Validation and Quantification of FFPE Antigen Retrieval by Proteome Analysis

通过蛋白质组分析验证和定量 FFPE 抗原修复

• 批准号: 7137367
• 负责人: Brian M Balgley
• 金额: $ 21.96万
• 依托单位: CALIBRANT BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7137367
• 关键词: antigens; proteins; tissues

DESCRIPTION (provided by applicant): Because of the long history of the use of formalin as the standard fixative for tissue processing in histopathology, there are a large number of archival formalin-fixed and paraffin-embedded (FFPE) tissue banks worldwide. These FFPE tissue collections, with the attached clinical and outcome information, present invaluable resources for conducting retrospective protein biomarker investigations. However, the high degree of covalently cross-linked proteins in FFPE tissues hinders efficient extraction of proteins from tissue sections and prevents subsequent bioanalytical efforts from opening the door to a veritable treasure trove of information sequestered in archival tissue banks. Thus, this project aims to address methodological optimization for achieving effective protein extraction from FFPE tissues together with technological development for performing comprehensive and comparative studies of protein expression profiles within FFPE tissue specimens. By combining Calibrant's ability to enable proteomic profiling from minute protein samples with the technology and expertise offered by Professor Clive R. Taylor (University of Southern California School of Medicine) in antigen retrieval (AR)-immunohistochemistry (IHC) and tumor pathology, the proposed research represents a synergistic effort toward the evaluation and validation of a novel biomarker discovery paradigm on the basis of years of archived FFPE tissue collections. The specific aims for R41 Phase and the respective scientific milestones are: Specific Aim 1. Evaluation and optimization of protein recovery from FFPE tissues using AR approaches coupled with Gemini proteome platform (Months 1-12). Scientific Milestone: Selection of an optimal protein recovery protocol to be employed in proteome investigations using a "test battery" AR approach and IHC staining for a panel of 10-20 proteins. Specific Aim 2. Validation and quantification of antigens retrieved from FFPE tissue specimens (Months 13-24). Scientific Milestone: Demonstration of a sample consumption of 5 microgram total protein or less for an average of 5 peptides in each protein identification and the identification of at least 3,000 high confidence proteins with greater than 80% reproducibility in identified proteins among triplicates of identical FFPE tissue using combined AR and Gemini technologies. Direct comparison of IHC results and proteomic display will be attempted for a panel of 10-20 proteins.

描述（由申请人提供）：由于福尔马林作为组织病理学中组织处理的标准固定剂的使用有着悠久的历史，因此全世界有大量的档案福尔马林固定石蜡包埋（FFPE）组织库。这些 FFPE 组织集合以及附加的临床和结果信息为进行回顾性蛋白质生物标志物研究提供了宝贵的资源。然而，FFPE 组织中高度共价交联的蛋白质阻碍了从组织切片中有效提取蛋白质，并阻止后续的生物分析工作打开档案组织库中隐藏的名副其实的信息宝库的大门。因此，该项目旨在解决方法优化问题，以实现从 FFPE 组织中有效提取蛋白质，并开发技术以对 FFPE 组织样本中的蛋白质表达谱进行全面和比较研究。通过将 Calibrant 能够对微小蛋白质样品进行蛋白质组分析的能力与 Clive R. Taylor 教授（南加州大学医学院）在抗原修复 (AR)-免疫组织化学 (IHC) 和肿瘤病理学方面提供的技术和专业知识相结合，提出了该研究代表了基于多年存档的 FFPE 组织收藏的协同努力，旨在评估和验证新型生物标志物发现范式。 R41 阶段的具体目标和各自的科学里程碑是： 具体目标 1. 使用 AR 方法结合 Gemini 蛋白质组平台评估和优化 FFPE 组织的蛋白质回收率（第 1-12 个月）。科学里程碑：使用“测试电池”AR 方法和对一组 10-20 种蛋白质进行 IHC 染色，选择用于蛋白质组研究的最佳蛋白质回收方案。具体目标 2. 对从 FFPE 组织样本中回收的抗原进行验证和定量（第 13-24 个月）。科学里程碑：证明每次蛋白质鉴定中平均 5 种肽的样品消耗量为 5 微克或更少，并鉴定至少 3,000 个高置信度蛋白质，在相同 FFPE 组织的三份重复中，所鉴定蛋白质的重现性大于 80%使用 AR 和 Gemini 技术相结合。将尝试对一组 10-20 种蛋白质进行 IHC 结果和蛋白质组展示的直接比较。