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Quantitative Systems Biology by Mass Spectrometry

质谱定量系统生物学

基本信息

批准号：
BB/F011067/1
负责人：
Kathryn Lilley
金额：
$ 13.85万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FF011067%2F1
关键词：
Quantitative Systems Biology Mass Spectrometry

项目摘要

This application addresses the need to extend the capabilities of the proteomics (CCP and PNAC) and metabolomics facilities (Griffin lab.) in the Dept. of Biochemistry to enable both targeted protein and metabolite quantification using multiple reaction monitoring (MRM) analyses, non-targetted metabolomics and the analyses of intact protein. Targeted proteomics using MRM analysis is becoming increasingly popular as a method for validating global quantitation studies, determining the stoichiometry of components of protein complexes and the phosphorylation status of known phosphorylation sites. Currently CCP has access to a combination of mass spectrometers with high sensitivity and mass accuracy, but none of the existing portfolio of CCP instruments are ideal for MRM analysis. The UPLC-MS Quattro-Premier XE system is an attractive package to allow highly multiplexed, sensitive MRM analysis. Its high scanning speed coupled with excellent selectivity allows robust quantification of specific proteins/peptides. This system represents good value for money with lower running costs in terms of service and maintenance compared with alternative mass spectrometric systems. To date PNAC has provided intact protein analyses for the Dept. of Biochemistry, particularly to support the large structural biology community. Currently PNAC is utilizing older mass spectrometers incapable of delivering useful data on proteins above 25 kDa. The QTof Ultima is an instrument ideally suited to fill this gap, providing both the resolution and sensitivity needed to analyse large molecules; access to this technology would greatly enhance the services offered by PNAC Facility. Interest has steadily increased in the area of metabolomics, both within the department and in the wider Cambridge area. This workload is set to increase further with the arrival of Prof. Steve Oliver as Professor of Systems Biology. This application addresses the increased demand for metabolomics in Cambridge as well as addressing two key technical challenges that hinder LC-MS based metabolomics. The upgrade of our QTof Micro to a QTof Ultima system will increase our coverage of the metabolome by providing a 4-fold increase in sensitivity and a >2-fold increase in resolution. In addition there is a growing realisation that data must be made quantitative, especially for many systems biology based approaches where data from different omic technologies are cross-correlated. The UPLC-MS Quattro-Premier XE system will address this issue, allowing the quantitative analysis of known metabolites by MRM based analyses. The principle applicants, Kathryn Lilley, Julian Griffin, Len Packman and Stephen Oliver and have an established track record with respect to collaboration in systems biology projects [Karp, 2005#2, Dunkley, 2004, 2006, Sadowski, 2007, Castrillo, 2007] and also with many of the co-applicants [numerous joint publications]. Moreover, the Griffin and Lilley groups successfully share mass spectrometric equipment already (LTQ) and work closely together with respect to methods of data analysis and integration of proteomics and metabolomics datasets. The proposal describes on-going projects and many new projects within the Dept. of Biochemistry that will be facilitated by the funding of the proposed equipment. These projects range from the absolute measurement of abundance of targeted proteins within complexes and pathways to metabolites within networks, flux through metabolic pathways and metabolites which are secreted in response to drug efflux in bacteria.

该应用程序满足了扩展蛋白质组学(CCP和PNAC)和代谢组学设施(Griffin实验室)的能力的需要。在系里。生物化学部使用多反应监测(MRM)分析、非靶向代谢组学和完整蛋白质分析来实现靶向蛋白质和代谢物的量化。使用MRM分析的靶向蛋白质组学作为一种验证全球定量研究、确定蛋白质复合体各组分的化学计量比和已知磷酸化位点的磷酸化状态的方法正变得越来越流行。目前CCP拥有高灵敏度和高质量精度的质谱计组合，但现有的CCP仪器组合中没有一种是用于MRM分析的理想仪器。UPLC-MS Quattro-Premier XE系统是一个极具吸引力的组件，可用于高度多路复用、高灵敏度的MRM分析。它的高扫描速度与出色的选择性相结合，使特定蛋白质/多肽的定量变得可靠。与其他质谱学系统相比，该系统在服务和维护方面具有更低的运行成本，具有很好的性价比。到目前为止，PNAC已经为国防部提供了完整的蛋白质分析。生物化学，特别是支持大型结构生物界。目前，PNAC使用的是较老的质谱仪，无法提供有关25 kDa以上蛋白质的有用数据。QTof Ultima是一种非常适合填补这一空白的仪器，它提供了分析大分子所需的分辨率和灵敏度；获得这项技术将极大地增强PNAC设施提供的服务。对代谢组学领域的兴趣稳步增加，无论是在系内还是在更广泛的剑桥地区都是如此。随着史蒂夫·奥利弗教授作为系统生物学教授的到来，这一工作量将进一步增加。这项应用解决了剑桥对代谢组学日益增长的需求，以及解决了阻碍基于LC-MS的代谢组学的两个关键技术挑战。通过将我们的QTof Micro升级到QTof Ultima系统，我们将提供4倍的灵敏度和2倍的分辨率，从而扩大我们对代谢组的覆盖范围。此外，人们越来越认识到，数据必须量化，特别是对于许多基于系统生物学的方法来说，其中来自不同基因组技术的数据是相互关联的。UPLC-MS Quattro-Premier XE系统将解决这一问题，允许通过基于MRM的分析对已知代谢物进行定量分析。主要申请人凯瑟琳·利利、朱利安·格里芬、伦·帕克曼和斯蒂芬·奥利弗，在系统生物学项目的合作方面有既定的记录[Karp，2005#2，Dunkley，2004,2006，Sadowski，2007，Castrillo，2007]，也与许多共同申请者[许多联合出版物]。此外，Griffin和Lilley集团成功地共享了质谱学设备(LTQ)，并在数据分析方法以及蛋白质组学和代谢组学数据集的整合方面进行了密切合作。该建议书描述了部门内正在进行的项目和许多新项目。通过为拟议的设备提供资金，将促进生物化学的发展。这些项目的范围从绝对测量复合体和途径中目标蛋白质的丰度，到网络中的代谢物、代谢途径中的流量和细菌对药物外流的反应而分泌的代谢物。