Analysis to enhance sensitivity for membrane proteins

描述：（申请人提供） 在相当长的一段时间里，全面分析 组织和细胞群中的蛋白质表达是二维的 (2-D)凝胶电泳和银染色，最近结合质量 用于蛋白质鉴定的光谱法。这种方法目前在 PI的实验室，用于NCI资助的项目，其目标是分子 分析肿瘤以设计新的癌症分类方案， 用于早期癌症诊断的新蛋白质标记物的鉴定。的 标准2-方法通常允许检测1000-2000种蛋白质同种型 在组织样本中，这远远低于蛋白质同种型的数量， 包含在细胞或组织类型中。特别是，整体的二维分析 细胞裂解物从细胞区室如膜区室产生很少的蛋白质 proteins.蛋白质B的标记，生物素化已经被广泛地应用于蛋白质的生物标记。 用于选择性捕获和分析单个蛋白质。 抗原，并有可能提供一个实质性的增加， 分析整个蛋白质子集的灵敏度。初步数据 申请人小组获得的结果表明， 灵敏度和低丰度膜蛋白的产量， approach.本应用程序的目标是制定程序 系统分离、定量分析、鉴定和数据库 来自细胞和组织的生物素化膜蛋白，其利用 这种方法的敏感性增加。R21阶段的目标是 评估膜的效用。 生物素化程序， 证明生物素化的蛋白质确实代表膜蛋白; 并且它们的二维模式在细胞谱系方面提供了信息 和分化状态;如果可行，将此程序应用于 完整组织中膜的选择性生物素化。这些成就 里程碑将导致R33阶段进一步优化生物素化 程序;开发已鉴定的膜蛋白数据库 他们的表达模式，作为;资源， 研究人员;和液相色谱法的发展为基础的 分离生物素化蛋白质及其质量的方法 基于光谱法的鉴定。

DESCRIPTION: (provided by applicant) For quite some time, the standard approach for comprehensive profiling of protein expression in tissues an cell populations has been two-dimensional (2-D) gel electrophoresis and silver staining, coupled more recently with mass spectrometry for protein identification. This approach is currently in use in the PI's laboratory, fo NCI funded projects whose objectives are the molecular analysis of tumors to devise novel classification schemes of cancer, and the identification of novel protein markers for early cancer diagnosis. The standard 2- approach generally allows detection of 1000-2000 protein isoforms in a tissue sample, which is far short of the number of protein isoforms contained in a cell or tissue type. In particular, the 2-D analysis of whole cell lysates yields few proteins from cellular compartments such as membrane proteins. The tagging of proteins b, biotinylation has been extensively utilized for the selective capture and analysis of individual proteins an. antigens and has the potential for providing a substantial increment in sensitivity for the analysis of whole subsets of proteins. Preliminary data obtained by the applicant group demonstrate a remarkable increase in sensitivity and in the yield of low abundance membrane proteins using this approach. The objectives of this application are to develop procedures for the systematic separation, quantitative analysis, identification anc databasing of biotinylated membrane proteins from cells and tissues, that take advantage of the increased sensitivity of this approach. The goals of the R21 phase are to evaluate the utility of the membrane. biotinylation procedure by demonstrating that biotinylated proteins indeed represent membrane proteins; anc that their 2-D patterns are informative with respect to cellular lineage and differentiation state; and that it if feasible to apply this procedure for the selective biotinylation of membranes in intact tissue. Achievement o these milestones will lead in the R33 phase to further optimization of biotinylation procedures; to the development of a database of identified membrane proteins and of their expression patterns, to serve as ; resource for other investigators; and to the development of liquid chromatography based procedures for the separation of biotinylated proteins coupled with their mass spectrometry based identification.