Proteomics of Cell Signaling in Embryogenesis.

胚胎发生中细胞信号转导的蛋白质组学。

• 批准号: 10112932
• 负责人: STEVEN P GYGI
• 金额: $ 60.54万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10112932
• 关键词: Address; Adult; Affinity; Aging; Animal Cap; Autoimmunity; Bayesian Method; Behavior; Biological Models; Biology; Cell Differentiation process; Cell physiology; Cells; Chemistry; Childhood; Collaborations; Communities; Complex; Confidence Intervals; Consensus; Data; Data Set; Databases; Decision Making; Defect; Detection; Development; Ectoderm; Embryo; Embryonic Development; Erinaceidae; Experimental Designs; Funding; Genetic Transcription; Genome; Genotype; Goals; Grant; Heritability; Human; Hybrids; Immune System Diseases; Individual; Knowledge; Label; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Mathematics; Measurement; Measures; Methods; Mitogen-Activated Protein Kinases; Modernization; Modification; Mutate; Neurons; Organism; Outcome; Pathway interactions; Patients; Peptides; Pharmacology; Phenotype; Phosphoproteins; Phosphorylated Peptide; Phosphorylation; Phosphorylation Site; Phosphotransferases; Pluripotent Stem Cells; Population; Post-Translational Protein Processing; Procedures; Process; Protein Dynamics; Proteins; Proteomics; RNA; Reagent; Regenerative Medicine; Regulatory Pathway; Resources; Sampling; Signal Pathway; Signal Transduction; Site; Specificity; System; Systems Biology; Technology; Testing; Time; Tissues; Translating; Translations; Xenopus; Xenopus laevis; base; developmental disease; differential expression; embryo stage 2; experimental study; genetic regulatory protein; genome-wide; improved; inhibitor/antagonist; innovation; insight; kinase inhibitor; knock-down; mathematical analysis; mathematical methods; novel strategies; phosphoproteomics; protein expression; protein profiling; receptor; relating to nervous system; response; scaffold; stem; tool; ubiquitin ligase; vertebrate embryology

Project Summary Most cellular processes in both the embryo and the adult are controlled by either differential expression or modification of regulatory proteins. We will develop proteomic tools to study quantitatively the effect of protein expression levels and post-translational modification on key signaling pathways in cells, and probe the complex behavior of these pathways in vertebrate embryonic development. Our premise is that highly reliable and very accurate quantitative profiling of protein levels and phosphorylation along with proper mathematical analysis will lead to insights into how signaling pathways communicate information and enable cells to make decisions on a cellular and tissue level. Phosphorylation is thought to be the major regulatory modification of proteins. At least half of all mammalian proteins are phosphorylated, yet for most phospho-sites we do not know which kinase is responsible for the phosphorylation, and hence how the target protein is regulated. We propose to dramatically advance this situation via a systems biology framework for studying phosphorylation cascades in mammalian cells in culture and in early vertebrate embryonic development, using Xenopus laevis embryos as a model system. We have shown how manipulation of kinase activity, with poly-specific kinase inhibitors, can identify the relationship between a phenotype and a specific kinase. By measuring phosphorylation sites quantitatively by MS (using each individual phosphosite as its own phenotype) we plan to provide a kinase- substrate map at a genome scale for both human and Xenopus, and use this to connect kinase activity to cellular phenotype. Our team is comprised of a close and well established collaboration between an expert embryologist/cell biologist/biochemist (Kirschner), an expert in systematic analysis and statistical inference (Peshkin) and an expert in proteomic mass spectrometry (Gygi). Our recently developed methods will allow us to reach unprecedented depth of quantification of proteins and protein post-translational modifications. In this grant we will develop further innovations in experimental design, analytical improvements and improvements in MS. The large size of Xenopus embryos permits organism-level studies of such sensitivity that they can be used on a relatively small number of experimentally manipulated embryos. Our efforts will produce systems- level knowledge of the dynamic protein states in cells and in cell populations in early vertebrate development. The resulting data sets and methods should be a powerful resource for vertebrate embryology and could be informative for heritable defects in human embryos. Many embryonic pathways, such as MAPkinase, Wnt, and hedgehog, are also very important in childhood development, tissue and cell turnover in adults, regenerative medicine, and diseases of the immune system and cancer. As many of these pathways and kinase relationships seem conserved across stages of development and across vertebrate species, there is promise of rapid transfer from model system to human patients. In particular we expect that our approach will also serve to facilitate systems-level pharmacology approaches to therapy.

