Exploring the Rampant Expansion of Ubiquitin Proteomics

  • Amalia Rose
  • Thibault MayorEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)


The ubiquitin proteasome system can arguably affect all cellular proteins with few exceptions. In addition to regulating many pathways such as cell cycle progression, inflammation, gene expression, DNA repair, and vesicle trafficking—to just name a few—ubiquitination can occur to any nascent or newly translated protein that misfolds. In the past years, substantial progress has been achieved in advancing our global understanding of the ubiquitinome—the ensemble of ubiquitinated proteins within a cell—using mass spectrometry-based proteomics. Notably, over 50,000 conjugation sites have now been reported. In this review, we discuss recent proteomics methods used to expand our knowledge of the ubiquitin proteasome system through the identification of ubiquitination sites, poly-ubiquitin chain types, and E3 ubiquitin ligase substrates.

Key words

Ubiquitin Proteasome E3 Proteomics Mass spectrometry diGly 



The authors would like to thank all lab members for the discussions and Cristen Molzahn for the comments on the manuscript. They also acknowledge support from the Canadian Institutes of Health Research (CIHR) and British Columbia Proteomics Network (BCPN); TM is a MSFHR new investigator.


  1. 1.
    Cohen P, Tcherpakov M (2010) Will the ubiquitin system furnish as many drug targets as protein kinases? Cell 143(5):686–693. Scholar
  2. 2.
    Huang X, Dixit VM (2016) Drugging the undruggables: exploring the ubiquitin system for drug development. Cell Res 26(4):484–498. Scholar
  3. 3.
    Weathington NM, Mallampalli RK (2014) Emerging therapies targeting the ubiquitin proteasome system in cancer. J Clin Invest 124(1):6–12. Scholar
  4. 4.
    Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81:203–229. Scholar
  5. 5.
    Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479. Scholar
  6. 6.
    Wilkinson KD (2000) Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Cell Dev Biol 11(3):141–148. Scholar
  7. 7.
    Rajalingam K, Dikic I (2016) SnapShot: expanding the ubiquitin code. Cell 164(5):1074–1074.e1. Scholar
  8. 8.
    Zhang Y, Fonslow BR, Shan B et al (2013) Protein analysis by shotgun/bottom-up proteomics. Chem Rev 113(4):2343–2394. Scholar
  9. 9.
    Peng J, Schwartz D, Elias JE et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21(8):921–926. Scholar
  10. 10.
    Lichti CF, Wildburger NC, Emmett MR et al (2014) Post-translational modifications in the human proteome. In: Genomics and proteomics for clinical discovery and development. Springer, Dordrecht, pp 101–136Google Scholar
  11. 11.
    Mayor T, Lipford JR, Graumann J et al (2005) Analysis of poly-ubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. Mol Cell Proteomics 4(6):741–751. Scholar
  12. 12.
    Mayor T, Graumann J, Bryan J et al (2007) Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway. Mol Cell Proteomics 6(11):1885–1895. Scholar
  13. 13.
    Meierhofer D, Wang X, Huang L, Kaiser P (2008) Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J Proteome Res 7(10):4566–4576. Scholar
  14. 14.
    Tagwerker C, Flick K, Cui M et al (2006) A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking. Mol Cell Proteomics 5(4):737–748. Scholar
  15. 15.
    Xu G, Paige JS, Jaffrey SR (2011) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28(8):868–873. Scholar
  16. 16.
    Kim W, Bennett EJ, Huttlin EL et al (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44(2):325–340. Scholar
  17. 17.
    Skaug B, Chen ZJ (2010) Emerging role of ISG15 in antiviral immunity. Cell 143(2):187–190. Scholar
  18. 18.
    Wagner SA, Beli P, Weinert BT et al (2012) Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol Cell Proteomics 11(12):1578–1585. Scholar
  19. 19.
    Wagner SA, Beli P, Weinert BT et al (2011) A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 10(10):M111.013284. Scholar
  20. 20.
    Hornbeck PV, Zhang B, Murray B et al (2015) PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations. Nucleic Acids Res 43(DI):D512–D520. Scholar
  21. 21.
    Udeshi ND, Mertins P, Svinkina T, Carr SA (2013) Large-scale identification of ubiquitination sites by mass spectrometry. Nat Methods 8(10):1950–1960. Scholar
  22. 22.
    Mann M (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7(12):952–959. Scholar
