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（由申请方提供）：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历史悠久，因此全球有大量福尔马林固定和石蜡包埋（FFPE）存档组织库。这些FFPE组织收集，以及所附的临床和结局信息，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，并阻止了随后的生物分析工作打开了通往封存在档案组织库中的名副其实的信息宝库的大门。因此，本项目旨在解决方法优化，实现有效的蛋白质提取FFPE组织连同技术开发进行全面和比较研究的蛋白质表达谱在FFPE组织标本。通过将Calibrant从微小蛋白质样品中进行蛋白质组分析的能力与克莱夫R. Taylor（南加州大学医学院）在抗原修复（AR）-免疫组织化学（IHC）和肿瘤病理学方面的研究表明，拟议的研究代表了在多年存档FFPE组织收集的基础上对新型生物标志物发现范式进行评估和验证的协同努力。R41阶段的具体目标和相应的科学里程碑是：具体目标1。使用AR方法结合Gemini蛋白质组平台评价和优化FFPE组织的蛋白质回收率（第1-12个月）。科学里程碑：选择最佳的蛋白质回收方案，用于蛋白质组研究，使用“测试组合”AR方法和10-20种蛋白质组的IHC染色。具体目标2。从FFPE组织标本中回收的抗原的验证和定量（第13-24个月）。科学里程碑：证明在每个蛋白质鉴定中平均5种肽的样品消耗为5微克总蛋白质或更少，并且使用组合的AR和Gemini技术在相同FFPE组织的三份重复样品中鉴定至少3，000种高置信度蛋白质，其中所鉴定的蛋白质的再现性大于80%。将尝试对一组10-20种蛋白质直接比较IHC结果和蛋白质组学显示。