Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells

eIF5-mimic protein (5MP) controls translation through binding to the ribosomal pre-initiation complex (PIC) and alters non-AUG translation rates for cancer oncogenes and repeat-expansions in neurodegenerative diseases. Here, we describe a semi-quantitative protocol for detecting 5MP-associated prote...

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Autores principales: Singh, Chingakham Ranjit, Tani, Naoki, Nakamura, Akira, Asano, Katsura
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9529597/
https://www.ncbi.nlm.nih.gov/pubmed/36181679
http://dx.doi.org/10.1016/j.xpro.2022.101739
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author Singh, Chingakham Ranjit
Tani, Naoki
Nakamura, Akira
Asano, Katsura
author_facet Singh, Chingakham Ranjit
Tani, Naoki
Nakamura, Akira
Asano, Katsura
author_sort Singh, Chingakham Ranjit
collection PubMed
description eIF5-mimic protein (5MP) controls translation through binding to the ribosomal pre-initiation complex (PIC) and alters non-AUG translation rates for cancer oncogenes and repeat-expansions in neurodegenerative diseases. Here, we describe a semi-quantitative protocol for detecting 5MP-associated proteins in cultured human and fly cells. We detail one-step anti-FLAG affinity purification and whole-lane mass spectrometry analysis of samples resolved by SDS-PAGE. This protocol allows for quantitative evaluation of the effect of 5MP mutations on its molecular interactions, to elucidate translational control by 5MP. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
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spelling pubmed-95295972022-10-05 Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells Singh, Chingakham Ranjit Tani, Naoki Nakamura, Akira Asano, Katsura STAR Protoc Protocol eIF5-mimic protein (5MP) controls translation through binding to the ribosomal pre-initiation complex (PIC) and alters non-AUG translation rates for cancer oncogenes and repeat-expansions in neurodegenerative diseases. Here, we describe a semi-quantitative protocol for detecting 5MP-associated proteins in cultured human and fly cells. We detail one-step anti-FLAG affinity purification and whole-lane mass spectrometry analysis of samples resolved by SDS-PAGE. This protocol allows for quantitative evaluation of the effect of 5MP mutations on its molecular interactions, to elucidate translational control by 5MP. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021). Elsevier 2022-09-30 /pmc/articles/PMC9529597/ /pubmed/36181679 http://dx.doi.org/10.1016/j.xpro.2022.101739 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Singh, Chingakham Ranjit
Tani, Naoki
Nakamura, Akira
Asano, Katsura
Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
title Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
title_full Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
title_fullStr Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
title_full_unstemmed Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
title_short Mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
title_sort mass spectrometry analysis of affinity-purified cytoplasmic translation initiation complexes from human and fly cells
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9529597/
https://www.ncbi.nlm.nih.gov/pubmed/36181679
http://dx.doi.org/10.1016/j.xpro.2022.101739
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