Proteomics

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Internally Tagged Ubiquitin: a Tool to Identify Linear Polyubiquitin-modified Proteins by Mass Spectrometry


ABSTRACT: Even though ubiquitination controls a plethora of cellular processes, modifications by linear polyubiquitin have so far only 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 substrates have been identified. The existing methods for the study of lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present a novel approach to discovering linear polyubiquitin-modified substrates by combining lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-Rex HEK293T cells and afterwards validated newly identified linear polyubiquitin targets. Moreover, we demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for the NFkappaB signalling. Overall, we have established a powerful method for the detection of linear polyubiquitin substrates.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Nicolas Nalpas  

LAB HEAD: Koraljka Husnjak

PROVIDER: PXD005878 | Pride | 2017-03-21

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
20121213_CO_0319IvDi_R08_01.raw Raw
20121213_CO_0319IvDi_R08_02.raw Raw
20121213_CO_0319IvDi_R08_03.raw Raw
20121213_CO_0319IvDi_R08_04.raw Raw
20121213_CO_0319IvDi_R08_05.raw Raw
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Publications

Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.

Kliza Katarzyna K   Taumer Christoph C   Pinzuti Irene I   Franz-Wachtel Mirita M   Kunzelmann Simone S   Stieglitz Benjamin B   Macek Boris B   Husnjak Koraljka K  

Nature methods 20170320 5


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 prote  ...[more]

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