Mapping Cellular Microenvironments – Proximity Labeling/MS within a Campus Core Facility Structure (GoeCoOp-PL/MS)

绘制细胞微环境 â 园区核心设施结构内的邻近标签/MS (GoeCoOp-PL/MS)

• 批准号: 502174128
• 金额: --
• 依托单位: Georg-August-Universität Göttingen
• 依托单位国家: 德国
• 项目类别: Core Facilities
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/502174128?language=en
• 关键词: Mapping; Cellular; Microenvironments; Proximity; Labeling

Proximity-dependent labeling with biotin in living cells followed by mass spectrometry analysis (PL/MS) advances to a key technology to study protein-protein interactions and molecular microenvironments (called Biotin IDentification, BioID). Genetically engineered biotin ligases and split variants thereof (Split-BioID) with improved versatility and speed are currently developed to enable the analysis of context-specific and short-lived protein-protein interactions. Demanding quantitative mass spectrometry approaches that consider appropriate control cell lines and that apply proven workflows for MS data analysis are prerequisites for high quality results generated with this methodology. The Core Facility Protein Analytics (CF-PA) did pioneering work with PL/MS in Saccharomyces cerevisiae resulting in a model study for the proximity labeling of a ribosomal microenvironment. Currently the application of this methodology is broadened within the CF-PA to further fungal and microbial organisms and plants. An open PL/MS service module named GoeCoOp-PL/MS will be provided on the basis of close collaboration with Campus colleagues, e.g. members of the Goettingen Proteomics Forum (GPF), and external scientists to boost the local expertise for this fundamental technology. Furthermore, the possibilities of proximity labeling of metabolites with biotin and MS analysis will be studied and tested for routine applicability.

在活细胞中用生物素进行邻近依赖性标记，然后进行质谱分析（PL/MS），这是研究蛋白质-蛋白质相互作用和分子微环境的关键技术（称为生物素鉴定，BioID）。目前开发了具有改进的通用性和速度的基因工程生物素连接酶及其分裂变体（Split-BioID），以能够分析背景特异性和短寿命的蛋白质-蛋白质相互作用。考虑适当的对照细胞系并将成熟的工作流程应用于MS数据分析的高要求定量质谱方法是使用该方法生成高质量结果的先决条件。核心设施蛋白质分析（CF-PA）在酿酒酵母中进行了PL/MS的开创性工作，从而为核糖体微环境的邻近标记进行了模型研究。目前，这种方法的应用在CF-PA中被扩大到进一步的真菌和微生物有机体和植物。一个名为GoeCoOp-PL/MS的开放式PL/MS服务模块将在与校园同事密切合作的基础上提供，例如哥廷根蛋白质组学论坛（GPF）的成员和外部科学家，以提高这一基础技术的本地专业知识。此外，还将研究用生物素和MS分析对代谢物进行邻近标记的可能性，并检测其常规适用性。