Analysis of dynamic protein-protein interactions in Chlamydomonas using QUICK-X, a novel quantitative mass spectrometry-based approach

使用 QUICK-X（一种基于定量质谱的新型方法）分析衣藻中的动态蛋白质-蛋白质相互作用

• 批准号: 135884105
• 负责人: Professor Dr. Michael Schroda
• 金额: --
• 依托单位: Fachgebiet Molekulare Biotechnologie und Systembiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/135884105?language=en
• 关键词: Analysis; dynamic; protein; interactions; Chlamydomonas

A generally fruitful approach towards the analysis of a protein’s function is the identification of its interaction partners. However, tools employed until now for analysing protein-protein interactions suffer from the high number of false positives. Hence, interactions identified need to be verified in a time-consuming, costly process. Only two years ago, a new protocol was published allowing for the unequivocal identification of protein-protein interactions at high sensitivity in mammalian cell cultures. This protocol - termed QUICK - is based on stable isotope labeling of cell cultures with 13C-labeled amino acids, immunoprecipitation, RNAi-mediated knock-down, and quantitative mass spectrometry. We have improved QUICK mainly by introducing a crosslinking step (QUICK-X) and have successfully applied it to Chlamydomonas reinhardtii. We use this unicellular alga as model organism to understand i) the mechanisms leading to the activation of silenced transgenes and ii) the roles of the chloroplast HSP70 and HSP90 chaperone systems in chloroplast biogenesis and signalling. We propose first to further improve QUICK-X by using stable isotope labeling with 15NH4Cl. Second, we want to expose Chlamydomonas to various environmental conditions and, using QUICK-X, identify novel protein-protein interactions and monitor their dynamics. Knowledge of these will provide the fundament for a deeper understanding of transgene activation and chloroplast chaperone function in Chlamydomonas.

一个通常富有成效的方法来分析蛋白质的功能是确定其相互作用的合作伙伴。然而，迄今为止用于分析蛋白质-蛋白质相互作用的工具遭受大量假阳性。因此，需要在一个耗时、费钱的过程中核实已查明的相互作用。仅在两年前，一项新的方案发表，允许在哺乳动物细胞培养中以高灵敏度明确鉴定蛋白质-蛋白质相互作用。该方案-称为QUICK -基于用13 C标记的氨基酸对细胞培养物进行稳定同位素标记、免疫沉淀、RNAi介导的敲低和定量质谱。我们主要通过引入交联步骤（QUICK-X）改进了QUICK，并成功地将其应用于莱茵衣藻。我们使用这种单细胞拟南芥作为模式生物来理解i）导致沉默的转基因激活的机制和ii）叶绿体HSP 70和HSP 90伴侣系统在叶绿体生物发生和信号传导中的作用。我们首先建议通过使用15 NH 4Cl的稳定同位素标记来进一步改进QUICK-X。其次，我们希望将衣原体暴露于各种环境条件下，并使用QUICK-X，识别新的蛋白质-蛋白质相互作用并监测其动态。这些知识将为深入了解衣原体的转基因激活和叶绿体伴侣功能提供基础。