Validation and Quantification of FFPE Antigen Retrieval by Proteome Analysis

描述(申请人提供)：由于在组织病理学中使用福尔马林作为组织处理的标准固定剂有很长的历史，世界各地有大量的档案福尔马林固定和石蜡包埋(FFPE)组织库。这些FFPE组织收集，以及随附的临床和结果信息，为进行回溯性蛋白质生物标记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地提取蛋白质，并阻止了随后的生物分析工作打开大门，打开了隔离在档案组织库中的名副其实的信息宝库。因此，该项目旨在解决从FFPE组织中实现有效蛋白质提取的方法优化，以及对FFPE组织样本中的蛋白质表达谱进行全面和比较研究的技术开发。通过将CALIBRANT能够从微小蛋白质样本中进行蛋白质组分析的能力与南加州大学医学院的克莱夫·R·泰勒教授在抗原检索(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，拟议的研究代表着一项协同努力，以多年存档的FFPE组织收集为基础，评估和验证一种新的生物标记物发现范式。R41阶段的具体目标和各自的科学里程碑是：具体目标1.结合双子蛋白质组平台的AR方法对FFPE组织中蛋白质回收的评估和优化(1-12个月)。科学里程碑：选择一种最佳的蛋白质回收方案，用于蛋白质组研究，使用“测试电池”AR方法，并对一组10-20种蛋白质进行IHC染色。具体目标2.从FFPE组织标本中提取的抗原的确认和量化(13-24个月)。科学里程碑：展示了在每个蛋白质鉴定中平均消耗5微克总蛋白质或更少的样品，并使用AR和Gemini相结合的技术在相同的FFPE组织的三个副本中鉴定出至少3,000个高置信度蛋白质，其识别蛋白质的重复性大于80%。将尝试对一组10-20个蛋白质进行IHC结果和蛋白质组展示的直接比较。

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.