Proteasomal Compositional and Conformational Landscapes Mapped By in-situ Cross-Linking Mass Spectrometry

The proteasome is a vital cellular machine responsible for degrading proteins and maintaining proteostasis. While cryo-EM and cross-linking mass spectrometry (XL-MS) have provided valuable structural insights, these investigations have primarily focused on purified proteasomes in vitro. Here, we perform XL-MS in-situ with an enrichable trifunctional cross-linker to characterize the proteasome within its native cellular environment. Leveraging a two-step enrichment strategy and the high effective protein concentrations within cellular compartments, we generated a comprehensive dataset of proteasome-centered cross-links, which unveiled novel interacting proteins and enabled the construction of dynamic structural models of the proteasome. Furthermore, by resolving nuclear and cytoplasmic fractions, we uncovered distinct proteasome compositions, structural dynamics, and interaction profiles in these two compartments. The cross-linking data also informed AI-based structural predictions and provided crucial restraints for refining docking models. Our integrated approach establishes a powerful workflow for investigating the architecture, functional dynamics, and regulation of large protein complexes in situ, offering deeper insights of their regulatory mechanisms and cellular functions.