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

STAR Protoc. 2022 Dec 16;3(4):101739. doi: 10.1016/j.xpro.2022.101739. Epub 2022 Sep 30.

Authors

Chingakham Ranjit Singh¹, Naoki Tani², Akira Nakamura³, Katsura Asano⁴

Affiliations

¹ Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. Electronic address: crs6361@psu.edu.
² Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan. Electronic address: naotani@kumamoto-u.ac.jp.
³ Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan. Electronic address: akiran@kumamoto-u.ac.jp.
⁴ Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan; Hiroshima Research Center for Healthy Aging, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan. Electronic address: kasano@ksu.edu.

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).

Keywords: Bioinformatics; Biotechnology and bioengineering; Cell culture; Mass spectrometry; Protein biochemistry; Protein expression and purification.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cytosol / metabolism
Humans
Mass Spectrometry
Protein Biosynthesis*
Proteins / metabolism
Ribosomes* / metabolism

Substances

Proteins

Grants and funding

P20 GM103418/GM/NIGMS NIH HHS/United States