Leijie Chen, Jinyou Mo, Yuyong Tan, Liang Lv, Jia Liu
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引用次数: 0
摘要
核糖体相关蛋白质质量控制（RQC）核心因子核输出介导因子（NEMF）将C端扩展序列（CES）附加到核糖体滞留的新生链（NC）上。特定的 CES 组成可被酶直接识别，并促进 NC 的降解。然而，NEMF介导的CES在很大程度上仍未被发现。在这里，我们介绍了一种通过串联质谱（MS/MS）分析鉴定和表征线粒体NC（mitoNCs）上由NEMF介导的C端修饰的方法。我们介绍了为未知 CES 构建定制 MS/MS 图谱数据库的策略，并详细说明了 CES 修饰样品的制备步骤。有关该方案使用和执行的完整细节，请参阅 Lv 等人的文章1。
Protocol for identification of NEMF-mediated C-terminal extensions on mitochondrial nonstop proteins via customized MS/MS spectra database searching.
The ribosome-associated protein quality control (RQC) core factor nuclear export mediator factor (NEMF) appends C-terminal extended sequences (CESs) to ribosome-stalled nascent chains (NCs). Specific CESs compositions could be directly recognized by enzymes and facilitate NC degradation. Yet, NEMF-mediated CESs remains largely unidentified. Here, we present a protocol for identifying and characterizing NEMF-mediated C-terminal modifications on mitochondrial NCs (mitoNCs) via tandem mass spectrometry (MS/MS) analysis. We describe strategies aimed at constructing a customized MS/MS spectra database for unknown CESs and detail the steps for CES-modified sample preparation. For complete details on the use and execution of this protocol, please refer to Lv et al.1.
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