Ubiquitin diGLY Proteomics as an Approach to Identify and Quantify the Ubiquitin-Modified Proteome

  • Amit Fulzele
  • Eric J. BennettEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1844)


Protein ubiquitylation is one of the most prevalent posttranslational modifications (PTM) within cells. Ubiquitin modification of target lysine residues typically marks substrates for proteasome-dependent degradation. However, ubiquitylation can also alter protein function through modulation of protein complexes, localization, or activity, without impacting protein turnover. Taken together, ubiquitylation imparts critical regulatory control over nearly every cellular, physiological, and pathophysiological process. Affinity purification techniques coupled with quantitative mass spectrometry have been robust tools to identify PTMs on endogenous proteins. A peptide antibody-based affinity approach has been successfully utilized to enrich for and identify endogenously ubiquitylated proteins. These antibodies recognize the Lys-ϵ-Gly-Gly (diGLY) remnant that is generated following trypsin digestion of ubiquitylated proteins, and these peptides can then be identified by standard mass spectrometry approaches. This technique has led to the identification of >50,000 ubiquitylation sites in human cells and quantitative information about how many of these sites are altered upon exposure to diverse proteotoxic stressors. In addition, the diGLY proteomics approach has led to the identification of specific ubiquitin ligase targets. Here we provide a detailed method to interrogate the ubiquitin-modified proteome from any eukaryotic organism or tissue.

Key words

Ubiquitin Proteomics diGLY Affinity purification Mass spectrometry SILAC 



We thank Marilyn Leonard and Danielle Garshott for providing a critical reading of this manuscript and Ruoyu (Lulu) Li for assistance in making Fig. 1. This work was supported by the NIH (DP2-GM119132, PGM085764) (E.J.B).


