Proteomics of Cell Signaling in Embryogenesis.

项目摘要 胚胎和成体的大多数细胞过程都是由差异表达或 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作用 细胞内关键信号通路的表达水平和翻译后修饰，并探索其复合体 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和非常 蛋白质水平和磷酸化的准确定量分析以及适当的数学分析 将导致深入了解信号通路如何传递信息并使细胞做出决定 在细胞和组织水平上。磷酸化被认为是蛋白质的主要调节修饰。在… 所有哺乳动物的蛋白质中至少有一半是磷酸化的，然而对于大多数的磷酸化位点，我们不知道是哪一个。 激酶负责磷酸化，因此目标蛋白是如何调节的。我们建议 通过研究磷酸化级联反应的系统生物学框架，极大地促进了这种情况的发生 哺乳动物细胞在培养和早期脊椎动物胚胎发育中，使用非洲爪哇胚胎作为 模型系统。我们已经展示了如何用多聚特定的激酶抑制剂来操纵激酶的活性 确定一种表型和一种特定的激酶之间的关系。通过测量磷酸化位点 通过MS定量(使用每个单独的亚磷酸盐作为其自己的表型)，我们计划提供一种激酶- 在人类和非洲爪哇的基因组尺度上绘制底物图，并使用它将激酶活性与 细胞表型。我们的团队是由一位专家之间密切而良好的合作组成的 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)和蛋白质组质谱学专家(Gygi)。我们最近开发的方法将使我们能够 以达到前所未有的蛋白质量化和蛋白质翻译后修饰的深度。在这 我们将在实验设计、分析改进和改进方面进行进一步的创新 在MS中，非洲爪哇胚胎的巨大尺寸使有机体水平的研究能够如此敏感，以至于它们可以 用于相对较少数量的实验操作的胚胎。我们的努力将产生系统- 了解脊椎动物早期发育过程中细胞和细胞群体中蛋白质的动态状态。 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，并可能 人类胚胎中可遗传缺陷的信息。许多胚胎途径，如MAPK、Wnt和 刺猬在儿童发育、成人的组织和细胞更新、再生等方面也非常重要 医学、免疫系统疾病和癌症。因为许多这些通路和蛋白激酶关系 看起来在发育阶段和脊椎动物物种之间是保守的，有希望快速转移 从模型系统到人类患者。特别是，我们期望我们的做法也将有助于促进 系统层面的药理学治疗方法。

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.