An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes.

摘要: Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study ubiquitin proteome we have established immunoaffinity purification method for proteomic analysis endogenously ubiquitinated complexes. A strong, specific enrichment factors was achieved using FK2 antibody bound to G-beaded agarose, which recognizes monoubiquitinated polyubiquitinated conjugates. Mass spectrometric two immunoprecipitations (IPs) resulted identification 296 FK2-specific proteins both experiments. The isolation ubiquitination-related confirmed by pathway analyses (using Ingenuity Pathway Analysis Gene Ontology-annotation enrichment). Additionally, comparing that specifically came down IP with databases showed a high percentage our enriched fraction indeed ubiquitinated. Finally, assessment protein-protein interactions revealed significantly more were residing complexes than random sets. This method, is capable isolating their interacting proteins, can be widely used unraveling ubiquitin-mediated various systems tissues when different states.