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Use of biotinylated ubiquitin for analysis of rat brain mitochondrial proteome and interactome.

Buneeva OA, Medvedeva MV, Kopylov AT, Zgoda VG, Medvedev AE - Int J Mol Sci (2012)

Bottom Line: Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated.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.

View Article: PubMed Central - PubMed

Affiliation: Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 10 Pogodinskaya street, Moscow 119121, Russia; E-Mails: olbun@yandex.ru (O.A.B.); a.t.kopylov@gmail.com (A.T.K.); vic@ibmh.msk.su (V.G.Z.).

ABSTRACT
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.

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SDS-PAGE of solution of biotinylated ubiquitin before (tracks 2, 4, 6) and after incubation (tracks 3, 5, 7) with avidin-agarose. 1. low molecular weight markers; 2. 0.375 μg of biotinylated ubiquitin; 4. 0.75 μg biotinylated ubiquitin; 6. 0.150 μg of biotinylated ubiquitin; 3, 5, and 7 are the same as in 2, 4, and 6 but after incubation with avidin-agarose.
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f1-ijms-13-11593: SDS-PAGE of solution of biotinylated ubiquitin before (tracks 2, 4, 6) and after incubation (tracks 3, 5, 7) with avidin-agarose. 1. low molecular weight markers; 2. 0.375 μg of biotinylated ubiquitin; 4. 0.75 μg biotinylated ubiquitin; 6. 0.150 μg of biotinylated ubiquitin; 3, 5, and 7 are the same as in 2, 4, and 6 but after incubation with avidin-agarose.

Mentions: Figure 1 shows that incubation of biotinylated ubiquitin (0.9 mg/mL) with avidin-agarose suspension (1:1) resulted in complete elimination of the protein from the incubation medium (Figure 1) thus suggesting its effective binding to the sorbent. This allowed us to use the biotinylated ubiquitin for evaluation of the effect of ubiquitin on mitochondrial proteomic profiling.


Use of biotinylated ubiquitin for analysis of rat brain mitochondrial proteome and interactome.

Buneeva OA, Medvedeva MV, Kopylov AT, Zgoda VG, Medvedev AE - Int J Mol Sci (2012)

SDS-PAGE of solution of biotinylated ubiquitin before (tracks 2, 4, 6) and after incubation (tracks 3, 5, 7) with avidin-agarose. 1. low molecular weight markers; 2. 0.375 μg of biotinylated ubiquitin; 4. 0.75 μg biotinylated ubiquitin; 6. 0.150 μg of biotinylated ubiquitin; 3, 5, and 7 are the same as in 2, 4, and 6 but after incubation with avidin-agarose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472765&req=5

f1-ijms-13-11593: SDS-PAGE of solution of biotinylated ubiquitin before (tracks 2, 4, 6) and after incubation (tracks 3, 5, 7) with avidin-agarose. 1. low molecular weight markers; 2. 0.375 μg of biotinylated ubiquitin; 4. 0.75 μg biotinylated ubiquitin; 6. 0.150 μg of biotinylated ubiquitin; 3, 5, and 7 are the same as in 2, 4, and 6 but after incubation with avidin-agarose.
Mentions: Figure 1 shows that incubation of biotinylated ubiquitin (0.9 mg/mL) with avidin-agarose suspension (1:1) resulted in complete elimination of the protein from the incubation medium (Figure 1) thus suggesting its effective binding to the sorbent. This allowed us to use the biotinylated ubiquitin for evaluation of the effect of ubiquitin on mitochondrial proteomic profiling.

Bottom Line: Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated.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.

View Article: PubMed Central - PubMed

Affiliation: Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 10 Pogodinskaya street, Moscow 119121, Russia; E-Mails: olbun@yandex.ru (O.A.B.); a.t.kopylov@gmail.com (A.T.K.); vic@ibmh.msk.su (V.G.Z.).

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus