Functional Proteomics Study of Ephrin Signaling

在功能蛋白质组学研究中使用质谱是鉴定参与重要细胞内信号转导系统的蛋白质的有力方法。我们建议开发和使用一种新的功能蛋白质组学方法的基础上同位素编码的亲和标签（ICAT）化学和液相色谱/质谱法，以确定参与信号转导复合物的形成，在响应配体刺激的蛋白质。这种策略应提高相对蛋白质定量的准确性，以及增加的速度，灵敏度和动态范围的蛋白质鉴定的策略的基础上，从SDS-PAGE凝胶的蛋白质的鉴定。肝配蛋白和肝配蛋白受体（EphR）参与中枢和外周神经系统的模式化。我们将使用我们的方法来研究通过EphRs的信号传导以及通过B ephrin配体在培养的神经元细胞中的“反向信号传导”，通过鉴定和定量与先前表征的响应于ephrinB 2或EphRs刺激的信号转导蛋白相关的蛋白质。将确定三组蛋白质：组成型结合蛋白，那些增加和那些减少与其他信号蛋白在响应配体刺激。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ephrin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，将使重点突出，假设驱动的实验，以阐明这些蛋白质在肝配蛋白信号传导中的作用。为研究ephrin信号而开发的功能蛋白质组学方法也可用于研究神经系统功能所必需的各种其他信号转导系统。最终，从这些实验中获得的信息可能会让我们更好地了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.