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

描述(由申请人提供)：布朗大学和罗德岛大学的一个富有成效的生物医学研究财团正在请求NCRR共享仪器计划的支持，以购买质谱仪(MS)升级，这将采取现有的Thermo LTQ MS并将其转换为Thermo LTQ Orbitrap XL ETD混合MS。值得注意的是，Orbitrap XL ETD包括两个碎裂区域，高能C陷阱解离(HCD)和电子转移解离(ETD)，这两个区域是LTQ碰撞诱导解离(CID)选项的补充。这些额外的、可供选择的碎片化能力对于参与研究人员积极开展的各种项目(磷酸蛋白质组学；翻译后修饰分析；“中间向下”蛋白质组学)的成功至关重要。要求对LTQ Orbitrap XL ETD MS进行新的升级，这对于收集和分析该联盟要求苛刻、在某些情况下具有高度技术挑战性的研究项目所需的高质量数据是必要的。Orbitrap XL ETD质谱仪的显著优点是其高灵敏度、分辨率和质量精度，以及快速扫描速度。参与这项提议的大多数拟议的主要用户正在进行从细胞或组织中提取的高度复杂的蛋白质混合物的分析。为了充分盘点复杂蛋白质组样本中的蛋白质组分，大多数高质量的数据都应该在与纳米液相色谱分离和洗脱一致的时间尺度上获得。Orbitrap是为本应用程序中描述的复杂样品提供最可靠、最高质量分析的理想位置。一旦安装，该质谱仪将通过实施内部开发的自动化数据管道而持续使用(即“全天候”)，该数据管道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SEQUEST搜索、多肽丰度量化、数据库搜索结果的统计验证以及将观察到的多肽与一系列公开可用的蛋白质组数据库进行交叉参考。蛋白质相互作用和动力学在细胞信号传递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育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。如本提案所述，此类研究目前是许多现有的和长期运行的NIH赞助的研究项目的组成部分，随着最近在布朗大学学术丰富计划中对新的教职员工队伍的投资，蛋白质组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学府正在进行的高质量生物医学研究产生直接和长期的影响。 公共卫生相关性：所要求的仪器升级将使我们的参与研究人员能够阐明在从哮喘到癌症再到神经元信号等一系列模型系统中参与调节细胞功能和生理反应的相互作用蛋白。这些研究构成了当前努力研究和利用我们不断扩大的蛋白质组知识的重要组成部分。目前正在将重点放在蛋白质及其在疾病和发育中的相互作用上，最终目标是确定药物治疗的新目标，并开发新的治疗方法。所要求的仪器将促进的基础研究将极大地扩展我们对在正常细胞和组织功能以及在各种人类疾病状态下各种信号通路之间的相互作用和串扰的知识。

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.