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Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking-Assisted Affinity Purification and Mass Spectrometry

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The Ubiquitin Proteasome System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1844))

Abstract

Protein-protein interactions are essential for protein complex formation and function. Affinity purification coupled with mass spectrometry (AP-MS) is the method of choice for studying protein-protein interactions at the systems level under different physiological conditions. Although effective in capturing stable protein interactions, transient, weak, and/or dynamic interactors are often lost due to extended procedures during conventional AP-MS experiments. To circumvent this problem, we have recently developed XAP (in vivo cross-linking (X)-assisted affinity purification)-MS strategy to better preserve dynamic protein complexes under native lysis conditions. In addition, we have developed XBAP (in vivo cross-linking (X)-assisted bimolecular tandem affinity purification)-MS method by incorporating XAP with bimolecular affinity purification to define dynamic and heterogeneous protein subcomplexes. Here we describe general experimental protocols of XAP- and XBAP-MS to study dynamic protein complexes and their subcomplexes, respectively. Specifically, we present their applications in capturing and identifying proteasome dynamic interactors and ubiquitin receptor (UbR)-proteasome subcomplexes.

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Acknowledgments

This work was supported by National Institutes of Health grants RO1GM074830 to L.H, R01GM106003 to L.H. and S. R.

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Authors and Affiliations

  1. Department of Physiology and Biophysics, University of California, Irvine, CA, USA

    Xiaorong Wang & Lan Huang

Authors
  1. Xiaorong Wang
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  2. Lan Huang
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Corresponding author

Correspondence to Lan Huang .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

    Thibault Mayor

  2. Department of Chemistry and Biochemistry, University of Nevada Las Vegas, Las Vegas, NV, USA

    Gary Kleiger

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Wang, X., Huang, L. (2018). Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking-Assisted Affinity Purification and Mass Spectrometry. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_25

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  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_25

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8705-4

  • Online ISBN: 978-1-4939-8706-1

  • eBook Packages: Springer Protocols

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