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

• Published in: STAR protocols 2022-12, Vol.3 (4), p.101739-101739, Article 101739
• Main Authors: Singh, Chingakham Ranjit, Tani, Naoki, Nakamura, Akira, Asano, Katsura
• Format: Article
• Language: English
• Subjects: Bioinformatics; Biotechnology and bioengineering; Cell culture; Cytosol - metabolism; Humans; Mass Spectrometry; Protein biochemistry; Protein Biosynthesis; Protein expression and purification; Proteins - metabolism; Protocol; Ribosomes - metabolism
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101739

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). [Display omitted] •Anti-FLAG-affinity purification of translation initiation complex via 5MP•Purification of proteins from human and fly cells after simple transfection procedures•Whole-lane mass spectrometry to analyze affinity-purified proteins•emPAI as a proxy for molecular amounts of identified proteins Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.