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

基本信息

批准号：
7289831
负责人：
Satya Prakash Saxena
金额：
$ 21.96万
依托单位：
CALIBRANT BIOSYSTEMS, INC.
依托单位国家：
美国
项目类别：
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/7289831
关键词：
Address Antibodies Antigens Archives Automobile Driving Back Behavior Bioinformatics Biological Biological Markers Buffers California Classification Clinical Comparative Study Complex Condition Consumption Coupled DNA Detection Development Diagnostic Early Diagnosis Electrospray Ionization Evaluation Fingerprint Fixatives Formalin Functional disorder Furuncles Gene Expression Genetic Transcription Hand Heating Histopathology Human Genome Immunohistochemistry Individual Investigation Isotopes Knowledge Laboratories Liquid Chromatography Literature Liver Loss of Heterozygosity Malignant Neoplasms Mass Spectrum Analysis Measurement Methods Molecular Molecular Abnormality Molecular Profiling Monitor Mus Normal tissue morphology Numbers Organ Outcome Paraffin Embedding Pattern Peptides Pharmaceutical Preparations Phase Polymorphism Analysis Post-Transcriptional Regulation Post-Translational Protein Processing Preclinical Drug Evaluation Preparation Prognostic Marker Proteins Proteome Proteomics Protocols documentation Publishing Range Recording of previous events Recovery Relative (related person)Reporting Reproducibility Research Resected Resources Retrieval Roche brand of trastuzumab Sampling Score Shotguns Signal Transduction Single Nucleotide Polymorphism Solutions Specimen Spleen Staining method Stains Standardization Standards of Weights and Measures System Techniques Technology Temperature Testing Therapeutic Time Tissue Banks Tissue Sample Tissues Tumor Pathology Two-Dimensional Polyacrylamide Gel Electrophoresis Universities Validation Variant Water anticancer research base cDNA Arrays cancer type clinical Diagnosis clinical application comparative genomic hybridization crosslink link protein malignant breast neoplasm medical schools neoplastic cell novel prevent professor prognostic protein expression response sample fixation surface enhanced laser desorption ionization tissue processing tool two-dimensional

项目摘要

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）：由于将福尔马林用作组织病理学组织加工的标准固定剂的历史悠久，因此在全球范围内有大量的福尔马林固定档案和石蜡包裹（FFPE）组织库。这些FFPE组织的收集以及附着的临床和结果信息提供了宝贵的资源，用于进行回顾性蛋白质生物标志物研究。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，并防止了随后的生物分析努力，无法打开门口，直到真正的档案库中隔离的信息宝库。因此，该项目旨在解决方法学优化，以从FFPE组织中获得有效的蛋白质提取，以及技术开发，以对FFPE组织样品内的蛋白质表达谱进行全面和比较研究。通过结合校准剂的能力，可以从微小蛋白样品中启用蛋白质组学分析以及克莱夫·泰勒教授（南加州大学医学院）在抗原检索（AR） - 免疫组织化学（IHC）和肿瘤病理学中提供的技术和专业知识，提议的研究代表了尚于评估和验证的尚无定论的尚无定义的研究。存档的FFPE组织收集。 R41相和各自的科学里程碑的具体目的是：特定目标1。使用AR方法与Gemini Proteome平台相结合的AR方法评估和优化蛋白质从FFPE组织中回收（1-12个月）。科学里程碑：使用“测试电池” AR方法和IHC染色用于10-20蛋白的最佳蛋白质回收方案，可用于蛋白质组研究中。具体目标2。从FFPE组织试样检索的抗原的验证和定量（13-24个月）。科学里程碑：在每种蛋白质鉴定中平均5肽的样品消耗量为5微克总蛋白，以及使用组合AR和Gemini技术在相同的FFPE组织中相同的一式三份蛋白质中鉴定出至少3,000个具有大于80％可重复蛋白的高置信蛋白的鉴定。 IHC结果和蛋白质组学显示的直接比较将尝试使用一组10-20蛋白。