  1. 1.
    Mann M, Ong S-E, Grønborg M, Steen H, Jensen ON, Pandey A (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol 20(6):261–268CrossRefGoogle Scholar
  2. 2.
    Steen H, Küster B, Fernandez M, Pandey A, Mann M (2001) Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode. Anal Chem 73(7):1440–1448CrossRefGoogle Scholar
  3. 3.
    Thalassinos K, Grabenauer M, Slade SE, Hilton GR, Bowers MT, Scrivens JH (2008) Characterization of phosphorylated peptides using traveling wave-based and drift cell ion mobility mass spectrometry. Anal Chem 81(1):248–254CrossRefGoogle Scholar
  4. 4.
    Carr SA, Huddleston MJ, Annan RS (1996) Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry. Anal Biochem 239(2):180–192CrossRefGoogle Scholar
  5. 5.
    Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20(3):301–305CrossRefGoogle Scholar
  6. 6.
    Ptacek J, Devgan G, Michaud G, Zhu H (2005) Global analysis of protein phosphorylation in yeast. Nature 438(7068):679CrossRefGoogle Scholar
  7. 7.
    Zhang H, Xiao-jun L, Martin DB, Aebersold R (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21(6):660CrossRefGoogle Scholar
  8. 8.
    Dell A, Morris HR (2001) Glycoprotein structure determination by mass spectrometry. Science 291(5512):2351–2356CrossRefGoogle Scholar
  9. 9.
    Apweiler R, Hermjakob H, Sharon N (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta 1473(1):4–8CrossRefGoogle Scholar
  10. 10.
    Brownlee M (1995) Advanced protein glycosylation in diabetes and aging. Annu Rev Med 46(1):223–234CrossRefGoogle Scholar
  11. 11.
    Spiro RG (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12(4):43R–56RCrossRefGoogle Scholar
  12. 12.
    Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, Marsischky G, Roelofs J, Finley D, Gygi SP (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21(8):921–926. Scholar
  13. 13.
    Scheffner M, Nuber U, Huibregtse JM (1995) Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373(6509):81CrossRefGoogle Scholar
  14. 14.
    Kirkpatrick DS, Denison C, Gygi SP (2005) Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat Cell Biol 7(8):750–757. Scholar
  15. 15.
    Xu P, Peng J (2006) Dissecting the ubiquitin pathway by mass spectrometry. Biochim Biophys Acta 1764(12):1940–1947. Scholar
  16. 16.
    Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J, Rush J, Hochstrasser M, Finley D, Peng J (2009) Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137(1):133–145. Scholar
  17. 17.
    Bennett EJ, Rush J, Gygi SP, Harper JW (2010) Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 143(6):951–965. Scholar
  18. 18.
    Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28(8):868–873. Scholar
  19. 19.
    Kim W, Bennett EJ, Huttlin EL, Guo A, Li J, Possemato A, Sowa ME, Rad R, Rush J, Comb MJ, Harper JW, Gygi SP (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44(2):325–340. Scholar
  20. 20.
    Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M, Choudhary C (2011) A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 10(10):M111 013284. Scholar
  21. 21.
    Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH (2001) Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol 3(2):193CrossRefGoogle Scholar
  22. 22.
    Clarke S (1993) Protein methylation. Curr Opin Cell Biol 5(6):977–983CrossRefGoogle Scholar
  23. 23.
    Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, Olsen JV, Mann M (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325(5942):834–840CrossRefGoogle Scholar
  24. 24.
    Ichimura Y, Kirisako T, Takao T, Satomi Y (2000) A ubiquitin-like system mediates protein lipidation. Nature 408(6811):488CrossRefGoogle Scholar
  25. 25.
    Mayor T, Deshaies RJ (2005) Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae. Methods Enzymol 399:385–392. Scholar
  26. 26.
    Aillet F, Lopitz-Otsoa F, Hjerpe R, Torres-Ramos M, Lang V, Rodríguez MS (2012) Isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. In: Ubiquitin family modifiers and the proteasome: reviews and protocols, pp 173–183CrossRefGoogle Scholar
  27. 27.
    Iconomou M, Saunders DN (2016) Systematic approaches to identify E3 ligase substrates. Biochem J 473(22):4083–4101. Scholar
  28. 28.
