Dataset Information

E2~dID: A versatile approach to identify substrates modified with ubiquitin and ubiquitin-like molecules by specific E3 enzymes

ABSTRACT: Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to most, if not all biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we have developed an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2 conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracto. This enables purification and identification of modified proteins by mass spectrometry under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of the APC/C in human cells. Finally, performing E2~dID with SUMO in S. cerevisiae we show that E2-dID can be easily adapted to other ubiquitin-like modifiers and experimental models.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD008624 | Pride | 2019-04-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Fusion_JorgCTMT8plex_P01.raw	Raw
	Fusion_JorgCTMT8plex_P02.raw	Raw
	Fusion_JorgCTMT8plex_P03.raw	Raw
	Fusion_JorgCTMT8plex_P04.raw	Raw
	Fusion_JorgCTMT8plex_P05.raw	Raw

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Publications

An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules.

Bakos Gábor G Yu Lu L Gak Igor A IA Roumeliotis Theodoros I TI Liakopoulos Dimitris D Choudhary Jyoti S JS Mansfeld Jörg J

Nature communications 20181114 1

Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and pro ...[more]

PMID: 30429481

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