  23. 23.
    Fang NN, Chan GT, Zhu M et al (2014) Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress. Nat Cell Biol 16(12):1227–1237. Scholar
  24. 24.
    Silva G, Finley D, Vogel C (2015) K63 polyubiquitination is a new modulator of the oxidative stress response. Nat Struct Mol Biol 22(2):116–123. Scholar
  25. 25.
    Rose CM, Isasa M, Ordureau A et al (2016) Highly multiplexed quantitative mass spectrometry analysis of ubiquitylomes. Cell Syst 3(4):395–403. Scholar
  26. 26.
    Rauniyar N, Yates JR (2014) Isobaric labeling-based relative quantification in shotgun proteomics. J Proteome Res 13(12):5293–5309. Scholar
  27. 27.
    Swaney DL, Beltrao P, Starita L et al (2014) Global analysis of phosphorylation and ubiquitylation crosstalk in protein degradation. Nat Methods 10(7):676–682. Scholar
  28. 28.
    Shi Y, Xu P, Qin J (2011) Ubiquitinated proteome: ready for global? Mol Cell Proteomics 10(5):R110.006882. Scholar
  29. 29.
    Chicooree N, Connolly Y, Tan C-T et al (2013) Enhanced detection of ubiquitin isopeptides. J Am Soc Mass Spectrom 24(3):421–430. Scholar
  30. 30.
    Griffiths JR, Connolly Y, Griffiths JR et al (2014) Mass spectral enhanced detection of Ubls using SWATH of SUMO and ubiquitin-derived isopeptides. J Am Soc Mass Spectrom 25(5):767–777. Scholar
  31. 31.
    Gillet LC, Navarro P, Tate S et al (2012) Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics 11(6):O111.016717. Scholar
  32. 32.
    Yau R, Rape M (2016) The increasing complexity of the ubiquitin code. Nat Cell Biol 18(6):579–586. Scholar
  33. 33.
    Kaliszewski P, Zoładek T (2008) The role of Rsp5 ubiquitin ligase in regulation of diverse processes in yeast cells. Acta Biochim Pol 55(4):649–662PubMedGoogle Scholar
  34. 34.
    Kirkpatrick DS, Hathaway NA, Hanna J et al (2006) Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology. Nat Cell Biol 8(7):700–710. Scholar
  35. 35.
    Phu L, Izrael-tomasevic A, Matsumoto ML et al (2011) Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals. Mol Cell Proteomics 10(5):M110.003756. Scholar
  36. 36.
    Liu C, Liu W, Ye Y, Li W (2017) Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chains. Nat Commun 8:14274. Scholar
  37. 37.
    Meyer H, Rape M (2014) Enhanced protein degradation by branched ubiquitin chains. Cell 157(4):910–921. Scholar
  38. 38.
    Valkevich EM, Sanchez NA, Ge Y, Strieter ER (2014) Middle-down mass spectrometry enables characterization of branched ubiquitin chains. Biochemistry 53(30):4979−4989. Scholar
  39. 39.
    Crowe SO, Rana ASJB, Deol KK et al (2017) Ubiquitin chain enrichment middle-down mass spectrometry enables characterization of branched ubiquitin chains in cellulo. J Anal Chem 89(8):4428–4434. Scholar
  40. 40.
    Ohtake F, Saeki Y, Ishido S et al (2016) The K48-K63 branched ubiquitin chain regulates NF-κB signaling. Mol Cell 64(2):251–266. Scholar
  41. 41.
    Hjerpe R, Aillet F, Lopitz-Otsoa F et al (2009) Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep 10(11):1250–1258. Scholar
  42. 42.
    Rahighi S, Ikeda F, Kawasaki M et al (2009) Specific recognition of linear ubiquitin chains by NEMO is important for NF-kB activation. Cell 136(6):1098–1109. Scholar
  43. 43.
    Kristariyanto YA, Rehman SAA, Campbell DG et al (2015) K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of K29 polyubiquitin. Mol Cell 58(1):83–94. Scholar
  44. 44.
    Kristariyanto YA, Rehman SAA, Weidlich S et al (2017) A single MIU motif of MINDY-1 recognizes K48-linked polyubiquitin chains. EMBO Rep 18(3):392–402. Scholar
  45. 45.
    Kulathu Y, Akutsu M, Bremm A et al (2009) Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain. Nat Struct Mol Biol 16(12):1328–1330. Scholar
  46. 46.
    Sims JJ, Scavone F, Cooper EM et al (2012) Polyubiquitin-sensor proteins reveal localization and linkage- type dependence of cellular ubiquitin signaling. Nat Methods 9(3):303–309. Scholar
  47. 47.
    Thorslund T, Ripplinger A, Hoffmann S et al (2015) Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage. Nature 527(7578):389–393. Scholar
  48. 48.
    Sims JJ, Cohen RE (2009) Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of Rap80. Mol Cell 33(6):775–783. Scholar
  49. 49.
    Yoshida Y, Saeki Y, Murakami A et al (2015) A comprehensive method for detecting ubiquitinated substrates using TR-TUBE. Proc Natl Acad Sci 112(15):4630–4635. Scholar
