Functional Proteomics Study of Ephrin Signaling

在功能蛋白质组学研究中使用质谱学是鉴定与重要的细胞内信号转导系统有关的蛋白质的有效方法。我们建议开发和使用一种新的基于同位素编码亲和标签(ICAT)化学和液相色谱/质谱仪的功能蛋白质组学方法来识别参与配体刺激反应的信号转导复合体形成的蛋白质。与基于从SDS-PAGE凝胶中鉴定蛋白质的策略相比，该策略将提高相对蛋白质定量的准确性，并提高蛋白质鉴定的速度、灵敏度和动态范围。肾上腺素和肾上腺素受体(EphRs)参与中枢和外周神经系统的模式形成。我们将使用我们的方法来研究在培养的神经细胞中通过EphRs的信号转导以及通过B ewitin配体的“反向信号”，方法是识别和定量与先前描述的信号转导蛋白相关的蛋白质，以响应ewitinB2或EphRs的刺激。将确定三组蛋白质：构成结合蛋白，那些增加的，以及那些在反应配体刺激时与其他信号蛋白结合减少的蛋白。提出的基于ICAT的功能蛋白质组学技术的目标是快速发现依赖于ephin信号的新的蛋白质-蛋白质相互作用。对这些相互作用进行全面的分类，特别是包括定量信息和对细胞信号状态的依赖，将使有重点的、假设驱动的实验能够阐明这些蛋白质在ePhin信号传递中的作用。功能蛋白质组学方法学是为研究肾上腺素信号而开发的，也可以用来研究神经系统功能所必需的其他各种信号转导系统。最终，从这些实验中获得的信息可能有助于更好地理解各种癌症和神经发育疾病的原因。

The use of mass spectrometry in functional proteomics studies is a powerful approach for identifying proteins involved in important intracellular signal transduction systems. We propose to develop and use a new functional proteomics method based on isotope-coded affinity tag (ICAT) chemistry and liquid chromatography/mass spectrometry to identify proteins that participate in signal transduction complex formation in response to ligand stimulation. This strategy should improve the accuracy of relative protein quantitation as well as increase the speed, sensitivity, and dynamic range of protein identification over strategies based on the identification of proteins from SDS-PAGE gels. Ephrins and ephrin receptors (EphRs) are involved in patterning of the central and peripheral nervous systems. We will use our method to study signaling through EphRs as well as "reverse signaling" through B ephrin ligands in cultured neuronal cells by identifying and quantitating proteins that associate with previously characterized signal transduction proteins in response to stimulation by ephrinB2 or EphRs. Three groups of proteins will be identified: constitutively binding proteins, those that increase and those that decrease in their association with other signaling proteins in response to ligand stimulation. The goal of the proposed ICAT-based functional proteomics technology is to rapidly discover new protein-protein interactions that depend on ephrin signaling. A comprehensive cataloging of these interactions, especially including quantitative information and dependence upon the signaling state of the cell, will enable focused, hypothesis-driven experiments to elucidate the roles of these proteins in ephrin signaling. The functional proteomics methodology developed for the study of ephrin signaling could also be used to study a variety of other signal transduction systems essential for the functioning of the nervous system. Ultimately, information gained from these experiments may lead to greater understanding of the causes of a variety of cancers and neurodevelopmental diseases.