A Novel Proteomics Technology for Protein Farnesylation

描述(由申请人提供)：法尼基转移酶抑制剂(FTI)是一种新引入的抗肿瘤药物家族，目前正在进行临床评估，鉴定和表征法尼基转移酶抑制剂(FTI)治疗后法尼化蛋白水平的变化是一项重大的科学挑战。现有的蛋白质组学方法仅限于对几千种最丰富的蛋白质进行定量，因此不适用于较少丰度的法尼化表达的靶向性分析。这项应用的主要目标是开发和验证一种强大的技术，通过叠氮底物(TAS)标记，以有效分离法尼化蛋白质。然后，这项技术将被应用于生理相关模型中法尼化蛋白质的蛋白质组学分析。TAS技术包括引入合成的叠氮修饰的法尼基底物，法尼基叠氮二磷酸(FPP-叠氮化)或法尼基叠氮醇(F-叠氮化氢-OH)，在细胞蛋白质法尼化过程中取代天然底物。最终得到的法尼基-叠氮(F-叠氮化物)修饰的蛋白质将通过叠氮特定的连接反应(Staudinger反应)被亲和纯化，该反应使用连接到可光裂解的珠子上的膦捕获试剂，然后通过紫外光诱导的光裂解释放。由于亲和纯化依赖于叠氮化合物和膦捕捉剂之间的特定结合反应产生的共价键，所以其他没有F-叠氮修饰的蛋白质可以通过彻底洗涤有效地去除。因此，TAS技术将允许以高产率、高特异性和低污染的方式分离法尼化蛋白质。 本提案R21部分的最初重点是TAS技术的发展及其在法尼化蛋白质分离中的应用。这些研究将随后扩展到香叶基香叶化蛋白。R33提案旨在将TAS技术应用于识别新的快速道行动目标。这些研究将为理解FTI功能的分子机制提供基础信息，并有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.