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.