To realize the heterogeneity analysis of the structure and interaction for human 26S proteasomes in the cytoplasm and nucleus, combined with living cell cross-linking and efficient cytoplasm and nucleus separation method, the conformation and interaction resolution of 26S proteasome in the cytoplasm and nucleus was realized. Besides, by ensemble refinement of the interaction conformation of proteasome and ubiquitin with the crosslinking restraints, the transport path of ubiquitin on the proteasome was depicted in the cytoplasm and nucleus. It is of great significance for gaining an intensive understanding of the ubiquitin proteasome system for degrading proteins in the cytoplasm and nucleus.