Table 1.

Comparison of different strategies for ubiquitination characterization: from ubiquitinated protein to ubiquitin chain architecture

Levels	Approaches	Advantages	Disadvantages	Application	Refs
At the protein level	Ub tagging-based approaches	• Can remove the majority of non-ubiquitinated proteins • Can identify ubiquitination sites and localize them to proteins	• Require expressing ubiquitin tag which may behave differently from endogenous ubiquitin and impair the identification accuracy of ubiquitylation • Low efficiency for ubiquitylation identification • Limit its application in tissues	Screen and validation of ubiquitinated substrates in cells	[34–36]
	Ub antibody-based approaches	• Can purify endogenous ubiquitinated proteins • Enrich the linkage specific ubiquitylated proteins by linkage-specific antibodies • Application in all samples	• High cost of antibodies • High background derived from binding proteins • Low efficiency for ubiquitylation identification	Validation of ubiquitinated substrates and their linkage types in all samples	[37–40]
	UBD-based approaches	• Do not require expressing ubiquitin tag and antibodies • Can purify endogenous ubiquitinated proteins • Enrich the linkage specific ubiquitylated proteins by linkage-specific UBDs	• lower affinity of monoubiquitylated proteins • Low efficiency for ubiquitylation identification • High background derived from UBAs and UBDs	Screen of ubiquitinated proteins and their linkage types in all samples	[47–55]
At the peptide level	Anti-diGly antibody-based approach	• Can identify large number of ubiquitination sites • High efficiency for ubiquitination identification	• High cost of antibody • False positive identification generated from ISG15 and NEDD8 modification • Cannot identify N-terminal ubiquitylation sites • Cannot reveal any information on ubiquitin chain topology	Profiling of ubiquitination sites in all samples	[57–65]
	UbiSite antibody-based approach	• Can identify large number of ubiquitination sites with high efficiency • Can identify N-terminal ubiquitination sites • Can avoid the interference of ISG15 and NEDD8 modification	• High cost of antibody • The longer ubiquitin remnants on the ubiquitination sites hamper the identification of the ubiquitinated peptides • Cannot reveal the information on ubiquitin chain topology	Profiling of ubiquitination sites in all samples	[70]
	Antibody-free approaches	• Do not require antibody • Low cost • Can identify large number of ubiquitination sites	• Low through-put compared with antibody-based approaches • Artifacts derived from ubiquitin mutation and chemical derivatization	Screen of ubiquitination sites in cells	[75–77]
At the topology level	Bottom-up proteomics, e.g. Ub-AQUA and UbiCRest	• Can dissect the ubiquitin chain architecture	• Cannot well distinguish branched from mixed ubiquitin chains • Low specificity in identifying linkage types • Cannot effectively analyze the heterotypic chains	Validation of the ubiquitin chain linkage in all samples	[79–86]
	Middle-down proteomics, e.g. Ub-clipping	• Can dissect the branched ubiquitin chains • Can reveal the ratio of branched to unbranched linkages	• Cannot dissect the chain linkage types at the branched point	Screen and validation of ubiquitination sites and its topologies in all samples	[91–96]
	Top-down proteomics	• Can dissect the branched ubiquitin chains • Can reveal the ratio of branched to unbranched linkages • Can dissect the chain linkage types at the branched point	• Low signal-to-noise (S/N) fragments with increasing molecular weight • Lack in in sample preparation and analytical approaches	Application in identifying ubiquitination sites and its topologies in all samples	[28, 97–99]