Platform for High-Throughput Analysis of Protein Adducts for Carcinogen Exposure

致癌物暴露蛋白质加合物高通量分析平台

• 批准号: 8079184
• 负责人: Harry Bermudez
• 金额: $ 20.5万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-20 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079184
• 关键词: Affinity; Amino Acid Sequence; Antibodies; Aromatic Amines; Bacteriophages; Binding; Biological Assay; Biological Markers; Blood Proteins; Blood specimen; Carcinogen exposure; Carcinogenesis Mechanism; Carcinogens; Cell Respiration; Chemicals; Clinical; DNA; DNA Adduction; DNA Adducts; DNA Modification Process; Data; Development; Diagnostic; Environmental Carcinogens; Enzyme-Linked Immunosorbent Assay; Epidemiology; Exposure to; Funding; Globin; Goals; Health; Human; In Vitro; Individual; Investigation; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediator of activation protein; Methods; Methylation; Microarray Analysis; Modeling; Modification; Mutation; Peptides; Persons; Phage Display; Phase; Pilot Projects; Post-Translational Protein Processing; Process; Proteins; Reaction; Risk Assessment; Role; Screening procedure; Sensitivity and Specificity; Serum Albumin; Site; Specificity; Specimen; Structure; System; Technology; Treatment Efficacy; Tumor Promotion; Validation; Xenobiotics; adduct; base; cancer therapy; carcinogenesis; chemical reaction; chemical synthesis; clinical Diagnosis; combinatorial; detector; high throughput analysis; high throughput technology; human subject; in vivo; innovation; macromolecule; satisfaction; scaffold; tool

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a high-throughput technology that can characterize covalent protein and DNA modifications ("adducts") induced by reactive carcinogens on a global basis with high sensitivity and specificity. Virtually all carcinogens, even the most inert, either contain an electrophilic center or undergo some level of oxidative metabolism to form reactive electrophiles. DNA and protein adducts are the products of the chemical reaction of such electrophiles with nucleophilic sites within these macromolecules. DNA adduction (including methylation and oxidative changes) has been widely implicated in mutation and carcinogenesis, while protein adducts (i.e., post-translational modifications) may have indirect roles in the mechanism of carcinogenesis if they involve DNA-related proteins or increase the rate of tumor promotion. In addition, such adducts are being explored as short- term, long-term, and/or cumulative markers of exposure to carcinogens. Current methods of adduct quantitation are expensive, highly chemical-specific, and labor-intensive. A technology for rapid and comprehensive profiling of macromolecular adduction in human subjects would be an important tool for carcinogen exposure and health risk assessment. This exploratory/developmental application proposes a proof-of-concept study to adapt three recently developed technologies, i.e., combinatorial chemical synthesis, antibody phage display, and microarray technology in a system to rapidly profile blood protein adducts of two important classes of carcinogens. Libraries of adducted peptides will be created to mimic structures formed in vivo in humans exposed to carcinogens. Adduct-specific scFv probes will be selected by screening with phage display libraries and validated with human blood specimens in an ELISA system. Results of ELISA-based adduct quantitation will be compared to those from conventional mass spectrometry-based analysis of blood protein adducts. If the proposed proof-of-concept studies are successful, expanded funding will be sought to develop a microarray-based global adduct detector for screening blood specimens from individual subjects. It is anticipated that the technology developed in this project will have wide applications for carcinogen exposure assessment, biomarker discovery, clinical diagnosis, and assessment of cancer treatment efficacies. PUBLIC HEALTH RELEVANCE: Many environmental carcinogens are able to chemically react with proteins and DNA in the body to form products called "adducts". These reaction products can be critical to the occurrence of cancer, or, alternatively, they can provide information on a person's exposure to these carcinogens. We propose to develop a rapid, reliable system to detect and measure these adducts in human specimens on a global basis, a technology that would be an important tool for carcinogen exposure assessment, clinical diagnosis, and assessment of cancer treatment efficacies.

描述（由申请人提供）：该项目的长期目标是开发一种高通量技术，该技术可以表征由反应性致癌物在全球基础上以高灵敏度和特异性为基础引起的共价蛋白质和DNA修饰（“加合物”）。几乎所有的致癌物，即使是最惰性的，要么都包含一个亲电中心，要么经历一定程度的氧化代谢以形成反应性电力。 DNA和蛋白质加合物是这些大分子内这种电物质与亲核位点的化学反应产物。 DNA内收（包括甲基化和氧化变化）已广泛与突变和致癌作用有关，而蛋白质加合物（即翻译后修饰）如果涉及DNA相关蛋白或增加肿瘤促进速率的癌变机制，可能在癌变机制中具有间接的作用。此外，这种加合物正在被探讨为接触致癌物的短期，长期和/或累积标记。当前的加合物定量方法昂贵，高度化学特异性且劳动密集型。用于快速，全面分析人类受试者大分子内收的技术将是致癌物暴露和健康风险评估的重要工具。该探索性/发育应用提出了一项概念验证研究，以适应三种最近开发的技术，即组合化学合成，抗体噬菌体显示和系统中的微阵列技术，以迅速介绍两种重要类癌症类的血液蛋白质加合物。加合肽的库将被创建，以模仿暴露于致癌物的人体中形成​​的模拟结构。将通过使用噬菌体显示库进行筛选，并用ELISA系统中的人体样本验证加合物特异性的SCFV探针。基于ELISA的加合物定量的结果将与传统质谱分析的血液蛋白加合物分析进行比较。如果拟议的概念验证研究成功，将寻求扩大的资金来开发基于微阵列的全球加合物检测器，以筛查各个受试者的血液标本。预计该项目中开发的技术将在致癌物的评估，生物标志物发现，临床诊断和癌症治疗效率评估中有广泛的应用。 公共卫生相关性：许多环境致癌物能够与体内的蛋白质和DNA化学反应，形成称为“加合物”的产品。这些反应产物对于癌症的发生至关重要，或者，它们可以提供有关某人接触这些致癌物的信息。我们建议开发一个快速，可靠的系统，以在全球范围内检测和测量人类标本中的这些加合物，该技术将是致癌物评估，临床诊断和癌症治疗效率评估的重要工具。