Quantitative Systems Biology by Mass Spectrometry

该应用程序解决了扩展蛋白质组学（CCP和PNAC）和代谢组学设施（格里芬实验室）的能力的需求。在部门。利用多反应监测（MRM）分析、非靶向代谢组学和完整蛋白质分析，实现靶向蛋白质和代谢物的定量。使用MRM分析的靶向蛋白质组学作为验证全局定量研究、确定蛋白质复合物组分的化学计量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可以使用具有高灵敏度和质量准确度的质谱仪组合，但现有的CCP仪器组合中没有一种是MRM分析的理想选择。UPLC-MS Quattro-Premier XE系统是一个有吸引力的软件包，可进行高度多路复用、灵敏的MRM分析。它的高扫描速度加上出色的选择性，可以对特定蛋白质/肽进行稳健的定量。与其他质谱系统相比，该系统在服务和维护方面的运行成本较低，具有良好的性价比。到目前为止，PNAC为该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学社区。目前，PNAC使用的是无法提供25 kDa以上蛋白质有用数据的较旧质谱仪。QTof Ultima是一种非常适合填补这一空白的仪器，提供分析大分子所需的分辨率和灵敏度;获得这项技术将大大增强PNAC设施提供的服务。在代谢组学领域，无论是在系内还是在更广泛的剑桥地区，人们的兴趣都在稳步增加。随着史蒂夫奥利弗教授担任系统生物学教授，这一工作量将进一步增加。该应用程序解决了剑桥代谢组学的需求增加，以及解决两个关键的技术挑战，阻碍了LC-MS代谢组学。我们的QTof Micro升级为QTof Ultima系统将通过提供4倍的灵敏度和>2倍的分辨率来增加我们对代谢组的覆盖范围。此外，人们越来越认识到，数据必须是定量的，特别是对于许多基于系统生物学的方法，其中来自不同组学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Kathryn Lilley、Julian Griffin、Len Packman和Stephen奥利弗在系统生物学项目合作方面有着良好的记录[Karp，2005#2，Dunkley，2004，2006，Sadowski，2007，Castrillo，2007]，并且与许多共同申请人[许多联合出版物]也有良好的合作记录。此外，Griffin和Lilley团队已经成功地共享了质谱设备（LTQ），并在数据分析方法和蛋白质组学与代谢组学数据集的整合方面密切合作。该提案介绍了正在进行的项目和许多新的项目在该部。这将通过拟议设备的资金来促进。这些项目的范围从绝对测量复合物和途径内靶向蛋白质的丰度到网络内的代谢物，通过代谢途径的通量和细菌中响应药物流出而分泌的代谢物。

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.