Proteostasis Core: Quantitative global proteomics

项目摘要/摘要(核心C) Core C将为项目参与者进行全球蛋白质组学实验。这项工作将在以下条件下进行 哈佛医学院(HMS)Steve Gygi博士和Daniel Finley博士的领导。这家著名的工厂位于 HMS可以一次提供10个样本的蛋白质水平的定量读数，使用TMT-MS3方法 由Gygi小组开发的全球蛋白质组学。10个样本的单次运行提供了详细的定量数据 在10个实验蛋白质组上。在定义的实验条件下对扰动的全面看法 条件产生了丰富的信息，包括蛋白质平衡的1000多个成分的水平 网络(PN)，如蛋白酶体和伴侣，这些酶的数千种底物的状态， 以及约3万个蛋白质修饰位点的占有率。重要的是，每次TMT-MS3运行都将提供 在给定时间和条件下PN状态的指纹。因此，新的表型特征 PN中的突变体，如具有增强的蛋白酶体活性的转基因突变体，将在 目前可能达到的最高分辨率。核心C还将为研究人员提供第二个蛋白质组学 签名：蛋白质泛素修饰的全球帐户。这将使用“GG”来完成 下拉式“方法，在这种方法中，泛素中含有二甘氨酸残基的胰蛋白肽通过 质谱仪前的免疫提纯。TMT蛋白质组学和泛素蛋白质组学并行应用， 将提供小区中PN状态的唯一详细且信息丰富的快照。这些分析性的 方法对于团队内的所有项目都将是无价的，包括侧重于生物传感器的核心项目 (核心B)和PN的化学调节器(核心D)。此外，蛋白酶体的蛋白质组学方法 和自噬项目(2和3)，涉及在小鼠体内的工作，将密切协调，以便 所得到的数据集可以被集成并受元数据分析。因此，Core C的方法论 将有助于团结项目的研究努力，并推动我们对PNS的了解更加量化 和系统层面。

Project Summary / Abstract (Core C) Core C will conduct global proteomics experiments for project participants. This work will be performed under the leadership of Drs. Steve Gygi and Daniel Finley at Harvard Medical School (HMS). The renowned facility at HMS can provide quantitative readouts of protein levels of 10 samples at once, using the TMT-MS3 method of global proteomics developed by the Gygi group. A single run of 10 samples provides detailed quantitative data on 10 experimental proteomes. This comprehensive view of perturbations under defined experimental conditions yield a wealth of information, including the levels of over 1000 components of the proteostasis network (PN), such as proteasomes and chaperones, the state of thousands of substrates of these enzymes, and the occupancy of ~30,000 protein modification sites. Importantly, each TMT-MS3 run will provide a fingerprint of the state of the PN at a given time and condition. Thus, the phenotypic characterization of new mutants in the PN, such as transgenic mutants with enhanced proteasome activity, will be accomplished at the highest resolution possible at present. Core C will also give researchers access to a second proteomics signature: a global account of ubiquitin modifications of proteins. This will be accomplished using the “GG pulldown” method, in which tryptic peptides bearing diglycine remnants from ubiquitin are enriched via immunopurification prior to mass spectrometry. TMT proteomics and ubiquitin proteomics, applied in parallel, will provide a uniquely detailed and informative snapshot of the state of the PN in a cell. These analytical approaches will be invaluable to all projects within the team, including core projects that focus on biosensors (Core B) and chemical regulators of the PN (Core D). In addition, proteomics approaches of the proteasome and autophagy projects (2 and 3), involving in vivo work in the mouse, will be closely coordinated so that the resulting datasets can be integrated and subjected to metadata analysis. Thus, the methodologies of Core C will help to unite the research efforts of the project and to drive our understanding of PNs to a more quantitative and systematic level.