Table 1.

Proteomic techniques used to identify substrates of E3 ligases using MS

Approach	Cell type	Substrate definition	Ligase	Validation approach/s	Substrates	References
IP-MS, in vitro ubiquitylation assay	HEK293	Co-interacting partners subsequently ubiquitylated in vitro	β-TrCP	In vitro ubiquitylation assay, CHX chase assay and β-TrCP1 and 2 gene silencing using dsRNA	4 known, 1 novel	[53]
IP-MS	HEK293	Strategic mutations that affect known substrate binding. Substrates are also defined by presence of phosphodegron	β-TrCP	In vitro ubiquitylation study, degron mining	27 known, 221 putative substrates	[61]
BioID	HEK293 Flp-in T-Rex	Biotinylated proteins that are stabilised by MG132 treatment	β-TrCP	CHX assays, in vivo ubiquitylation assay	17 previously reported substrates or interaction partners and > 50 novel putative substrate	[44]
Ubiquitin trapping	Yeast	Polyubiquitylated proteins that are trapped	8 F-box proteins	Cycloheximide (CHX) chase assay, in vivo ubiquitylation assays	18 known, 17 new	[46]
Ubiquitin trapping	HEK293 Flp-in T-Rex	Proteins that are enriched after substrate trapping	β-TrCP	CHX chase assays	12 known, 11 new	[48]
TR-TUBE	HEK293	TUBE-binding proteins that decreased in abundance when FBXO21 was inactive	FBXO21	In vivo ubiquitylation assay, siRNA knockdown of protein of FBXO21, CHX chase assays	3 highly reproducible targets were selected from MS experiments	[49]
NEDDylator	Jurkat cells	Substrates are NEDD8 modified by the fusion protein. Lists are also filtered for substrates containing a known binding motif	Inhibitors of apoptosis proteins (IAPs)	In vitro ubiquitylation assay	Over 50 putative IAP substrates.	[50]