RAPID DETECTION OF CELLULAR PROTEIN DIFFERENCES

描述：（申请人摘要）蛋白质组成的差异和 修饰是细胞差异的主要决定因素， 结构、功能和行为。 在许多情况下，这些差异 由差异基因表达产生。 然而，许多细胞变化 特征是译后修饰差异的结果。 了解细胞差异的分子基础是一个主要的驱动力 现代生物学的力量。 大量的重点放在了 差异基因表达 为了配合这些努力，我们采取了 基于蛋白质的方法，用于识别蛋白质差异， 细胞差异的基础。 典型的真核细胞由5,000多种不同的蛋白质组成， 其中许多是生物体所有细胞共有的。 述区分 不同细胞类型的特征是由少数蛋白质产生的。 的 用于展示细胞蛋白质阵列的标准方法是分离 根据它们的电荷和质量，通过二维聚丙烯酰胺 凝胶电泳（2DE），一种允许人们可视化 大约2,000种最丰富的细胞蛋白质。 2DE遭受 因为没有两种凝胶是完全相同的。 因此，我们认为， 必须求助于基于计算机的方法来排列不同的凝胶， 并不能完美地解决异质性问题。 我们已经开发出一种 一种绕过这些问题的技术， 在相同的凝胶上。 这是通过共价偶联每个 蛋白质样品与不同颜色的荧光染料，设计， 对标记蛋白的相对迁移没有影响， 电泳 荧光标记的蛋白质通过 荧光凝胶成像仪，可以区分两种荧光 染料。 普通蛋白质呈现为由两种荧光染料组成的斑点; 而两个样品之间不同的蛋白质，称为 “差异蛋白质”，显示偏向于一种或另一种染料。 这 差异凝胶电泳（DIGE）。 目前，DIGE 比银染法更敏感，可以检测出不同蛋白质 低至全细胞提取物中总蛋白质的0.01%。 该提案描述了改进DIGE的三个具体目标， 可视化几乎所有的细胞蛋白质，并检测到非常低的丰度 差异 用于自动检测和分离差异的软件 还将描述蛋白质。 一旦检测到差异蛋白， 质谱仪分析与基因组数据库相结合， 确定编码不同蛋白质的基因的同一性。

DESCRIPTION: (Applicant's abstract) Differences in protein composition and modification are the primary determinants of differences in cellular structure, function and behavior. In many cases, these differences are generated by differential gene expression. However, many cellular changes and features are the result of differences in post-translation modification. Understanding the molecular basis for cell differences is a major driving force in modern biology. A great deal of emphasis has been placed on differential gene expression. To complement these efforts, we have taken a protein-based approach for identifying the protein differences that are the basis for cellular differences. Typical eukaryotic cells are composed of more than 5,000 different proteins, many of which are common to all cells of the organism. The distinguishing features of different cell types are created by a minority of proteins. The standard method for displaying the array of cellular proteins is to separate them according to their charge and mass by two-dimensional polyacrylamide gel electrophoresis (2DE), a technique that allows one to visualize approximately 2,000 of the most abundant cellular proteins. 2DE suffers from a reproducibility problem-no two gels are perfectly alike. Therefore, one must resort to computer-based methods to align different gels, which does not perfectly resolve the heterogeneity problem. We have developed a technique that bypasses these problems by running the different protein samples on the same gel. This is accomplished by covalently coupling each protein sample with a different colored fluorescent dye that was designed to have no effect on the relative migration of labeled proteins during electrophoresis. The fluorescently tagged proteins are visualized by a fluorescent-gel imager that can discriminate between the two fluorescent dyes. Common proteins appear as spots composed of both fluorescent dyes; while proteins that differ between the two samples, referred to as "difference proteins", display a bias toward one or the other dye. This technique is called Difference Gel Electrophoresis (DIGE). Currently, DIGE is more sensitive than silver staining and can detect difference proteins down to 0.01% of total protein in a whole cell extract. This proposal describes three specific aims to improve DIGE so that one can visualize nearly all cellular proteins and detect very low abundance differences. Software for automated detection and isolation of difference proteins will also be described. Once a difference protein is detected, mass spectrometer analysis coupled to the genome data base will be used to determine the identity of the genes encoding the difference proteins.