Chapter title |
Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking-Assisted Affinity Purification and Mass Spectrometry
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Chapter number | 25 |
Book title |
The Ubiquitin Proteasome System
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Published in |
Methods in molecular biology, September 2018
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DOI | 10.1007/978-1-4939-8706-1_25 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8705-4, 978-1-4939-8706-1
|
Authors |
Xiaorong Wang, Lan Huang, Wang, Xiaorong, Huang, Lan |
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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Other | 1 | 8% |
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