The developmental proteome of retinal progenitor

描述（由申请人提供）：视网膜营养不良是一种涉及光感受器变性的疾病，光感受器是视网膜上唯一的感光细胞。对视网膜发育的深入了解有助于对抗视网膜营养不良的新疗法。干细胞研究的最新进展可能使视网膜神经元替代成为治疗许多视网膜退行性疾病的可行疗法。在这方面，目前对祖细胞/干细胞的组成部分只有很差的理解，当它成为一种特定类型的视网膜神经元时，它会发生变化。本研究的目的是将蛋白质组学作为一种新的研究工具应用于视网膜发育的研究。由于祖细胞对环境信号的反应能力反映在质膜“蛋白质组”中存在的蛋白质中，因此本项目将研究3个发育年龄视网膜祖细胞质膜蛋白表达的变化。该方案的具体目的是：(1)从发育中的视网膜（胚胎15天和18天，出生后0天）中分离视网膜祖细胞，纯化和分离质膜蛋白。(2)对这些发育年龄的祖细胞膜蛋白进行分类，并量化其表达水平。利用基质辅助激光解吸电离飞行时间质谱法对蛋白质进行鉴定，并利用生成的光谱对蛋白质序列数据库进行检索。(3)根据蛋白质表达水平的变化将蛋白质聚类为理论功能基团。这项研究将是第一次将蛋白质组学工具应用于视网膜发育，并将为使用这一强大技术的未来发育研究铺平道路。此外，该分析将确定数十种对视网膜发育重要的蛋白质和信号通路，并有助于更有效地关注视网膜祖细胞分化的进一步研究。

DESCRIPTION (provided by applicant): Retinal dystrophies are diseases that involve degeneration of photoreceptors, the sole light-sensing cells in the retina. A solid understanding of retinal development can contribute to new therapies for combating retinal dystrophies. Recent advances in stem-cell research may make replacement of retinal neurons a feasible therapy for the treatment of many retinal degenerative disorders. In this regard there is currently only a poor understanding of the components of a progenitor cell/stem cell that change as it becomes a particular type of retinal neuron. The purpose of the proposed studies is to apply the powerful techniques of proteomics as a novel research tool in the study of retinal development. Because the ability of a progenitor cell to respond to cues from its environment are reflected in the proteins present in the 'proteome' of the plasma membrane, this project will investigate changes in expression among plasma membrane proteins of retinal progenitor cells from 3 developmental ages. The specific aims of the proposal are (1) To isolate retinal progenitor cells from the developing retina (embryonic days 15 and 18, postnatal day 0), and purify and separate proteins of the plasma membrane. (2) To catalog the proteins of the progenitor cell plasma membrane at these developmental ages, and quantify their levels of expression. Proteins will be identified by using matrix assisted laser desorption ionization-time of flight mass spectrometry, and using generated spectra to search against protein sequence databases. (3) To cluster proteins into theoretical functional groups based on how their level of protein expression changes. This study will be the first to apply the tools of proteomics to retinal development and will pave the way for future developmental studies using this powerful technique. Further, this analysis will identify dozens of proteins and signaling pathways of importance for retinal development, and help to more efficiently focus further studies of differentiation of retinal progenitors.