Ubiquitination is an intricately regulated post-translational modification that involves the covalent attachment of ubiquitin to a substrate protein. The complex dynamic nature of the ubiquitination process regulates diverse cellular functions including targeting proteins for degradation, cell cycle, deoxyribonucleic acid (DNA) damage repair, and numerous cell signaling pathways. Ubiquitination also serves as a crucial mechanism in protein quality control. Dysregulation in ubiquitination could result in lethal disease conditions such as cancers and neurodegenerative diseases. Therefore, the ubiquitination cascade has become an attractive target for therapeutic interventions. Enormous efforts have been made to detect ubiquitination involving different detection techniques to better grasp the underlying molecular mechanisms of ubiquitination. This review discusses a wide range of techniques stretching from the simplest assays to real-time assays. This includes western blotting/immunoblotting, fluorescence assays, chemiluminescence assays, spectrophotometric assays, and nanopore sensing assays. This review compares these applications, and the inherent advantages and limitations.
BuetowLHuangDT. Structural insights into the catalysis and regulation of E3 ubiquitin ligases.Nat Rev Mol Cell Biol2016;17:626–42
9.
WenzelDMStollKEKlevitRE. E2s: structurally economical and functionally replete.Biochem J2011;433:31–42
10.
SchulmanBAHarperJW. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways.Nat Rev Mol Cell Biol2009;10:319–31
11.
DittmarGWinklhoferKF. Linear ubiquitin chains: cellular functions and strategies for detection and quantification.Front Chem2020;7:1–16
12.
de BiePCiechanoverA. Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms.Cell Death Differ2011;18:1393–402
13.
HeMZhouZShahAAZouHTaoJChenQWanY. The emerging role of deubiquitinating enzymes in genomic integrity, diseases, and therapeutics.Cell Biosci2016;6:62
14.
LoveKRCaticASchliekerCPloeghHL. Mechanisms, biology and inhibitors of deubiquitinating enzymes.Nat Chem Biol2007;3:697–705
15.
Reyes-TurcuFEVentiiKHWilkinsonKD. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.Annu Rev Biochem2009;78:363–97
16.
WingSS. Deubiquitinating enzymes: the importance of driving in reverse along the ubiquitin-proteasome pathway.Int J Biochem Cell Biol2003;35:590–605
17.
AmerikAYHochstrasserM. Mechanism and function of deubiquitinating enzymes.Biochim Biophys Acta – Mol Cell Res2004;1695:189–207
18.
LoisLMLimaCDChuaNH. Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis.Plant Cell2003;15:1347–59
19.
CappadociaLLimaCD. Ubiquitin-like protein conjugation: structures, chemistry, and mechanism.Chem Rev2018;118:889–918
20.
YangYHeYWangXLiangZHeGZhangPZhuHXuNLiangS. Protein SUMOylation modification and its associations with disease.Open Biol2017;7:170167
21.
ZhaoQMaYLiZZhangKZhengMZhangS. The function of SUMOylation and its role in the development of cancer cells under stress conditions: a systematic review.Stem Cells Int2020;2020:8835714
22.
KlizaKHusnjakK. Resolving the complexity of ubiquitin networks.Front Mol Biosci2020;7:21
23.
WangYSWuKPJiangHKKurkutePChenRH. Branched ubiquitination: detection methods, biological functions and chemical synthesis.Molecules2020;25:1–20
24.
YauRRapeM. The increasing complexity of the ubiquitin code.Nat Cell Biol2016;18:579–86
25.
KulathuYKomanderD. Atypical ubiquitylation-the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.Nat Rev Mol Cell Biol2012;13:508–23
26.
DengLMengTChenLWeiWWangP. The role of ubiquitination in tumorigenesis and targeted drug discovery.Signal Transduct Target Ther2020;5:11
27.
SchwartzALCiechanoverA. Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology.Annu Rev Pharmacol Toxicol2009;49:73–96
28.
EldridgeAGO’BrienT. Therapeutic strategies within the ubiquitin proteasome system.Cell Death Differ2010;17:4–13
29.
BedfordLLoweJDickLRMayerRJBronellJE. Ubiquitin-like protein conjugation and the ubiquiting-proteasome system as drug targets.Nat Rev Drug Discov2011;10:29–46
30.
YuHLuoHChangLZhaoBH. The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice.Proc Natl Acad Sci USA2022;119:1–11
31.
