Proteostasis Core: Quantitative global proteomics

蛋白质稳态核心：定量全局蛋白质组学

• 批准号: 10432029
• 负责人: Daniel J Finley
• 金额: $ 25.01万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10432029
• 关键词: Affect; Aging; Autophagocytosis; Bioinformatics; Biosensor; Brain; Cells; Chemicals; Complex; Coupled; Data; Data Set; Development; Disease; Ensure; Exposure to; Fingerprint; Fluorescence Microscopy; Genetic; Genetic Transcription; Glycylglycine; Goals; Human; Huntington Disease; Individual; Leadership; Longevity; Mass Spectrum Analysis; Measures; Messenger RNA; Metadata; Methodology; Methods; Modeling; Molecular Chaperones; Monitor; Motor Neurons; Mus; Neurodegenerative Disorders; Neurons; PGRN gene; Participant; Pathway interactions; Patients; Peptides; Pharmacology; Phenotype; Play; Post-Translational Protein Processing; Preparation; Proteins; Proteome; Proteomics; Reporter; Reporting; Research; Research Personnel; Resolution; Ribosomes; Role; Running; Sampling; Signal Pathway; Site; Spinal Cord; Stress; System; Tauopathies; Technology; Time; Transgenic Organisms; Ubiquitin; Work; base; biological adaptation to stress; cell age; data management; enzyme substrate; experimental study; in vivo; large datasets; link protein; medical schools; mouse model; multicatalytic endopeptidase complex; mutant; proteomic signature; proteostasis; response; sensor; transcriptome; transcriptome sequencing; transcriptomics; vector

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.

项目概要/摘要（核心C） 核心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 哈佛医学院（HMS）的史蒂夫·吉吉博士和丹尼尔·芬利博士的领导。著名的设施， HMS可以使用TMT-MS 3方法一次提供10个样品的蛋白质水平的定量读数。 Gygi小组开发的全球蛋白质组学。单次运行10个样品可提供详细的定量数据 10个实验蛋白质组。这一全面的看法扰动下定义的实验 条件产生了丰富的信息，包括超过1000种蛋白质抑制成分的水平， 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30，000个蛋白质修饰位点的占用。重要的是，每次TMT-MS 3运行将提供 在给定时间和条件下PN状态的指纹。因此，新的表型表征 PN中的突变体，例如具有增强的蛋白酶体活性的转基因突变体，将在 目前可能的最高分辨率。核心C还将使研究人员获得第二个蛋白质组学 签名：蛋白质泛素修饰的全球帐户。这将通过使用“GG pulldown”方法，其中带有来自泛素的双甘氨酸残基的胰蛋白酶肽通过 在质谱法之前进行免疫纯化。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状态的唯一详细和信息性快照。这些分析 这种方法对于团队中的所有项目都是非常宝贵的，包括关注生物传感器的核心项目 (Core B）和PN的化学调节剂（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，使 所得到的数据集可以被整合并经受元数据分析。因此，核心C 将有助于统一该项目的研究工作，并推动我们对PN的理解更加量化 系统层面。