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: 6742863
• 负责人: JERZY OLEJNIK
• 金额: $ 10万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742863
• 关键词: binding proteins; biotechnology; biotin; chemical synthesis; mass spectrometry; protein quantitation /detection; proteomics; reagent /indicator; stable isotope; technology /technique development; ultraviolet radiation

DESCRIPTION (provided by applicant): Proteomics is set to have a profound impact on disease diagnosis, treatment and monitoring. Protein expression profiling is one of the important parameters that can be monitored in the course of neoplastic disease. While several fast and efficient proteomic tools have been developed for protein identification, such as two-dimensional gel electrophoresis and mass spectrometry, quantitative proteomics has many limitations and is not widely applicable. The recently introduced Istotope Coded Affinity Tags (ICAT) technique, which when used in conjunction with mass spectrometry allows changes in the abundance of specific proteins in a complex mixture to be identified and monitored. However, there are several existing limitations to the ICAT technology including limited accuracy and proteome coverage. The aim of this proposal is to overcome these limitations by developing a new class of PhotoCleavable ICAT (PC-ICAT) reagents based on AmberGen's proprietary PC-biotins. These novel PC-ICATs are composed of four distinct elements: a biotin affinity tag, a photocleavable linker, which exhibits rapid and efficient photocleavage, a variable mass linker labeled with stable isotopes and a reactive group. During Phase I, several PC-ICAT reagents with various reactive groups will be synthesized and evaluated using model peptides, peptide mixtures, proteins and cell lysates. Performance of novel PC-ICAT reagents will be compared to conventional ICAT reagents. During evaluation, several critical parameters will be compared, including quantitation accuracy and overall sensitivity. During Phase II, the best performing PC-ICAT reagents will be further optimized and evaluated for the discovery of biomarkers in a variety of normal and malignant cell lysates. Quantitative proteomic kits for research and clinical markets will be developed. Dr. Steven Gygi, one of the developers of ICAT approach, will serve as a consultant on all Phases of this project.

描述（由申请人提供）：蛋白质组学将对疾病诊断、治疗和监测产生深远影响。蛋白质表达谱是肿瘤疾病过程中可监测的重要参数之一。虽然已经开发了几种快速有效的蛋白质组学工具用于蛋白质鉴定，例如二维凝胶电泳和质谱，但定量蛋白质组学具有许多局限性并且不广泛适用。最近引入的Istotope编码亲和标签（ICAT）技术，当与质谱法结合使用时，可以识别和监测复杂混合物中特定蛋白质丰度的变化。然而，ICAT技术存在几个现有的限制，包括有限的准确性和蛋白质组覆盖。该提案的目的是通过开发一类基于AmberGen专有PC-生物素的新型光可裂解ICAT（PC-ICAT）试剂来克服这些限制。这些新的PC-ICAT由四个不同的元素组成：生物素亲和标签，可光裂解的接头，其表现出快速和有效的光裂解，可变质量接头标记的稳定同位素和反应基团。在第一阶段，将使用模型肽、肽混合物、蛋白质和细胞裂解物合成和评价具有各种反应基团的几种PC-ICAT试剂。将新型PC-ICAT试剂的性能与常规ICAT试剂进行比较。在评价期间，将比较几个关键参数，包括定量准确度和总体灵敏度。在第二阶段，性能最好的PC-ICAT试剂将进一步优化和评估，以发现各种正常和恶性细胞裂解物中的生物标志物。将开发用于研究和临床市场的定量蛋白质组学试剂盒。ICAT方法的开发者之一Steven Gygi博士将担任该项目所有阶段的顾问。