SoucyTASmithPGMilhollenMABergerAJGavinJMAdhikariSBrownellJEBurkeKECardinDPCritchleySCullisCADoucetteAGarnseyJJGaulinJLGershmanRELublinskyARMcDonaldAMizutaniHNarayananUOlhavaEJPelusoSRezaeiMSintchakMDTalrejaTThomasMPTraoreTVyskocilSWeatherheadGSYuJZhangJDickLRClaiborneCFRolfeMBolenJBLangstonSP. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature2009;458:732–6
32.
MimnaughEGBonviniPNeckersL. The measurement of ubiquitin and ubiquitinated proteins.Electrophoresis1999;20:418–28
33.
DeverauxQWellsRRechsteinerM. Ubiquitin metabolism in ts85 cells, a mouse carcinoma line that contains a thermolabile ubiquitin activating enzyme.J Biol Chem1990;265:6323–9
34.
TakadaKKandaTOhkawaKMatsudaM. Ubiquitin and ubiquitin-protein conjugates in PC12h cells: changes during neuronal differentiation.Neurochem Res1994;19:391–8
35.
TakadaKNasuHHibiNTsukadaYOhkawaKFujimuroMSawadaHYokosawaH. Immunoassay for the quantification of intracellular multi-ubiquitin chains.Eur J Biochem1995;233:42–7
36.
BaarendsWMHoogerbruggeJWRoestHPOomsMVreeburgJHoeijmakersJHGrootegoedJA. Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.Dev Biol1999;207:322–33
37.
YanZZakRLuxtonGWRitchieTCBantel-SchaalUEngelhardtJF. Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors.J Virol2002;76:2043–53
38.
GirnitaLGirnitaALarssonO. Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor.Proc Natl Acad Sci USA2003;100:8247–52
39.
YamauchiKWadaKTanjiKTanakaMKamitaniT. Ubiquitination of E3 ubiquitin ligase TRIM5α and its potential role.FEBS J2008;275:1540–55
40.
MatsumotoMLCastellanosERZengYJKirkpatrickDS. Interpreting the language of polyubiquitin with linkage-specific antibodies and mass spectrometry.Methods Mol Biol2018;1844:385–400
41.
NewtonKMatsumotoMLWertzIEKirkpatrickDSLillJRTanJDuggerDGordonNSidhuSSFellouseFAKomuvesLFrenchDMFerrandoRELamCCompaanDYuCBosanacIHymowitzSGKelleyRFDixitVM. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.Cell2008;134:668–78
42.
YinQHanTFangBZhangGZhangCRobertsERIzumiVZhengMJiangSYinXKimMCaiJHauraEBKoomenJMSmalleyKSMWanL. K27-linked ubiquitination of BRAF by ITCH engages cytokine response to maintain MEK-ERK signaling.Nat Commun2019;10:1870
43.
MinMMevissenTETDe LucaMKomanderDLindonC. Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages.Mol Biol Cell2015;26:4325–32
44.
NakazawaSOikawaDIshiiRAyakiTTakahashiHTakedaHIshitaniRKameiKTakeyoshiIKawakamiHIwaiKHatadaISawasakiTItoHNurekiOTokunagaF. Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis.Nat Commun2016;7:12547
45.
YauRGDoernerKCastellanosERHaakonsenDLWernerAWangNYangXWMartinez-MartinNMatsumotoMLDixitVMRapeM. Assembly and function of heterotypic ubiquitin chains in cell-cycle and protein quality control.Cell2017;171:918–33
46.
GuvenAWuWSPatilSGokulKTekumallaPSharmaSDiersAGestaSVishnudasVSarangarajanRKelloggMDNarainNRKiebishMA. Diablo ubiquitination analysis by sandwich immunoassay.J Pharm Biomed Anal2019;173:40–6
47.
YoshidaYSaekiYTsuchiyaHTanakaK. Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE.Methods Enzymol2019;618:135–47
48.
HuZLiHWangXUllahKXuG. Proteomic approaches for the profiling of ubiquitylation events and their applications in drug discovery.J Proteomics2021;231:103996
49.
RahighiSIkedaFKawasakiMAkutsuMSuzukiNKatoRKenscheTUejimaTBloorSKomanderDRandowFWakatsukiSDikicI. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation.Cell2009;136:1098–109
50.
KulathuYAkutsuMBremmAHofmannKKomanderD. Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain.Nat Struct Mol Biol2009;16:1328–30
51.
KristariyantoYAAbdul RehmanSACampbellDGMorriceNAJohnsonCTothRKulathuY. K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of K29 polyubiquitin.Mol Cell2015;58:83–94
52.
