letters could not be included due to space cons