A Novel Proteomics Technology for Protein Farnesylation

蛋白质法呢基化的新型蛋白质组学技术

基本信息

批准号：
6785160
负责人：
YINGMING ZHAO
金额：
$ 14.04万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-07 至 2006-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6785160
关键词：
affinity chromatography alkyltransferase antineoplastics azides biotechnology cell line chemical cleavage chemical synthesis clinical research enzyme inhibitors mass spectrometry phosphines prenylation protein purification protein quantitation /detection proteomics technology /technique development

项目摘要

DESCRIPTION (provided by applicant): The identification and characterization of changes in the level of farnesylated proteins in response to the treatment of farnesyltransferase inhibitors (FTIs), a newly introduced family of antitumor agents currently undergoing clinical evaluation, represents a major scientific challenge. Extant proteomics methods are limited to quantifying a few thousands of the most abundant proteins and, therefore, are unsuitable for the targeted profiling of less abundant farnesylated expression. The major goal of this application is to develop and validate a powerful technology, Tagging via Azido Substrate (TAS), for the efficient isolation of farnesylated proteins. This technology will then be applied to the proteomics analysis of farnesylated proteins in physiologically relevant models. The TAS technology involves the introduction of a synthetic azide-modified farnesyl substrate, either farnesyl azide diphosphate (FPP-azide) or farnesyl azide alcohol (F-azide-OH), which replaces the natural substrate during cellular protein farnesylation. The resulting farnesyl-azide (F-azide)-modified proteins will be affinity-purified through an azide-specific conjugation reaction (Staudinger reaction) using a phosphine capture reagent linked to photo-cleavable beads, which can then be released by UV light-induced photo-cleavage. Since affinity purification relies on covalent bonding resulting from a specific conjugation reaction between an azide and phosphine capture reagent, other proteins without the F-azide modification can be effectively removed by thorough washing. Thus, the TAS technology will allow farnesylated proteins to be isolated with high yield, high specificity, and low contamination. The initial focus of the R21 portion of this proposal is on the development of TAS technology and its application to the isolation of farnesylated proteins. These studies will be extended subsequently to geranylgeranylated proteins. The R33 proposal will aim at applications of the TAS technology to the identification of novel FTI targets. These studies will provide fundamental information for the understanding of molecular mechanisms of FTI functions and are likely to identify novel targets for antitumor drug design.

描述（由申请人提供）：鉴定和表征Farnesylsylansferase抑制剂（FTIS）（FTIS）的鉴定和表征，这是一种新引入的抗肿瘤药物，目前正在接受临床评估，这是一项重大科学挑战。现有的蛋白质组学方法仅限于量化数千种最丰富的蛋白质，因此不适合针对较少丰富的法尼化表达的靶向分析。该应用程序的主要目的是开发和验证通过Azido底物（TAS）标记的强大技术，以有效地隔离法尼蛋白。然后，该技术将应用于生理相关模型中Farnesylation蛋白的蛋白质组学分析。 TAS技术涉及引入合成叠氮化物修饰的Farnesyl底物，即Farnesyl叠氮化物二磷酸（FPP-氮杂）或Farnesyl叠氮化物酒精（F-azide-OH），该醇（F-azide-OH）取代了在farnesylation farnesylation过程中的天然底物。所得的Farnesyl-Azide（F-氮化酶）修饰的蛋白将通过叠氮化物特异性共轭反应（Staudinger反应）使用磷蛋白捕获的试剂链接到与照片可裂解的珠相关，然后可以通过UV光诱导的光诱导的照片释放。由于亲和力纯化依赖于叠氮化物和磷酸捕获试剂之间特定的共轭反应产生的共价键，因此可以通过彻底洗涤有效去除其他没有F-氮化量的蛋白质。因此，TAS技术将允许以高产量，高特异性和低污染物分离出法尼基化的蛋白质。该提案的R21部分的最初重点是TAS技术的开发及其在隔离Farnesyal化蛋白上的应用。这些研究将随后扩展到黄烷基凝聚酰化蛋白。 R33提案将旨在将TAS技术应用于新型FTI目标的识别。这些研究将为理解FTI功能的分子机制提供基本信息，并可能确定抗肿瘤药物设计的新靶标。