Journal
NATURE METHODS
Volume 14, Issue 5, Pages 504-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.4228
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Funding
- UPStream grant (EU) [290257]
- Cancer Research UK
- The Francis Crick Institute [10015] Funding Source: researchfish
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Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INTUb.7KR) with SILAC-based mass spectrometry. We applied our approach in TNF alpha-stimulated T-REx HEK293T cells and validated several newly identified linear polyubiquitin targets. We demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for NF kappa B signaling.
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