    Beaudette P, Popp O, Dittmar G (2016) Proteomic techniques to probe the ubiquitin landscape. Proteomics 16(2):273–287. Scholar
  29. 29.
    Ordureau A, Munch C, Harper JW (2015) Quantifying ubiquitin signaling. Mol Cell 58(4):660–676. Scholar
  30. 30.
    Goldknopf IL, Busch H (1977) Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24. Proc Natl Acad Sci 74(3):864–868CrossRefGoogle Scholar
  31. 31.
    Bustos D, Bakalarski CE, Yang Y, Peng J, Kirkpatrick DS (2012) Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment. Mol Cell Proteomics 11(12):1529–1540. Scholar
  32. 32.
    Sylvestersen KB, Young C, Nielsen ML (2013) Advances in characterizing ubiquitylation sites by mass spectrometry. Curr Opin Chem Biol 17(1):49–58. Scholar
  33. 33.
    Carrano AC, Bennett EJ (2013) Using the ubiquitin-modified proteome to monitor protein homeostasis function. Mol Cell Proteomics 12(12):3521–3531CrossRefGoogle Scholar
  34. 34.
    Povlsen LK, Beli P, Wagner SA, Poulsen SL, Sylvestersen KB, Poulsen JW, Nielsen ML, Bekker-Jensen S, Mailand N, Choudhary C (2012) Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nat Cell Biol 14(10):1089–1098. Scholar
  35. 35.
    Udeshi ND, Mani D, Eisenhaure T, Mertins P, Jaffe JD, Clauser KR, Hacohen N, Carr SA (2012) Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition. Mol Cell Proteomics 11(5):148–159CrossRefGoogle Scholar
  36. 36.
    Ng AH, Fang NN, Comyn SA, Gsponer J, Mayor T (2013) System-wide analysis reveals intrinsically disordered proteins are prone to ubiquitylation after misfolding stress. Mol Cell Proteomics 12(9):2456–2467. Scholar
  37. 37.
    Iesmantavicius V, Weinert BT, Choudhary C (2014) Convergence of ubiquitylation and phosphorylation signaling in rapamycin-treated yeast cells. Mol Cell Proteomics 13(8):1979–1992CrossRefGoogle Scholar
  38. 38.
    Kronke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, Svinkina T, Heckl D, Comer E, Li X, Ciarlo C, Hartman E, Munshi N, Schenone M, Schreiber SL, Carr SA, Ebert BL (2014) Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343(6168):301–305. Scholar
  39. 39.
    Elia AE, Boardman AP, Wang DC, Huttlin EL, Everley RA, Dephoure N, Zhou C, Koren I, Gygi SP, Elledge SJ (2015) Quantitative proteomic atlas of ubiquitination and acetylation in the DNA damage response. Mol Cell 59(5):867–881. Scholar
  40. 40.
    Higgins R, Gendron JM, Rising L, Mak R, Webb K, Kaiser SE, Zuzow N, Riviere P, Yang B, Fenech E, Tang X, Lindsay SA, Christianson JC, Hampton RY, Wasserman SA, Bennett EJ (2015) The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins. Mol Cell 59(1):35–49. Scholar
  41. 41.
    Kronke J, Fink EC, Hollenbach PW, MacBeth KJ, Hurst SN, Udeshi ND, Chamberlain PP, Mani DR, Man HW, Gandhi AK, Svinkina T, Schneider RK, McConkey M, Jaras M, Griffiths E, Wetzler M, Bullinger L, Cathers BE, Carr SA, Chopra R, Ebert BL (2015) Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature 523(7559):183–188. Scholar
  42. 42.
    Gendron JM, Webb K, Yang B, Rising L, Zuzow N, Bennett EJ (2016) Using the ubiquitin-modified proteome to monitor distinct and spatially restricted protein homeostasis dysfunction. Mol Cell Proteomics 15(8):2576–2593. Scholar
  43. 43.
    Sundaramoorthy E, Leonard M, Mak R, Liao J, Fulzele A, Bennett EJ (2017) ZNF598 and RACK1 regulate mammalian ribosome-associated quality control function by mediating regulatory 40S ribosomal ubiquitylation. Mol Cell 65(4):751–760 e754. Scholar
  44. 44.
    Emanuele MJ, Elia AE, Xu Q, Thoma CR, Izhar L, Leng Y, Guo A, Chen YN, Rush J, Hsu PW, Yen HC, Elledge SJ (2011) Global identification of modular cullin-RING ligase substrates. Cell 147(2):459–474. Scholar
  45. 45.
    Lee KA, Hammerle LP, Andrews PS, Stokes MP, Mustelin T, Silva JC, Black RA, Doedens JR (2011) Ubiquitin ligase substrate identification through quantitative proteomics at both the protein and peptide levels. J Biol Chem 286(48):41530–41538. Scholar
  46. 46.
    Sarraf SA, Raman M, Guarani-Pereira V, Sowa ME, Huttlin EL, Gygi SP, Harper JW (2013) Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496(7445):372–376. Scholar
  47. 47.
