Ocular proteomics of retina

描述(摘自申请人的摘要)： 这个项目的长期目标是确定所有表达的蛋白质 在啮齿动物的视网膜上为视觉研究人员提供信息 关于视网膜细胞中实际表达的蛋白质。蛋白质分析方法 双向(2-D)凝胶电泳法的结合 光谱分析和基因组数据库搜索已经影响了 生物医学科学。这种新型技术通常被称为 “蛋白质组学”，并能够对从细胞或 纸巾。在过去的二十年里，对视觉传导的理解 途径及其潜在的分子机制得到了实质性的改善 通过使用来自各种动物模型的杆状外段制剂 包括牛、蛙等动物。最新的转基因小鼠模型 也充实和扩展了许多以前关于 视觉背后的分子机制。在这项提案中，蛋白质组学方法 将被用来表征在视网膜中表达的蛋白质 实验室啮齿动物，大鼠和小鼠。选择啮齿动物作为动物的原因 研究视网膜蛋白表达的模型有三个方面。首先是 人与啮齿动物基因序列的相似性。第二个是 转基因动物模型的可获得性及相关文献的积累 环境对啮齿动物视网膜的生理和病理影响 或者是遗传干扰。最后，更低的成本和更容易的实验 操纵使啮齿动物模型变得实用。有三个具体的目标 这项提议。目标1是对几乎所有的视网膜蛋白进行分析 从啮齿动物的视网膜中提取，并在2-D凝胶上分离。蛋白质图谱将会 通过研究不同时期的视网膜，沿着发育时间轴进行研究 出生后的日子。为了实现这一目标，还将努力开发一种技术 从蛋白质提取液中去除丰富的蛋白质以研究 少量表达的蛋白质，也可以形成大量 稳定同位素标记法定量蛋白质的光谱技术。目标2 是为了描述所识别的选定蛋白质的免疫组织化学定位 在目标1中。在这一目标中，将使用一套明确的标准，以便 选择的蛋白质将被分成生理上的组 截然不同。目标3是使蛋白质组信息在基于网络的 数据库，以便视觉研究人员可以在需要时检索数据。 该项目的完成将帮助研究人员研究视力和 利用啮齿动物模型研究其在蛋白质水平上的紊乱。

DESCRIPTION (From the Applicant's Abstract): The long aboutterm goal of this project is to define all the proteins expressed in rodent retinas in order to provide vision researchers with information regarding the proteins actually expressed in retinal cells. Protein analysis by the combination of two-dimensional (2-D) gel electrophoresis, mass spectrometry, and genome database search has impacted on the progress of biomedical sciences. This new breed of technology is generally called "proteomics" and enables massive analysis of proteins extracted from cells or tissues. In the past two decades the understanding of visual transduction pathway and its underlying molecular mechanisms has substantially been improved by the use of rod outer segment preparations from various animal models including bovine, frog, and other animals. The recent transgenic mouse model also has substantiated and extended much of the previous knowledge on the molecular mechanisms underlying vision. In this proposal, a proteomic approach will be used to characterize the proteins expressed in the retinas of laboratory rodents, rats and mice. The reasons for the choice of rodents as a model to study retinal protein expression are three-fold. First is the similarity in gene sequence between human and rodents. Second is the availability of transgenic animal models and the accumulated literatures on the physiology and pathological changes of rodent retinas induced by environmental or genetic perturbation. Finally, the lower costs and ease of experimental manipulation makes a rodent model practical. There are three specific aims for this proposal. Aim 1 is to profile virtually all the retinal proteins that are extracted from rodent retinas and separated on 2-D gels. Protein profiling will be made along the developmental time axis by investigating retinas at different postnatal days. In this aim, efforts will also be made to develop a technique to remove abundant proteins from the protein extract in order to investigate proteins expressed in minor quantities, and also to develop a mass spectrometric technique to quantify proteins by stable isotope labeling. Aim 2 is to profile immunohistochemical localization of selected proteins identified in Aim 1. In this aim, a defined set of criteria will be used so that the proteins chosen will be classified into groups that are physiologically distinct. Aim 3 is to make the proteomic information accessible on a web-based database so that vision researchers can retrieve data when needed. Accomplishment of this project will help researchers investigating vision and its disorders at the protein level using rodent models.