Abstract
We have succeeded in purifying the 20S core proteasome particle from less than 1 g of skeletal muscle in a rapid process involving two chromatographic steps. The individual subunits were readily resolved by two-dimensional PAGE, and the identities of each of the 14 subunits were assigned by a combination of peptide mass fingerprinting and MS/MS/de novo sequencing. To assess the dynamics of proteasome biogenesis, chicks were fed a diet containing stable isotope-labeled valine, and the rate of incorporation of label into valine-containing peptides derived from each subunit was assessed by mass spectrometric analysis after two-dimensional separation. Peptides containing multiple valine residues from the 20S proteasome and other soluble muscle proteins were analyzed to yield the relative isotope abundance of the precursor pool, a piece of information that is essential for calculation of turnover parameters. The rates of synthesis of each subunit are rather similar, although there is evidence for high turnover subunits in both the alpha (nonproteolytic) and beta (proteolytic) rings. The variability in synthesis rate for the different subunits is consistent with a model in which some subunits are produced in excess, whereas others may be the rate-limiting factor in the concentration of 20S subunits in the cell. The ability to measure turnover rates of proteins on a proteome-wide scale in protein assemblies and in a complex organism provides a new dimension to the understanding of the dynamic proteome.
Highlights
We have succeeded in purifying the 20S core proteasome particle from less than 1 g of skeletal muscle in a rapid process involving two chromatographic steps
Because the ubiquitin-proteasome system is considered to play a critical role in skeletal muscle protein breakdown [11, 12], we have characterized the 20S proteasome core particle from chick skeletal muscle
The 20S Proteasome in Chicken Skeletal Muscle combination of two-dimensional (2D)1 electrophoresis, in-gel protein digestion, MALDI-TOF peptide mass fingerprinting (PMF), and de novo sequencing by MS/MS
Summary
Animals—Layer (ISA Brown) chicks were obtained immediately post-hatching and grown to 6 days on a synthetic diet with valine as the limiting amino acid (see below). For each gram of tissue, 1 ml of buffer B (20 mM MES, 50 mM NaCl, pH 6.0) was added, and the soluble protein fraction was isolated by centrifugation at 25,000 ϫ g, 4 °C for 45 min. The column was equilibrated in 20 mM Tris, 20 mM KCl, 10 mM magnesium acetate, 2 mM DTT, 10% glycerol, pH 7.6, and bound proteins were eluted in a linear salt gradient (0 –1 M NaCl) over 30 min. The proteins present in each fraction were separated by SDS-PAGE, and those containing the 20S proteasome, identified by peptidolytic activity, were stored at Ϫ20 °C. For the 20S proteasome labeled with [2H8]valine, 250 l (ϳ5 g protein) of the ion exchange fraction containing the 20S complex was used. The relative isotope abundance (RIA) of the precursor pool was calculated from peptides derived from these proteins and containing two or three valine residues. Once the RIA of the precursor pool was established, this parameter is constant for that entire tissue, and it was possible to calculate the relative rates of synthesis of each proteasome subunit
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