20S and 26S Proteasome-binding proteins of the rabbit brain: a proteomic dataset

Published: 1 June 2021| Version 1 | DOI: 10.17632/g9tpsdctt4.1
Contributors:
,
,
Arthur T Kopylov

Description

Fractions of 26S and 20S proteasomes isolated from the rabbit brain by the method of salt fractionation (salt-induced precipitation) contain intrinsic proteasome proteins responsible for assembly of the core particle and regulatory particle of proteasome and also proteasome-binding proteins. These proteasome-binding proteins include components of the ubiquitin-proteasome system, some ubiquitinated proteins, as well as cytoskeleton components, protective proteins, regulators of gene expression, cell division, and differentiation, and multifunctional proteins (mainly, glycolytic enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase, etc.). The multifunctional proteins are also known as “moonlighting proteins” are involved in various (regulatory) processes in the cell and this suggests that they represent important components of the proteasome interactome rather than contaminants of the 26S and 20S proteasome fractions. Data obtained by high-resolution mass spectrometry in Ubiquitine-tag triggered MS3-mode with synchronous fragment ions selection, and nano-flow liquid chromatography (see "Steps to reproduce for details). The presented dataset comprised of two subsets: (1) Data of proteomes obtained for each animal under the study; these data are in Excel format and contain necessary information about proteins, peptides, fragments, and their abundance, and other related meta-data. Data contained specific modifications identified in peptides by the PSMs. (2) Combined data for the complete cohort of animals with protein distribution. Both subsets also contain information about semi-quantitative protein representation estimated by the NSAF algorithm in the Search GUI/Peptide Shaker search engine.

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Steps to reproduce

ISOLATION OF RABBIT BRAIN 26S AND 20S PROTEASOMES Adult male albino New Zealand White rabbits (body mass of 3.5–5.0 kg) were decapitated after intramuscular injection of Zoletil (10 mg/kg). The extracted brain tissue was homogenized in Buffer A (30 mМ Tris-HCl, pH 7.5, 100 mМ NaCl, 1 mМ EDТА, 1 mМ dithiothreitol, 10% glycerol (v/v), 5 mМ MgCl2, 2 mМ АТP, 10 mМ Na2S2O5, a protease inhibitor cocktail (1 ml/l), tissue: buffer ratio 1:3 (w/v). The homogenate was centrifuged (90 min, 105,000 g, 4°C). After the addition of phosphocreatine and creatine kinase (final concentrations of 10 mM and 10 µg/ml respectively), the resultant supernatant was incubated for 30 min at 37°С. The mixture was fractionated with ammonium sulfate in three stages. Within 30 min ammonium sulfate was added by portions upon stirring at 4°С up to 38% saturation, kept stirred for 30 minutes at 4°С and centrifuged (10,000 g, 30 min, 4°C) to separate the pellet, containing a pool of 26S proteasomes. This pellet was resuspended in Buffer В (20 mМ Tris-HCl, pH 7.5, 1 mМ EDТА, 1 mМ dithiothreitol, 50% glycerol (v/v), 5 mМ MgCl2, 2 mМ АТP, 10 mМ Na2S2O5, a protease inhibitor cocktail (1 ml/l). After pellet resuspension and addition of phosphocreatine and creatine kinase (final concentrations of 10 mM and 10 µg /ml, respectively) the mixture was incubated for 30 min at 37°С. The resultant fraction of 26S proteasomes (protein content of approximately 2 mg/ml) was also stored at -20°С. The supernatant from the previous step (38% saturated ammonium sulfate supernatant) was used for the isolation of 20S proteasomes. Ammonium sulfate was gradually added to supernatant up to 42% saturation, kept stirred for 30 minutes, and centrifuged under the same conditions to separate the pellet containing contaminant proteins. The resultant supernatant was used to obtain the 20S proteasome fraction which was isolated gradually adding ammonium sulfate up to 70% saturation followed by centrifugation (10000 g, 30 min, 4°C). The final pellet was resuspended in Buffer C (20 mМ Tris-HCl, pH 7.5, 1 mМ EDТА, 1 mМ dithiothreitol, 50% glycerol (v/v), 10 mМ Na2S2O5, a protease inhibitor cocktail (1 ml/l). The 20S proteasome fraction (protein content of approximately 2 mg/ml) was also stored at -20°С. MASS SPECTROMETRY High-resolution mass spectrometry was performed using an Orbitrap Fusion with an NSI ion source in a positive ionization mode. Surveyed in a range of 400–1200 m/z of precursor ions with charge states z = 2+ to z = 6+ were isolated in the quadrupole mass analyzer within ±1.5 Th and triggered to fragmentation. Fragmentation and tandem scanning were performed in MS3 synchronous precursor ions (secondary ions) selection conditioned by the mass difference between the fragment ions of either ΔM = 114.0429 u (the ubiquitin tag GG) or ΔM = 383.2281 u (the ubiquitin tag LRGG) detected with an asymmetric mass tolerance of −3 ppm/+7 ppm. Only two ions were allowed for synchronous selection in the MS3 mode.

Institutions

Naucno-issledovatel'skij institut biomedicinskoj himii imeni V N Orehovica

Categories

Proteasome, Brain, Rabbit, Ubiquitylation

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