Acquisition of an Orbitrap XL ETD Mass Spectrometer through Upgrading an LTQ

描述（由申请人提供）：生产生物医学研究人员组成的一个财团和大学的罗德岛的布朗大学请求NCRR共享仪器的支持计划购买的质谱(MS)升级将现有热LTQ女士并将其转换成热LTQ Orbitrap XL等混合产生的女士LTQ Orbitrap高性能质和MSn系统,结合快速LTQ离子阱与高质量准确性Orbitrap质量数据采集分析。值得注意的是，Orbitrap XL ETD包括两种分裂机制，高能c阱离解（HCD）和电子转移离解（ETD），这是对LTQ碰撞诱导离解（CID）选项的补充。这些额外的、可选择的片段化能力对于参与研究者积极追求的各种项目（磷酸化蛋白质组学、翻译后修饰分析、“中下游”蛋白质组学）的成功至关重要。LTQ Orbitrap XL ETD MS的新升级对于收集和分析高质量数据是必要的，这些数据是该联盟要求很高的，在某些情况下，技术上具有挑战性的研究项目所需要的。Orbitrap XL ETD质谱仪的独特优点是其高灵敏度，分辨率和质量精度，以及快速扫描速率。参与本提案的大多数拟议主要用户都在从事来自细胞或组织的高度复杂的蛋白质混合物的分析。为了在复杂的蛋白质组学样品中充分列出蛋白质成分，高质量的数据大多数是在与纳米液相色谱分离和洗脱相一致的时间尺度上获得的。Orbitrap的理想定位是为本应用程序中描述的复杂样品提供最强大，最高质量的分析。安装后，该质谱仪将通过内部开发的自动化数据管道持续使用（即“24/7”），其中包括多肽的自动多维毛细管分离，纳米喷雾LC/ESI-MS，自动数据采集和后处理。我们目前的内部技术平台提供全自动测序搜索，肽丰度定量，数据库搜索结果的统计验证，以及与一系列公开可用的蛋白质组学数据库交叉引用观察到的肽。蛋白质相互作用和动力学在细胞信号传导中起着至关重要的作用，是目前探索和利用蛋白质组的重要组成部分。系统网络生物学将是我们关注蛋白质及其在疾病和发展中的相互作用的重要组成部分，最终目标是确定药物治疗的新靶点和开发新的治疗方法。正如本提案所详述的那样，这些研究目前是许多现有的和长期运行的美国国立卫生研究院资助的研究项目的组成部分，并且随着布朗大学最近在学术充实计划中对新教员线的投资，蛋白质组学研究的数量和种类将在未来几年继续增长。很明显，所要求的仪器将对进一步推动布朗大学和罗德岛州其他高等院校正在进行的高质量生物医学研究产生直接和持久的影响。

DESCRIPTION (provided by applicant): A consortium of productive biomedical investigators at Brown University and the University of Rhode Island is requesting support from the NCRR Shared Instrumentation Program for purchase of a mass spectrometer (MS) upgrade which would take an existing Thermo LTQ MS and transform it into a Thermo LTQ Orbitrap XL ETD hybrid MS. The resulting LTQ Orbitrap will be a high performance LC-MS and MSn system, combining rapid LTQ ion trap data acquisition with high mass accuracy Orbitrap mass analysis. Significantly, the Orbitrap XL ETD includes two fragmentation regimes, higher-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD), that are complementary to the LTQ's collision induced dissociation (CID) option. These additional, alternative fragmentation capabilities are essential for the success of the diverse projects (phosphoproteomics; posttranslational modification analyses; "middle-down" proteomics) being actively pursued by the participating investigators. The requested new upgrade to the LTQ Orbitrap XL ETD MS is necessary for the collection and analysis of the high quality data required by the consortium's demanding and in some cases highly technically challenging research projects. The distinct benefits of the Orbitrap XL ETD mass spectrometer are its high sensitivity, resolution and mass accuracy, coupled to a fast scan rate. Most of the proposed major users participating in this proposal are pursuing the analysis of highly complex mixtures of proteins derived from cells or tissues. To adequately inventory the protein constituents within a complex proteomic sample, high quality data most be acquired on a time scale consistent with nano-liquid-chromatographic separation and elution. The Orbitrap is ideally positioned to provide the most robust, highest quality analysis of the complex samples described in this application. Once installed, this mass spectrometer will be utilized continuously (i.e., "24/7") through implementation of an in-house developed automated data pipeline which includes automated multidimensional capillary separations of peptides, nanospray LC/ESI-MS, automated data acquisition, and post-processing. Our current in-house technology platform provides for fully automated Sequest searching, quantitation of peptide abundance, statistical validation of database search results, and cross-referencing of observed peptides against an array of publicly available proteomic databases. Protein interactions and dynamics play a critical role in cell signaling, and form an important part of current efforts to explore and exploit the proteome. Systems network biology will be a vital component of our focus on proteins and their interactions in disease and development with the eventual goal of identification of new targets for drug therapy and the development of new approaches to therapeutics. As detailed in this proposal, such studies are presently an integral part of a number of existing and long-running NIH-sponsored research projects and with the recent investments in new faculty lines at Brown University in the Academic Enrichment Plan, the number and variety of proteomic studies will continue to grow over the next several years. It is very clear that the requested instrumentation will have an immediate and long-lasting impact in further propelling the high-quality biomedical research being pursued at Brown University and at the other institutions of higher learning in the state of Rhode Island. PUBLIC HEALTH RELEVANCE: The requested instrumentation upgrade will allow our participating investigators to elucidate the interacting proteins that are involved in the regulation of cell function and physiological response in a number of model systems ranging from asthma to cancer to neuronal signaling. These studies form an important part of current efforts to investigate and capitalize on our expanding knowledge of the proteome. This focus on proteins and their interactions in disease and development is being pursued with the eventual goal of identification of new targets for drug therapy and the development of new approaches to therapeutics. The basic research that will be facilitated by the requested instrumentation will greatly expand our knowledge of the interplay and cross-talk between various signaling pathways both in normal cellular and tissue function and in a variety of human disease states.