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

Chingakham Ranjit Singh; Naoki Tani; Akira Nakamura; Katsura Asano

doi:10.1016/j.xpro.2022.101739

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

Chingakham Ranjit Singh
, Naoki Tani
, Akira Nakamura
, Katsura Asano

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

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).

Original language	English (US)
Journal	STAR Protocols
DOIs	https://doi.org/10.1016/j.xpro.2022.101739
State	Published - Dec 16 2022

All Science Journal Classification (ASJC) codes

General Neuroscience
General Immunology and Microbiology
General Biochemistry, Genetics and Molecular Biology

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10.1016/j.xpro.2022.101739

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

abstract = "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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doi = "10.1016/j.xpro.2022.101739",

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AU - Singh, Chingakham Ranjit

AU - Tani, Naoki

AU - Nakamura, Akira

AU - Asano, Katsura

PY - 2022/12/16

Y1 - 2022/12/16

N2 - 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).

AB - 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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UR - https://www.scopus.com/pages/publications/85139039626#tab=citedBy

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DO - 10.1016/j.xpro.2022.101739

M3 - Article

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AN - SCOPUS:85139039626

SN - 2666-1667

JO - STAR Protocols

JF - STAR Protocols

ER -

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

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