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