Photocleavable ICAT Reagents for Quantitative Proteomics

描述（申请人提供）：蛋白质组学将对疾病的诊断、治疗和监测产生深远的影响。蛋白表达谱是肿瘤病程中可监测的重要参数之一。虽然已经开发了几种快速高效的蛋白质组学工具，如二维凝胶电泳和质谱分析，但定量蛋白质组学有许多局限性，不能广泛应用。最近引入的同位素编码亲和标签（ICAT）技术，当与质谱结合使用时，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技术存在一些现有的限制，包括有限的准确性和蛋白质组覆盖。本提案的目的是通过基于AmberGen专有的pc -生物素开发一类新的光可切割ICAT （PC-ICAT）试剂来克服这些限制。这些新型pc - icat由四个不同的元素组成：生物素亲和标签，具有快速和有效的光裂解能力的光裂解连接体，稳定同位素标记的可变质量连接体和活性基团。在第一阶段，将合成几种具有不同反应基团的PC-ICAT试剂，并使用模型肽、肽混合物、蛋白质和细胞裂解物进行评估。新型PC-ICAT试剂的性能将与传统的ICAT试剂进行比较。在评估过程中，将比较几个关键参数，包括定量精度和总体灵敏度。在II期，性能最好的PC-ICAT试剂将被进一步优化和评估，用于在各种正常和恶性细胞裂解物中发现生物标志物。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博士将担任该项目的所有阶段的顾问。

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.