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Analysis of Ubiquitinated Proteome by Quantitative Mass Spectrometry

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Book cover Quantitative Methods in Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 893))

Abstract

Protein modification by ubiquitin (Ub) is one of the most common posttranslational events in eukaryotic cells. Ubiquitinated proteins are destined to various fates such as proteasomal degradation, protein trafficking, DNA repair, and immune response. In the last decade, vast improvements of mass spectrometry make it feasible to analyze the minute amount of ubiquitinated components in vivo. When combined with quantitative strategies, such as stable isotope labeling with amino acids in cell culture (SILAC), it is capable of profiling ubiquitinated proteome under different experimental conditions. Here, we describe a procedure to perform such a study, including differential protein labeling by the SILAC method, enrichment of ubiquitinated species, mass spectrometric analysis, and quality control to reduce false positives. The potential challenges and limitations of the procedure are also discussed.

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Acknowledgements

This work was partially supported by the National Institutes of Health grants (RR025822, and NS055077), and the American Cancer Society grant (RSG-09-181).

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Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Chan Hyun Na & Junmin Peng

  2. Department Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Chan Hyun Na & Junmin Peng

Authors
  1. Chan Hyun Na
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  2. Junmin Peng
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Corresponding author

Correspondence to Junmin Peng .

Editor information

Editors and Affiliations

  1. Fak. Medizin, Medizinische Proteom Center, Universität Bochum, Universitätsstraße 150, Bochum, 44801, Germany

    Katrin Marcus

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Na, C.H., Peng, J. (2012). Analysis of Ubiquitinated Proteome by Quantitative Mass Spectrometry. In: Marcus, K. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 893. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-885-6_26

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  • DOI: https://doi.org/10.1007/978-1-61779-885-6_26

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-884-9

  • Online ISBN: 978-1-61779-885-6

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