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

基本信息

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

项目摘要

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.

分析蛋白质功能的一种普遍富有成果的方法是鉴定其相互作用伙伴。但是，到目前为止，用于分析蛋白质蛋白质相互作用的工具遭受了大量假阳性的影响。因此，确定的相互作用需要在耗时，昂贵的过程中进行验证。仅在两年前，就发布了一项新方案，允许在哺乳动物细胞培养物中高灵敏度下明确鉴定蛋白质蛋白质相互作用。该方案（称为快速）基于具有13C标记氨基酸，免疫沉淀，RNAi介导的敲低的固定氨基酸和定量质谱法的稳定的同位素标记。我们主要通过引入交联步（Quick-X），成功地将其应用于Chlamydomonas Reinhardtii，取得了迅速的进步。我们使用这种单细胞藻类作为模型生物来理解i）导致沉默翻译激活的机制，ii）叶绿体HSP70和HSP90伴侣蛋白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15NH4CL使用稳定的同位素标记进一步改善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识别新型蛋白质 - 蛋白质相互作用并监测其动力学。对这些知识的知识将为对衣原体中的转化激活和叶绿体伴侣功能的更深入的了解为基础提供了基础。