Updated project metadata. Within the cell, chemical reactions are often confined and organized through a modular architecture. In this project, a cell-free nanoscale model of the proteasome was analyzed that exploits this compartmentalization principle to perform regulated protein unfolding and degradation. To this end, the protein unfolding machine p97 and the protease chymotrypsin were encapsulated in different DNA nanocompartments, and the sequential unfolding and proteolytic degradation of a model substrate, I3mEos were monitored at different time points via LC-MS/MS-based proteomics using LFQ quantification.