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  Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry

Meierhofer, D., Wang, X., Huang, L., & Kaiser, P. (2008). Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. Journal of Proteome Research, 7(10), 4566-4576. doi:10.1021/pr800468j.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-808F-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-8090-A
Genre: Journal Article

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© 2008 American Chemical Society
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 Creators:
Meierhofer, David1, 2, Author              
Wang, Xiaorong, Author
Huang, Lan, Author
Kaiser, Peter2, Author
Affiliations:
1Mass Spectrometry (Head: David Meierhofer), Scientific Service (Head: Christoph Krukenkamp), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479669              
2Department of Biological Chemistry, University of California, Irvine, California 92697, USA, ou_persistent22              

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Free keywords: HB-ubiquitin • ubiquitin profiling • SILAC • MG132 • tandem affinity purification
 Abstract: Ubiquitination regulates a host of cellular processes by labeling proteins for degradation, but also by functioning as a regulatory, nonproteolytic posttranslational modification. Proteome-wide strategies to monitor changes in ubiquitination profiles are important to obtain insight into the various cellular functions of ubiquitination. Here we describe generation of stable cell lines expressing a tandem hexahistidine-biotin tag (HB-tag) fused to ubiquitin for two-step purification of the ubiquitinated proteome under fully denaturing conditions. Using this approach we identified 669 ubiquitinated proteins from HeLa cells, including 44 precise ubiquitin attachment sites on substrates and all seven possible ubiquitin chain-linkage types. To probe the dynamics of ubiquitination in response to perturbation of the ubiquitin/proteasome pathway, we combined ubiquitin profiling with quantitative mass spectrometry using the stable isotope labeling with amino acids in cell culture (SILAC) strategy. We compared untreated cells and cells treated with the proteasome inhibitor MG132 to identify ubiquitinated proteins that are targeted to the proteasome for degradation. A number of proteasome substrates were identified. In addition, the quantitative approach allowed us to compare proteasome targeting by different ubiquitin chain topologies in vivo. The tools and strategies described here can be applied to detect changes in ubiquitination dynamics in response to various changes in growth conditions and cellular stress and will contribute to our understanding of the ubiquitin/proteasome system.

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Language(s): eng - English
 Dates: 2008-09-102008-10
 Publication Status: Published in print
 Pages: -
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 Rev. Method: -
 Identifiers: DOI: 10.1021/pr800468j
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Title: Journal of Proteome Research
  Other : J. Proteome Res.
Source Genre: Journal
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Publ. Info: Washington, D.C. : American Chemical Society
Pages: - Volume / Issue: 7 (10) Sequence Number: - Start / End Page: 4566 - 4576 Identifier: ISSN: 1535-3893
CoNE: https://pure.mpg.de/cone/journals/resource/111019664290000