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ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

ABSTRACT: Proximity labeling coupled to mass spectrometry enables in situ mapping of protein-protein interactions. Here, we have developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and a newly generated mouse model to monitor the interactome of proteasomes in vivo. We demonstrate that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on proteasome activity. Analysis of proteins labeled by tagged proteasomes retrieved more than half of the known proteasome-interacting proteins in a single mass spectrometry analysis, including assembly factors, activators and ubiquitin-cycle related proteins. We optimized the protocol for processing of proximity labeled samples to minimize contamination from streptavidin and implemented Data Independent Acquisition (DIA) mass spectrometry for label-free analysis. We demonstrate the utility of our workflow by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling can be used to identify both endogenous and small molecule-induced proteasome substrates.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293 Cell

SUBMITTER: Emilio Cirri

LAB HEAD: Alessandro Ori

PROVIDER: PXD034965 | Pride | 2024-07-05

REPOSITORIES: Pride

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			Action	DRS
	20211227_214903_271221_AB_mTurboID_EXP_Whole_proteome_analysis_dDIA.sne	Other
	20211227_215254_271221_AB_mTurboID_EXP_Whole_proteome_analysis_dDIA_Peptide_Report.xls	Xls
	20211227_215254_271221_AB_mTurboID_EXP_Whole_proteome_analysis_dDIA_Report.xls	Xls
	211221_AB_WP_DIA_A4_mT_MG_wp_10.raw	Raw
	211221_AB_WP_DIA_A4_mT_MG_wp_13.raw	Raw

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ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

Project description:Proximity labeling coupled to mass spectrometry enables in situ mapping of protein-protein interactions. Here, we have developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and a newly generated mouse model to monitor the interactome of proteasomes in vivo. We demonstrate that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on proteasome activity. Analysis of proteins labeled by tagged proteasomes retrieved more than half of the known proteasome-interacting proteins in a single mass spectrometry analysis, including assembly factors, activators and ubiquitin-cycle related proteins. We optimized the protocol for processing of proximity labeled samples to minimize contamination from streptavidin and implemented Data Independent Acquisition (DIA) mass spectrometry for label-free analysis. We demonstrate the utility of our workflow by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling can be used to identify both endogenous and small molecule-induced proteasome substrates.

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