High Resolution Mass Spectrometry System

描述（由申请人提供）：需要高分辨率和质量精确的Orbitrap Velos混合FT质谱仪系统来帮助发现“智能药物”的新靶点。靶向药物疗法提供优于全身化疗的优势，因为靶向特定蛋白质/基因最大限度地减少副作用。基于质谱的蛋白质组学非常适合于以高灵敏度、高通量和高特异性来阐明有缺陷的癌细胞信号传导途径。我们将使用稳定同位素标记（例如，SILAC、iTRAQ、TMT）和无标签策略（例如，光谱计数、MS/MS平均TIC峰面积积分、MS峰分析）。此外，蛋白质-蛋白质相互作用（PPI）网络将在癌症信号通路中建立。将使用市售和免费软件（Sequest via Protein Discoverer、Mascot Browser、Scaffold、Scaffold PTM、MaxQuant和Cytoscape）和内部开发的软件工具（PTM Quantifier和NakedQuant）处理和分析数据。 与上一代LTQ Orbitrap相比，Velos Orbitrap的扫描速度提高了2倍，灵敏度提高了3倍，肽和蛋白质覆盖深度提高了2倍。质谱仪将耦合到微毛细管LC/MS/MS模式下的无分流纳米流HPLC，以获得癌症研究的最佳灵敏度。Velos Orbitrap系统不允许开放使用，仅由经过培训的专业人员操作。服务合同、工资和用品将通过BIDMC的担保运营账户提供资金，并将通过NIH资助的项目赠款收回。 Orbitrap Velos将主要由七个主要用户和三个次要用户共享，此外还将成为Beth Israel Deaconess Medical Center（BIDMC）设备齐全的质谱核心设施的一部分，并可由Longwood医疗区内的其他PI访问。对将利用该技术的五个主要项目的描述是：（1）评估 有助于AKT活化的磷酸肌醇-3-激酶（PI 3 K）复合物，（2）果蝇细胞中胰岛素信号传导途径的蛋白质-蛋白质相互作用组的剖析，（3）涉及贫血的疾病模型中核糖体的蛋白质组分的定量，（4）阐明结节性硬化症（TSC）肿瘤抑制因子的功能作用（TSC 1和TSC 2）对TORC复合物的影响和（5）阿尔茨海默病脑斑块的蛋白质组学表征。我们预计，拟议的实验将发现几个“可药物化”的蛋白质靶点，并最终导致一个或多个临床试验。 公共卫生相关性：一个高分辨率的Thermo Scientific Orbitrap Velos质谱仪与一个无分裂的纳米HPLC相结合，将用于发现癌症等疾病所需的新的“智能”药物靶标，以减少副作用，并根据细胞的缺陷途径有效地攻击和杀死癌细胞。基于质谱的蛋白质组学是通过定量和定性实验阐明蛋白质信号通路在病变细胞中的功能作用的理想方法。

DESCRIPTION (provided by applicant): A high resolution and mass accurate Orbitrap Velos hybrid FT mass spectrometer system is required to help uncover novel targets for "smart drugs". Targeted drug therapies offer advantages over systemic chemotherapies since targeting specific proteins/genes minimizes side effects. Mass spectrometry based proteomics is well-suited to elucidate defective cancer cell signaling pathways with high sensitivity, throughput and specificity. We will perform both identification and quantification of peptides/proteins and modified peptides/proteins from cell lines and tumor tissues using stable isotope labeling (e.g., SILAC, iTRAQ, TMT) and label-free strategies (e.g., spectral counting, MS/MS average TIC peak area integration, MS peak profiling) through discovery and targeted experiments. In addition, protein-protein interaction (PPI) networks will be established in cancer signaling pathways. Data will be processed and analyzed using commercially available and freely available software (Sequest via Protein Discoverer, Mascot Browser, Scaffold, Scaffold PTM, MaxQuant and Cytoscape) and in-house developed software tools (PTM Quantifier and NakedQuant). Compared to the previous generation LTQ Orbitrap, the Velos Orbitrap represents a 2-fold improvement in scan speed and 3-fold sensitivity improvement resulting in a 2-fold improvement in depth of peptide and protein coverage. The mass spectrometer will be coupled to a splitless nanoflow HPLC in microcapillary LC/MS/MS mode for optimal sensitivity for cancer research. The Velos Orbitrap system will not be allowed open access and operated by trained professionals only. Service contracts, salaries, and supplies will be funded through a guaranteed operating account through BIDMC and will be recaptured through charge-backs from the NIH funded project grants. The Orbitrap Velos will be primarily shared among seven major users and three minor users in addition to being part of the fully equipped mass spectrometry core facility at Beth Israel Deaconess Medical Center (BIDMC) and accessible by additional PIs within the Longwood medical area subject to availability. Descriptions for five main projects that will utilize the technology are the (1) Evaluation of the functional role of the phosphoinositide-3-kinase (PI3K) complex contributing to AKT activation, (2) Dissection of the protein-protein interact-ome of the insulin signaling pathway in drosophila cells, (3) Quantification of protein components of the ribosome in disease models involving anemia, (4) Elucidation of the functional role of the tuberous sclerosis complex (TSC) tumor suppressors (TSC1 and TSC2) effects on the TORC complexes and (5) Proteomics characterization of Alzheimer's Disease brain plaques. We anticipate that the proposed experiments will uncover several "druggable" protein targets and eventually lead to one or more clinical trials. PUBLIC HEALTH RELEVANCE: A high resolution Thermo Scientific Orbitrap Velos mass spectrometer coupled to a splitless nano-HPLC will be used to discover new "smart" drug targets that are needed in diseases such as cancer to reduce side effects and efficiently attack and kill cancer cells based on the cell's defective pathways. Mass spectrometry based proteomics is ideal for elucidating the functional role of protein signaling pathways in diseased cells through quantitative and qualitative experimentation.