KristariyantoYAAbdul RehmanSAWeidlichSKnebelAKulathuY. A single MIU motif of MINDY-1 recognizes K48-linked polyubiquitin chains.EMBO Rep2017;18:392–402
TongaonkarPMaduraK. Reconstituting ubiquitination reactions with affinity-purified components and 32P-ubiquitin.Anal Biochem1998;260:135–41
56.
SelvinPR. Fluorescence resonance energy transfer.Methods1995;246:300–34
57.
SzöllosiJDamjanovichSMátyusL. Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research.Commun Clin Cytom1998;34:159–79
58.
Földes-PappZDemelUTilzGP. Laser scanning confocal fluorescence microscopy: an overview.Int Immunopharmacol2003;3:1715–29
59.
ShyuYJHuCD. Fluorescence complementation: an emerging tool for biological research.Trends Biotechnol2008;26:622–30
60.
GururajaTLPrayTRLoweRDongGHuangJDaniel-IssakaniSPayanDG. A homogeneous FRET assay system for multiubiquitin chain assembly and disassembly.Methods Enzymol2005;399:663–82
61.
KoszelaJPhamNTEvansDMannSPerez-PiIShaveSCeccarelliDFJSicheriFTyersMAuerM. Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.BMC Biol2018;16:1–13
62.
WeeKELaiZAugerKRMaJHoriuchiKYDowlingRLDoughertyCSCormanJIWynnRCopelandRA. Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate.J Protein Chem2000;19:489–98
63.
MacdonaldJMHaasALLondonRE. Novel mechanism of surface catalysis of protein adduct formation. NMR studies of the acetylation of ubiquitin.J Biol Chem2000;275:31908–13
64.
Vijay-KumarSBuggCECookWJ. Structure of porin refined at 1.8 Å resolution.J Mol Biol1987;194:531–44
65.
DavydovIVWoodsDSafiranYJOberoiPFearnheadHOFangSJensenJPWeissmanAMKentenJHVousdenKH. Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2.J Biomol Screen2004;9:695–703
66.
CleggRM. Fluorescence resonance energy transfer.Curr Opin Biotechnol1995;6:103–10
67.
RoyerCAScarlataSF. Chapter 5 fluorescence approaches to quantifying biomolecular interactions.Methods Enzymol2008;450:79–106
68.
ToselandCP. Fluorescent labeling and modification of proteins.J Chem Biol2013;6:85–95
69.
SekarRBPeriasamyA. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations.J Cell Biol2003;160:629–33
70.
BossisGChmielarskaKGärtnerUPichlerAStiegerEMelchiorF. A fluorescence resonance energy transfer-based assay to study SUMO modification in solution.Methods Enzymol2005;398:20–32
71.
TangZLiBBharadwajRZhuHOzkanEHakalaKDeisenhoferJYuH. APC2 cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex.Mol Biol Cell2001;12:3839–51
72.
BoisclairMDMcClureCJosiahSGlassSBottomleySKamerkarSHemmiläI. Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer.J Biomol Screen2000;5:319–28
73.
GruberSJCorneaRLLiJPetersonKCSchaafTMGillispieGDDahlRZseboKMRobiaSLThomasDD. Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.J Biomol Screen2014;19:215–22
74.
KlostermeierDMillarDP. Time-resolved fluorescence resonance energy transfer: a versatile tool for the analysis of nucleic acids.Biopolymers2001;61:159–79
75.
VisserAJWGLaptenokSPVisserNVvan HoekABirchDJSBrochonJCBorstJW. Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein Pairs.Eur Biophys J2010;39:241–53
76.
HongCASwearingenEMallariRGaoXCaoZNorthAYoungSWHuangSG. Development of a high throughput time-resolved fluorescence resonance energy transfer assay for TRAF6 ubiquitin polymerization.Assay Drug Dev Technol2003;1:175–80
77.
CarlsonCBHortonRAVogelKW. A toolbox approach to high-throughput TR-FRET-based SUMOylation and DeSUMOylation assays.Assay Drug Dev Technol2009;7:348–55
78.
MartinSFTathamMHHayRTSamuelID. Quantitative analysis of multi-protein interactions using FRET: application to the SUMO pathway.Protein Sci2008;17:777–84
79.
TagitOHildebrandtN. Fluorescence sensing of circulating diagnostic biomarkers using molecular probes and nanoparticles.ACS Sensors2017;2:31–45
MoerkeNJ. Fluorescence polarization (FP) assays for monitoring peptide-protein or nucleic acid-protein binding.Curr Protoc Chem Biol2009;1:1–15
82.
MotACPrellEKleckerMNaumannCFadenFWestermannBDissmeyerN. Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes.New Phytol2018;217:613–24
83.
