The 20S proteasome is a large multisubunit proteolytic machine that is central to intracellular protein degradation. It is found in all three kingdoms of life and is ubiquitous in archaea and eukaryotes. Since its discovery, much effort employing a diverse array of structural biology methods has been applied to help understand its structure/function relationships. Here, we will specifically focus on the application of native mass spectrometry (MS) approaches for structural investigations of the 20S proteasome. Native MS is a method that examines intact protein assemblies, without disturbing the noncovalent interactions that govern the overall structure. This method is ideally suited to revealing the intrinsic heterogeneity of a given sample and provides insight into the composition, stoichiometry, subunit architecture, and topology of the protein assembly. Initially, we describe native MS-oriented protocols for the isolation of endogenous 20S proteasomes from yeast, rat liver, and human cells. We then highlight the applicability of native MS methodologies, using different instrumental platforms, for structural investigations of the complex. In particular, by means of proteasome biology, we highlight the different approaches used to analyze both intact complexes-their natural heterogeneity and interactions with substrates and regulators-and their individual constituent subunits.
Keywords: 20S proteasome; Native mass spectrometry; Structural analysis.
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