PTMs serve as key regulatory mechanisms for 20S proteasome functions. Alterations in 20S PTMs have been previously observed with changes in modified protein degradation patterns and altered cellular phenotypes. Despite decades of investigation, our knowledge pertaining to the various PTMs of 20S complexes and their biological significance remain limited. In this investigation, we show that 2-DE offers an analytical tool with high resolution and reproducibility. Accordingly, it has been applied for the characterization of PTMs including glycosylation, phosphorylation, oxidation, and nitrosylation. The PTMs of murine cardiac 20S proteasomes and their associating proteins were examined. Our 2-DE analyses displayed over 25 spots for the 20S complexes (17 subunits), indicating multiply modified subunits of cardiac proteasomes. The identification of specific PTM sites subsequent to 2-DE was supported by MS. These PTMs included phosphorylation and oxidation. Most of the PTMs occurred in low stoichiometry and required enrichment to enhance the detection sensitivity. In conclusion, our studies support 2-DE as a central tool in the analyses of 20S proteasome PTMs. The approaches utilized in this investigation demonstrate their application in mapping the PTMs of the 20S proteasomes in cardiac tissue, which are applicable to other samples and biological conditions.