项目摘要 胚胎和成人中的大多数细胞过程均由差分表达或 调节蛋白的修饰。我们将开发蛋白质组学工具来定量研究蛋白质的影响 在细胞中关键信号通路上的表达水平和翻译后修饰，并探测复合物 这些途径在脊椎动物胚胎发育中的行为。我们的前提是高度可靠，非常 蛋白质水平和磷酸化的准确定量分析以及正确的数学分析 将导致有关信号通路如何传达信息并使细胞做出决策的见解 在细胞和组织水平上。磷酸化被认为是蛋白质的主要调节性修饰。在 所有哺乳动物蛋白的至少一半是磷酸化的，但是对于大多数磷酸化的位置，我们不知道哪个 激酶负责磷酸化，因此如何调节靶蛋白。我们建议 通过系统生物学框架急剧推进这种情况，以研究磷酸化级联 培养物和早期脊椎动物胚胎发育中的哺乳动物细胞，使用爪蟾Laevis胚胎作为一种 模型系统。我们已经展示了用多特异性激酶抑制剂对激酶活性的操纵如何可以 确定表型与特定激酶之间的关系。通过测量磷酸化位点 通过MS定量（使用每个单独的磷材料作为其自身表型），我们计划提供激酶 - 针对人和Xenopus的基因组量表的底物图，并使用它将激酶活性连接到 细胞表型。我们的团队由专家之间的紧密合作组成 胚胎学家/细胞生物学家/生物化学家（Kirschner），系统分析和统计推断专家 （Peshkin）和蛋白质组学质谱法专家（Gygi）。我们最近开发的方法将使我们 要达到蛋白质定量和蛋白质后修饰的前所未有的深度。在这个 授予我们将在实验设计，分析改进和改进方面发展进一步的创新 在MS中。大尺寸的爪蟾胚胎允许生物级研究这种敏感性，以至于它们可以是 用于相对少量的实验操纵胚胎。我们的努力将产生系统 - 早期脊椎动物发育中细胞和细胞种群中动态蛋白态的水平知识。 由此产生的数据集和方法应该是脊椎动物胚胎学的强大资源，可以是 人类胚胎中可遗传的缺陷提供了信息。许多胚胎途径，例如mapkinase，wnt和 刺猬，在成年人的儿童发育，组织和细胞更新中也非常重要，再生 医学和免疫系统和癌症的疾病。这些途径和激酶关系中的许多 在开发阶段和跨脊椎动物物种之间似乎是保守的，有望快速转移 从模型系统到人类患者。特别是我们期望我们的方法也有助于促进 系统级药理学治疗方法。

项目成果

Quantitative Proteomics Reveals Remodeling of Protein Repertoire Across Life Phases of Daphnia pulex.

定量蛋白质组学揭示了溞生命阶段蛋白质库的重塑。

• DOI: 10.1002/pmic.201900155
• 发表时间: 2019
• 期刊: Proteomics
• 影响因子: 3.4
• 作者: Peshkin,Leonid;Boukhali,Myriam;Haas,Wilhelm;Kirschner,MarcW;Yampolsky,LevY
• 通讯作者: Yampolsky,LevY

A cell type annotation Jamboree-Revival of а communal science forum.

• DOI: 10.1002/dvg.23383
• 发表时间: 2020-09
• 期刊: Genesis (New York, N.Y. : 2000)
• 作者: Peshkin L;Kirschner MW
• 通讯作者: Kirschner MW

Identification of kinases activated by multiple pro-angiogenic growth factors.

• DOI: 10.3389/fphar.2022.1022722
• 发表时间: 2022
• 期刊: Frontiers in pharmacology
• 影响因子: 5.6

On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development.

• DOI: 10.1016/j.devcel.2015.10.010
• 发表时间: 2015-11-09
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Peshkin L;Wühr M;Pearl E;Haas W;Freeman RM Jr;Gerhart JC;Klein AM;Horb M;Gygi SP;Kirschner MW
• 通讯作者: Kirschner MW

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

• DOI: 10.1016/j.cell.2015.04.044
• 发表时间: 2015-05-21
• 期刊: Cell
• 影响因子: 64.5
• 作者: Klein AM;Mazutis L;Akartuna I;Tallapragada N;Veres A;Li V;Peshkin L;Weitz DA;Kirschner MW
• 通讯作者: Kirschner MW