A Novel Proteomics Technology for Protein Farnesylation

描述（由申请方提供）：法尼基转移酶抑制剂（FTIs）（一种新引入的抗肿瘤药物家族，目前正在进行临床评价）治疗后法尼基化蛋白水平变化的鉴别和表征代表了一项重大科学挑战。现存的蛋白质组学方法仅限于定量数千种最丰富的蛋白质，因此不适合于对丰度较低的法尼基化表达进行靶向分析。本申请的主要目标是开发和验证一种强大的技术，通过叠氮基底物（TAS）标记，用于有效分离法尼基化蛋白质。该技术将应用于生理相关模型中法尼基化蛋白质的蛋白质组学分析。 TAS技术涉及引入合成的叠氮化物修饰的法呢基底物，法呢基叠氮二磷酸（FPP-叠氮化物）或法呢基叠氮醇（F-叠氮化物-OH），其在细胞蛋白法呢基化期间替代天然底物。所得的法尼基-叠氮化物（F-叠氮化物）-修饰的蛋白质将通过叠氮化物特异性缀合反应（施陶丁格反应）使用连接至光可裂解珠的膦捕获试剂进行亲和纯化，所述光可裂解珠然后可以通过UV光诱导的光裂解释放。由于亲和纯化依赖于由叠氮化物和膦捕获试剂之间的特异性缀合反应产生的共价键合，因此可以通过彻底洗涤有效地去除没有F-叠氮化物修饰的其他蛋白质。因此，TAS技术将允许以高产率、高特异性和低污染分离法尼基化蛋白质。 该提案的R21部分的最初重点是TAS技术的开发及其在分离法尼基化蛋白质中的应用。这些研究将随后扩展到香叶基香叶基化蛋白质。R33提案旨在将TAS技术应用于识别新的FTI目标。这些研究将为理解FTI功能的分子机制提供基础信息，并可能为抗肿瘤药物设计确定新的靶点。

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.