Photocleavable ICAT Reagents for Quantitative Proteomics

用于定量蛋白质组学的光裂解 ICAT 试剂

• 批准号: 7216411
• 负责人: Mark Lim
• 金额: $ 37.45万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7216411
• 关键词: Affinity; Amines; Area; Avidin; Benchmarking; Binding; Biological Markers; Biological Models; Biotin; Buffers; Cell Line; Cells; Condition; Development; Diagnostic; Digestion; Disease; Elements; Evaluation; Exhibits; Family; Goals; Iodoacetamide; Isotopically-Coded Affinity Tagging; Label; Light; Maleimides; Malignant - descriptor; Mass Spectrum Analysis; Measures; Methods; Modeling; Molecular Profiling; Monitor; Normal Horse Serum; Numbers; PC3 cell line; Peptides; Performance; Phase; Procedures; Prostate; Proteins; Proteome; Proteomics; Purpose; Reagent; Recovery; Relative (related person); Research; Sampling; Series; Solid; Speed; Streptavidin; Surveys; TimeLine; Tissues; Two-Dimensional Gel Electrophoresis; base; clinical application; design; neoplastic; novel; novel strategies; protein expression; tool; user-friendly

DESCRIPTION (provided by applicant): Protein expression profiling is becoming one of the most attractive tools for monitoring the course of neoplastic disease as well as identifying diagnostic biomarkers associated with the disease. While several proteomic tools have been extensively investigated for this purpose, including two-dimensional gel electrophoresis and mass spectrometry, quantitative proteomics is still difficult to achieve due to the limited accuracy and inability of these methods to survey the entire proteome. The goal of this project is to overcome these limitations by developing a new approach to quantitative proteomics based on the use of PhotoCleavable Isotope Coded Affinity Tags (PC-ICAT) along with mass spectrometry. PC-ICAT reagents are composed of four distinct elements: a biotin affinity tag, a photodeavable linker which exhibits rapid and efficient photocleavage, a variable mass linker and a chemically reactive group. The light and heavy versions of the PC-ICAT reagents are used to tag two different samples, for example extracts from normal and disease related tissues. After digestion, capture and photorelease, the relative abundance of the tagged peptides in each sample is measured using LC-MS. During Phase I of this project, a sulfhydryl reactive PC-ICAT maleimide reagent has been synthesized and extensively evaluated using both model peptides and proteins. Significantly, the performance of PC-ICAT exhibits superior performance compared with the conventional (CAT reagent. During Phase II, we will extend the usefulness of PC-ICAT in proteomic research and clinical applications by: 1) synthesizing and evaluating an amine reactive version of the PC-ICAT reagent, 2) evaluating a solid supported PC-ICAT reagent and 3) exploring the use of PC-ICAT reagents for protein expression profiling and biomarker discovery. For this purpose, we will apply PC-ICAT reagents and LC-MS to study cell lysates derived from normal and malignant human prostate cell lines. Dr. Cathy Costello, an expert in the area of mass spectrometry and proteomics will serve as a consultant during Phase II of this project.

描述（由申请人提供）：蛋白质表达谱正在成为监测肿瘤疾病过程以及鉴定与疾病相关的诊断生物标志物的最有吸引力的工具之一。虽然已经为此目的广泛研究了几种蛋白质组学工具，包括二维凝胶电泳和质谱，但由于这些方法的准确性有限且无法调查整个蛋白质组，定量蛋白质组学仍然难以实现。本项目的目标是克服这些限制，开发一种新的方法，定量蛋白质组学的基础上使用的光裂解同位素编码的亲和标签（PC-ICAT）沿着与质谱。PC-ICAT试剂由四种不同的元素组成：生物素亲和标签、表现出快速和有效光切割的可光切割接头、可变质量接头和化学反应基团。轻和重版本的PC-ICAT试剂用于标记两种不同的样品，例如来自正常和疾病相关组织的提取物。消化，捕获和光释放后，在每个样品中的标记肽的相对丰度测量使用LC-MS。在第一阶段的这个项目中，巯基反应PC-ICAT马来酰亚胺试剂已合成和广泛的评估使用模型肽和蛋白质。值得注意的是，与常规的（CAT试剂相比，PC-ICAT的性能表现出上级性能。在第二阶段，我们将通过以下方式扩展PC-ICAT在蛋白质组学研究和临床应用中的有用性：1）合成和评估PC-ICAT试剂的胺反应型，2）评估固体支持的PC-ICAT试剂，3）探索PC-ICAT试剂在蛋白质表达谱分析和生物标志物发现中的用途。为此，我们将应用PC-ICAT试剂和LC-MS来研究来自正常和恶性人前列腺细胞系的细胞裂解物。Cathy Costello博士是质谱和蛋白质组学领域的专家，将在该项目的第二阶段担任顾问。