Table 2.

Comparison of three different approaches in the isolation of ubiquitinated proteins for proteomic identification of ubiquitination sites.

#	Tags	Advantages	Disadvantages	Examples
1	Enrichment with affinity-tagged ubiquitin	Versatile small affinity tags are available to label ubiquitin Cannot isolate different types of ubiquitination, such as monoubiquitination, multi-monoubiquitination, polyubiquitination Can reduce non-specifically bound proteins by using harsh washing conditions	Free ubiquitin is also isolated Tagged ubiquitin has to be expressed in cells Not appropriate for samples from tissues and patients	See Table 1.
2	Immunoprecipitation with anti-ubiquitin antibodies	Can selectively isolate ubiquitinated proteins with specific ubiquitin chain linkages Can eliminate free ubiquitin Compatible with samples from tissues and patients without genetic manipulation	Washing conditions may not be sufficient to completely remove non-specifically bound proteins Antibody-derived peptides may be isolated, which may affect the detection of ubiquitinated peptides Relative high cost	(Matsumoto et al., 2005; Vasilescu et al., 2005; Vasilescu et al., 2007)
3	Isolation with UBDs	Compatible with samples from tissues and patients without genetic manipulation Different UBDs can be used to generate affinity proteins that interact with polyubiquitinated proteins with specific ubiquitin chain linkages	Cannot isolate mono- or multi-monoubiquitinated proteins Washing conditions may not be harsh enough to completely remove non-specifically bound proteins. UBDs are large proteins and must be purified prior to the experiments	(Maor et al., 2007; Shi et al., 2011; Tan et al., 2008)