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Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome
Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Molecular Diversity Preservation International (MDPI)
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472765/ https://www.ncbi.nlm.nih.gov/pubmed/23109873 http://dx.doi.org/10.3390/ijms130911593 |
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author | Buneeva, Olga A. Medvedeva, Marina V. Kopylov, Arthur T. Zgoda, Victor G. Medvedev, Alexei E. |
author_facet | Buneeva, Olga A. Medvedeva, Marina V. Kopylov, Arthur T. Zgoda, Victor G. Medvedev, Alexei E. |
author_sort | Buneeva, Olga A. |
collection | PubMed |
description | Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination under norm and various brain pathologies. |
format | Online Article Text |
id | pubmed-3472765 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Molecular Diversity Preservation International (MDPI) |
record_format | MEDLINE/PubMed |
spelling | pubmed-34727652012-10-29 Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome Buneeva, Olga A. Medvedeva, Marina V. Kopylov, Arthur T. Zgoda, Victor G. Medvedev, Alexei E. Int J Mol Sci Article Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination under norm and various brain pathologies. Molecular Diversity Preservation International (MDPI) 2012-09-14 /pmc/articles/PMC3472765/ /pubmed/23109873 http://dx.doi.org/10.3390/ijms130911593 Text en © 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. http://creativecommons.org/licenses/by/3.0 This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
spellingShingle | Article Buneeva, Olga A. Medvedeva, Marina V. Kopylov, Arthur T. Zgoda, Victor G. Medvedev, Alexei E. Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome |
title | Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome |
title_full | Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome |
title_fullStr | Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome |
title_full_unstemmed | Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome |
title_short | Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome |
title_sort | use of biotinylated ubiquitin for analysis of rat brain mitochondrial proteome and interactome |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472765/ https://www.ncbi.nlm.nih.gov/pubmed/23109873 http://dx.doi.org/10.3390/ijms130911593 |
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