FranklinTGPrunedaJN. A high-throughput assay for monitoring ubiquitination in real time.Front Chem2019;7:816
84.
KerppolaTK. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.Nat Protoc2006;1:1278–86
85.
KerppolaTK. Visualization of molecular interactions by fluorescence complementation.Nat Rev Mol Cell Biol2006;7:449–56
86.
LvYZhaoXLiuLDuGZhouJChenJ. A simple procedure for protein ubiquitination detection in Saccharomyces cerevisiae: Gap1p as an example.J Microbiol Methods2013;94:25–9
87.
ChenZZhongYWangYXuSLiuZBaskakovIVMonteiroMJKarbowskiMShenYFangS. Ubiquitination-induced fluorescence complementation (UiFC) for detection of K48 ubiquitin chains in vitro and in live cells.PLoS ONE2013;8:e73482
88.
WeidemannTWachsmuthMKnochTAMüllerGWaldeckWLangowskiJ. Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging.J Mol Biol2003;334:229–40
89.
HausteinESchwilleP. Trends in fluorescence imaging and related techniques to unravel biological information.HFSP J2007;1:169–80
90.
BaeyensWRGSchulmanSGCalokerinosACZhaoYCampañaAMGNakashimaKDe KeukeleireD. Chemiluminescence-based detection: principles and analytical applications in flowing streams and in immunoassays.J Pharm Biomed Anal1998;17:941–53
91.
KrickaLJThorpeGHG. Chemiluminescent and bioluminescent methods in analytical chemistry a review.Analyst1983;108:1274–96
92.
RouleauNWangJKarrasLAndrewsEBielefeld-SevignyMChenY. Highly sensitive assays for SUMOylation and small ubiquitin-like modifier-dependent protein-protein interactions.Anal Biochem2008;375:364–6
93.
YasgerAJadhavASimenovACoussensNP. AlphaScreen-based assays: ultra-high-throughput screening for small-molecule inhibitors of challenging enzymes and protein-protein interaction.Methods Mol Biol2016;176:77–98
94.
ZhouXZhuDLiaoYLiuWLiuHMaZXingD. Synthesis, labeling and bioanalytical applications of a tris(2,2’-bipyridyl)ruthenium(II)-based electrochemiluminescence probe.Nat Protoc2014;9:1146–59
ShenJZhouTHuangR. Recent advances in electrochemiluminescence sensors for pathogenic bacteria detection.Micromachines2019;10:8
97.
SyedAJAndersonJC. Applications of bioluminescence in biotechnology and beyond.Chem Soc Rev2021;50:5668–705
98.
NakajimaYOhmiyaY. Bioluminescence assays: multicolor luciferase assay, secreted luciferase assay and imaging luciferase assay.Expert Opin Drug Discov2010;5:835–49
99.
ZegzoutiHZdanovskaiaMHsiaoKGoueliSA. ADP-Glo: a bioluminescent and homogeneous ADP monitoring assay for Kinases.Assay Drug Dev Technol2009;7:560–72
100.
MondalSHsiaoKGoueliSA. A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins.Anal Biochem2016;510:41–51
101.
XuYPistonDWJohnsonCH. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins.Proc Natl Acad Sci USA1999;96:151–6
102.
XieQSouttoMXuXZhangYJohnsonCH. Bioluminescence resonance energy transfer (BRET) imaging in plant seedlings and mammalian cells.Methods Mol Biol2011;680:3–28
103.
PflegerKDEidneKA. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET).Nat Methods2006;3:165–74
104.
WojcikiewiczRJ. Regulated ubiquitination of proteins in GPCR-initiated signaling pathways.Trends Pharmacol Sci2004;25:35–41
105.
ShenoySKMcDonaldPHKohoutTALefkowitzRJ. Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin.Science2001;294:1307–13
106.
PerroyJPontierSCharestPGAubryMBouvierM. Real-time monitoring of ubiquitination in living cells by BRET.Nat Methods2004;1:203–8
107.
PassosMLCSaraivaMLMFS. Detection in UV-visible spectrophotometry: detectors, detection systems, and detection strategies.Meas J Int Meas Confed2019;135:896–904
108.
BerndsenCEWolbergerC. A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins.Anal Biochem2011;418:102–10
NirIHuttnerDMellerA. Direct sensing and discrimination among ubiquitin and ubiquitin chains using solid-state nanopores.Biophys J2015;108:2340–9
112.
WlokaCVan MeerveltVVan GelderDDandaNJagerNWilliamsCPMagliaG. Label-free and real-time detection of protein ubiquitination with a biological nanopore.ACS Nano2017;11:4387–94
