Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome

Olga A Buneeva,Marina V Medvedeva,Victor G Zgoda,Alexei E Medvedev,Arthur T Kopylov

doi:10.3390/ijms130911593

Olga A Buneeva, Marina V Medvedeva + Show 3 more

Open Access

https://doi.org/10.3390/ijms130911593

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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.

Highlights

Ubiquitin is a 76-residue protein, which is widely distributed in all eukaryotic cells [1,2,3,4,5]
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
Incubation of rat brain mitochondrial fraction with biotinylated ubiquitin followed by loading of cleared Triton X-100 lysates onto the avidin-agarose, intensive washing of the affinity sorbent and subsequent proteomic analysis of eluted proteins resulted in identification of 50 individual proteins (Table 1)

Summary

Introduction

Ubiquitin is a 76-residue protein, which is widely distributed in all eukaryotic cells [1,2,3,4,5]. Taking into consideration the regulatory role of protein-protein interactions controlling the ubiquitination process [1,2,3,4,5,6,7,8,9], interactome, it is clear that ubiquitin and other components of the ubiquitn conjugating machinery as well as ubiquitinated proteins do potentially interact with many protein partners and form a particular ubiquitin. Trypsinolysis of ubiquitinated proteins yields a unique peptide from the ubiquitination site containing a lysine residue with an isopeptide-linked glycine-glycine sequence [9]. This signature peptide can be identified by mass analysis due to its mass shift of 114.1 Da and the lack of proteolytic cleavage of modified lysine residues

Results

Discussion

Conclusion