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Updated: Jun 12, 2025

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A
Published on: June 10, 2020
Protocol for identification of NEMF-mediated C-terminal extensions on mitochondrial nonstop proteins via customized
Leijie Chen1, Jinyou Mo2, Yuyong Tan1
1Department of Gastroenterology, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China; Center for Medical Research, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China; Clinical Research Center for Digestive Diseases in Hunan Province, Changsha, Hunan 410011, China.
Abstract:
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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