Monitoring mitochondrial translation by pulse SILAC

Imami, Koshi; Selbach, Matthias; Ishihama, Yasushi

doi:10.1016/j.jbc.2022.102865

Cited by 7 publications

(2 citation statements)

References 73 publications

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“…On the other hand, trypsin alone can be a limiting factor in peptide-centric proteomics, and thus multiple proteases, such as AspN, CluC, LysN and chymotrypsin, have been explored to expand the peptide sequence coverage within proteins ( 56 ) . In particular, chymotrypsin cleaves peptide bonds at the C-termini of hydrophobic amino acids (Tyr, Phe, Trp and Leu) and therefore is effective for the identification of membrane proteins ( 57 ) . Indeed, Tsumgari et al demonstrated that the combination of three digestion patterns (trypsin/LysC, GluC, chymotrypsin) boosted the identification of N -myristoylated peptides approximately 1.4-fold in comparison to trypsin/LysC alone ( 20 ) .…”

Section: Global Analysis Of Endogenous Lipidated Peptides/sitesmentioning

confidence: 99%

Exploring protein lipidation by mass spectrometry-based proteomics

Tsumagari,

Isobe,

Imami

et al. 2023

The Journal of Biochemistry

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Protein lipidation is a common co- or post-translational modification that plays a crucial role in regulating the localization, interaction, and function of cellular proteins. Dysregulation of lipid modifications can lead to various diseases, including cancer, neurodegenerative diseases, and infectious diseases. Therefore, the identification of proteins undergoing lipidation and their lipidation sites should provide insights into many aspects of lipid biology, as well as providing potential targets for therapeutic strategies. Bottom-up proteomics using liquid chromatography/tandem mass spectrometry is a powerful technique for the global analysis of protein lipidation. Here, we review proteomic methods for profiling protein lipidation, focusing on the two major approaches: the use of chemical probes, such as lipid alkyne probes, and the use of enrichment techniques for endogenous lipid-modified peptides. The challenges facing these methods and the prospects for developing them further to achieve a comprehensive analysis of lipid modifications are discussed.

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Section: Global Analysis Of Endogenous Lipidated Peptides/sitesmentioning

confidence: 99%

Exploring protein lipidation by mass spectrometry-based proteomics

Tsumagari,

Isobe,

Imami

et al. 2023

The Journal of Biochemistry

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“…For mass spectrometry analysis, beads were washed twice using buffer D (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 15 mM MgCl2) after washing with buffer B. We followed the original protocol (Imami et al 2018(Imami et al , 2023 with some modifications. Beads were resuspended in PTS buffer (12 mM sodium deoxycholate (SDC), 12 mM sodium Nlauroylsarcosinate (SLS) in 100 mM Tris-HCl pH 8.0) followed by incubation for 30 min at 37°C under constant agitation.…”

Section: Immunoprecipitationmentioning

confidence: 99%

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development

Ugajin,

Imami,

Takada

et al. 2023

RNA

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Ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ribosome ubiquitination is crucial for translational regulation and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To explore ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ribosomal protein Rpl36/eL36 from its endogenous locus. We examined ribosome ubiquitination during zebrafish development by combining affinity purification of ribosomes from rpl36-FLAG zebrafish embryos with immunoblotting analysis. Our findings showed that ubiquitination of ribosomal proteins dynamically changed as development proceeded. We also showed that during zebrafish development, the ribosome was ubiquitinated by Znf598, an E3 ubiquitin ligase that activates RQC. Ribosomal protein Rps10/eS10 was found to be a key ubiquitinated protein during development. Furthermore, we showed that Rps10/eS10 ubiquitination-site mutations reduced the overall ubiquitination pattern of ribosome. These results demonstrate the complexity and dynamics of ribosome ubiquitination during zebrafish development.

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