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 [Display omitted] •Construction of customized MS/MS database for NEMF-mediated CESs•Isolation of mitoGFPns carrying CESs via immunoprecipitation and in-gel digestion•Identification of CESs via MS/MS spectra searching Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The ribosome-associated protein quality control (RQC) core factor NEMF appends C-terminal extended sequences (CESs) to ribosome-stalled nascent chains (NCs). Specific CES compositions could be directly recognized by enzymes and facilitate NC degradation. Yet, NEMF-mediated CES 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.