    Fang NN, Chan GT, Zhu M, Comyn SA, Persaud A, Deshaies RJ, Rotin D, Gsponer J, Mayor T (2014) Rsp5/Nedd4 is the major ubiquitin ligase that targets cytosolic misfolded proteins upon heat-stress. Nat Cell Biol 16(12):1227CrossRefGoogle Scholar
  48. 48.
    Tong Z, Kim M-S, Pandey A, Espenshade PJ (2014) Identification of candidate substrates for the Golgi Tul1 E3 ligase using quantitative diGly proteomics in yeast. Mol Cell Proteomics 13(11):2871–2882CrossRefGoogle Scholar
  49. 49.
    Garzia A, Jafarnejad SM, Meyer C, Chapat C, Gogakos T, Morozov P, Amiri M, Shapiro M, Molina H, Tuschl T (2017) The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs. Nat Commun 8:16056CrossRefGoogle Scholar
  50. 50.
    Na CH, Jones DR, Yang Y, Wang X, Xu Y, Peng J (2012) Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. J Proteome Res 11(9):4722–4732. Scholar
  51. 51.
    Wagner SA, Beli P, Weinert BT, Scholz C, Kelstrup CD, Young C, Nielsen ML, Olsen JV, Brakebusch C, Choudhary C (2012) Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol Cell Proteomics 11(12):1578–1585. Scholar
  52. 52.
    Buckley SM, Aranda-Orgilles B, Strikoudis A, Apostolou E, Loizou E, Moran-Crusio K, Farnsworth CL, Koller AA, Dasgupta R, Silva JC, Stadtfeld M, Hochedlinger K, Chen EI, Aifantis I (2012) Regulation of pluripotency and cellular reprogramming by the ubiquitin-proteasome system. Cell Stem Cell 11(6):783–798. Scholar
  53. 53.
    Rose CM, Isasa M, Ordureau A, Prado MA, Beausoleil SA, Jedrychowski MP, Finley DJ, Harper JW, Gygi SP (2016) Highly multiplexed quantitative mass spectrometry analysis of ubiquitylomes. Cell Syst 3(4):395–403 e394. Scholar
  54. 54.
    Sap KA, Bezstarosti K, Dekkers DHW, Voets O, Demmers JAA (2017) Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA-mediated subunit knockdown in drosophila. J Proteome Res 16(8):2848–2862. Scholar
  55. 55.
    Udeshi ND, Svinkina T, Mertins P, Kuhn E, Mani D, Qiao JW, Carr SA (2013) Refined preparation and use of anti-diglycine remnant (K-ε-GG) antibody enables routine quantification of 10,000 s of ubiquitination sites in single proteomics experiments. Mol Cell Proteomics 12(3):825–831CrossRefGoogle Scholar
  56. 56.
    Swaney DL, Beltrao P, Starita L, Guo A, Rush J, Fields S, Krogan NJ, Villén J (2013) Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation. Nat Methods 10(7):676–682CrossRefGoogle Scholar
  57. 57.
    Mertins P, Qiao JW, Patel J, Udeshi ND, Clauser KR, Mani DR, Burgess MW, Gillette MA, Jaffe JD, Carr SA (2013) Integrated proteomic analysis of post-translational modifications by serial enrichment. Nat Methods 10(7):634–637. Scholar
  58. 58.
    Theurillat JP, Udeshi ND, Errington WJ, Svinkina T, Baca SC, Pop M, Wild PJ, Blattner M, Groner AC, Rubin MA, Moch H, Prive GG, Carr SA, Garraway LA (2014) Prostate cancer. Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer. Science 346(6205):85–89. Scholar
  59. 59.
    Villen J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc 3(10):1630–1638. Scholar
  60. 60.
    Udeshi ND, Mertins P, Svinkina T, Carr SA (2013) Large-scale identification of ubiquitination sites by mass spectrometry. Nat Protoc 8(10):1950–1960. Scholar
  61. 61.
    Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896CrossRefGoogle Scholar
  62. 62.
    Satpathy S, Wagner SA, Beli P, Gupta R, Kristiansen TA, Malinova D, Francavilla C, Tolar P, Bishop GA, Hostager BS, Choudhary C (2015) Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation. Mol Syst Biol 11(6):810. Scholar
  63. 63.
    Beltrao P, Albanese V, Kenner LR, Swaney DL, Burlingame A, Villen J, Lim WA, Fraser JS, Frydman J, Krogan NJ (2012) Systematic functional prioritization of protein posttranslational modifications. Cell 150(2):413–425. Scholar
  64. 64.
    Thompson JW, Nagel J, Hoving S, Gerrits B, Bauer A, Thomas JR, Kirschner MW, Schirle M, Luchansky SJ (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
  65. 65.
    Fiskin E, Bionda T, Dikic I, Behrends C (2016) Global analysis of host and bacterial ubiquitinome in response to salmonella typhimurium infection. Mol Cell 62(6):967–981. Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Section of Cell and Developmental Biology, Division of Biological SciencesUniversity of California, San DiegoLa JollaUSA

Personalised recommendations