  50. 50.
    Yau RG, Doerner K, Castellanos ER et al (2017) Assembly and function of heterotypic ubiquitin chains in cell-cycle and protein quality control. Cell 171(4):918–933.e20. Scholar
  51. 51.
    Michel MA, Swatek KN, Hospenthal MK, Komander D (2017) Ubiquitin linkage-specific affimers reveal insights into K6-linked ubiquitin signaling. Mol Cell 68(1):233–246.e5. Scholar
  52. 52.
    Xu M, Skaug B, Zeng W, Chen ZJ (2009) A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFα and IL-1β. Mol Cell 36(2):302–314. Scholar
  53. 53.
    Ordureau A, Heo J-M, Duda DM et al (2015) Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy. Proc Natl Acad Sci 112(21):6637–6642. Scholar
  54. 54.
    Rittinger K, Ikeda F (2017) Linear ubiquitin chains: enzymes, mechanisms and biology. Open Biol 7(4):170026. Scholar
  55. 55.
    Kliza K, Taumer C, Pinzuti I et al (2017) Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry. Nat Methods 14(5):504–512. Scholar
  56. 56.
    Fang N, Ng A, Measday V, Mayor T (2011) Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turn over of cytosolic misfolded proteins. Nat Cell Biol 13(11):1344–1352. Scholar
  57. 57.
    Fang NN, Zhu M, Rose A et al (2016) Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress. Nat Commun 7:12907. Scholar
  58. 58.
    Thompson JW, Nagel J, Hoving S et al (2014) Quantitative Lys-∈−Gly-Gly (diGly) proteomics coupled with inducible RNAi reveals ubiquitin-mediated proteolysis of DNA damage-inducible transcript 4 (DDIT4) by the E3 Ligase HUWE1. J Biol Chem 289(42):28942–28955. Scholar
  59. 59.
    Krönke J, Udeshi ND, Narla A et al (2014) Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343(6168):301–305. Scholar
  60. 60.
    Krönke J, Fink EC, Hollenbach PW et al (2015) Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523(7559):183–188. Scholar
  61. 61.
    An J, Ponthier CM, Sack R et al (2017) PSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4 CRBN ubiquitin ligase. Nat Commun 8:15398. Scholar
  62. 62.
    Peschiaroli A, Dorrello NV, Guardavaccaro D et al (2006) SCFβTrCP-mediated degradation of claspin regulates recovery from the DNA replication checkpoint response. Mol Cell 23(3):319–329. Scholar
  63. 63.
    Dorrello NV, Peschiaroli A, Guardavaccaro D et al (2006) S6K1- and bTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 314(5798):467–472. Scholar
  64. 64.
    Ko HW, Jiang J, Edery I et al (2007) SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science 316(5826):900–904. Scholar
  65. 65.
    Busino L, Millman SE, Scotto L et al (2012) Fbxw7α- and GSK3-mediated degradation of p100 is a pro-survival mechanism in multiple myeloma. Nat Cell Biol 14(4):375–385. Scholar
  66. 66.
    Harper JW, Tan M-KM (2012) Understanding cullin-RING E3 biology through proteomics-based substrate identification. Mol Cell Proteomics 11(12):1541–1550. Scholar
  67. 67.
    Tan MKM, Lim HJ, Bennett EJ et al (2013) Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover. Mol Cell 52(1):9–24. Scholar
  68. 68.
    Watanabe K, Yumimoto K, Nakayama KI (2015) FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1. Genes Cells 20(8):667–674. Scholar
  69. 69.
    Roux KJ, Kim DI, Burke B (2013) BioID: a screen for protein-protein interactions. Curr Protoc Protein Sci 74:Unit 19.23. Scholar
  70. 70.
    Coyaud E, Mis M, Laurent EMN et al (2015) BioID-based identification of Skp cullin F-box (SCF) β-TrCP1/2 E3 ligase substrates. Mol Cell Proteomics 14(7):1781–1795. Scholar
  71. 71.
    Zhuang M, Guan S, Wang H et al (2013) Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator. Mol Cell 49(2):273–282. Scholar
  72. 72.
    Mark KG, Simonetta M, Maiolica A et al (2014) Ubiquitin ligase trapping identifies an SCFSaf1 pathway targeting unprocessed vacuolar/lysosomal proteins. Mol Cell 53(1):148–161. Scholar
  73. 73.
    O’Connor HF, Lyon N, Leung JW et al (2015) Ubiquitin-activated interaction traps (UBAITs) identify E3 ligase binding partners. EMBO Rep 16(12):1699–1712. Scholar
  74. 74.
    MacDonald C, Winistorfer S, Pope RM et al (2017) Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases. Traffic 18(7):465–484. Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, Michael Smith LaboratoriesUniversity of British ColumbiaVancouverCanada

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