Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling

Nature Biotechnology volume 28, pages 868–873 (2010)Cite this article

Subjects

Abstract

Protein ubiquitination is a post-translational modification (PTM) that regulates various aspects of protein function by different mechanisms. Characterization of ubiquitination has lagged behind that of smaller PTMs, such as phosphorylation, largely because of the difficulty of isolating and identifying peptides derived from the ubiquitinated portion of proteins. To address this issue, we generated a monoclonal antibody that enriches for peptides containing lysine residues modified by diglycine, an adduct left at sites of ubiquitination after trypsin digestion. We use mass spectrometry to identify 374 diglycine-modified lysines on 236 ubiquitinated proteins from HEK293 cells, including 80 proteins containing multiple sites of ubiquitination. Seventy-two percent of these proteins and 92% of the ubiquitination sites do not appear to have been reported previously. Ubiquitin remnant profiling of the multi-ubiquitinated proteins proliferating cell nuclear antigen (PCNA) and tubulin α-1A reveals differential regulation of ubiquitination at specific sites by microtubule inhibitors, demonstrating the effectiveness of our method to characterize the dynamics of lysine ubiquitination.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Generation of monoclonal antibodies that selectively recognize diglycine-modified lysines.
Figure 2: Profiling immunopurified ubiquitin remnant–containing peptides to identify ubiquitinated proteins.
Figure 3: Bioinformatic analysis of ubiquitinated proteins and ubiquitin-modified lysines.
Figure 4: Colchicine differentially regulates the ubiquitination of two lysines in PCNA.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Xu, P. & Peng, J. Dissecting the ubiquitin pathway by mass spectrometry. Biochim. Biophys. Acta 1764, 1940–1947 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ericsson, C., Goldknopf, I.L. & Daneholt, B. Inhibition of transcription does not affect the total amount of ubiquitinated histone 2A in chromatin. Exp. Cell Res. 167, 127–134 (1986).

    Article  CAS  PubMed  Google Scholar 

  4. Galluzzi, L., Paiardini, M., Lecomte, M.C. & Magnani, M. Identification of the main ubiquitination site in human erythroid alpha-spectrin. FEBS Lett. 489, 254–258 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Tomlinson, E., Palaniyappan, N., Tooth, D. & Layfield, R. Methods for the purification of ubiquitinated proteins. Proteomics 7, 1016–1022 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Beers, E.P. & Callis, J. Utility of polyhistidine-tagged ubiquitin in the purification of ubiquitin-protein conjugates and as an affinity ligand for the purification of ubiquitin-specific hydrolases. J. Biol. Chem. 268, 21645–21649 (1993).

    CAS  PubMed  Google Scholar 

  7. Peng, J. et al. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21, 921–926 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Srikumar, T., Jeram, S.M., Lam, H. & Raught, B. A ubiquitin and ubiquitin-like protein spectral library. Proteomics 10, 337–342 (2010).

    Article  CAS  PubMed  Google Scholar 

  9. Hershko, A., Heller, H., Elias, S. & Ciechanover, A. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258, 8206–8214 (1983).

    CAS  PubMed  Google Scholar 

  10. Denis, N.J., Vasilescu, J., Lambert, J.P., Smith, J.C. & Figeys, D. Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry. Proteomics 7, 868–874 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Rechsteiner, M. Ubiquitin-mediated pathways for intracellular proteolysis. Annu. Rev. Cell Biol. 3, 1–30 (1987).

    Article  CAS  PubMed  Google Scholar 

  12. Bonifacino, J.S. & Weissman, A.M. Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu. Rev. Cell Dev. Biol. 14, 19–57 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kirkpatrick, D.S., Denison, C. & Gygi, S.P. Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics. Nat. Cell Biol. 7, 750–757 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sun, L. & Chen, Z.J. The novel functions of ubiquitination in signaling. Curr. Opin. Cell Biol. 16, 119–126 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Etlinger, J.D., Li, S.X., Guo, G.G. & Li, N. Phosphorylation and ubiquitination of the 26S proteasome complex. Enzyme Protein 47, 325–329 (1993).

    Article  CAS  PubMed  Google Scholar 

  16. Peters, J.M. Subunits and substrates of the anaphase-promoting complex. Exp. Cell Res. 248, 339–349 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Schwartz, D. & Gygi, S.P. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat. Biotechnol. 23, 1391–1398 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Ahmad, S. & Gromiha, M.M. NETASA: neural network based prediction of solvent accessibility. Bioinformatics 18, 819–824 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Catic, A., Collins, C., Church, G.M. & Ploegh, H.L. Preferred in vivo ubiquitination sites. Bioinformatics 20, 3302–3307 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Gnad, F. et al. PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol. 8, R250 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Kim, S.C. et al. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol. Cell 23, 607–618 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Zhao, S. et al. Regulation of cellular metabolism by protein lysine acetylation. Science 327, 1000–1004 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Basu, A. et al. Proteome-wide prediction of acetylation substrates. Proc. Natl. Acad. Sci. USA 106, 13785–13790 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yang, X.J. & Seto, E. Lysine acetylation: codified crosstalk with other posttranslational modifications. Mol. Cell 31, 449–461 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Prosperi, E. Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control. Prog. Cell Cycle Res. 3, 193–210 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Mayer, A. et al. The prognostic significance of proliferating cell nuclear antigen, epidermal growth factor receptor, and mdr gene expression in colorectal cancer. Cancer 71, 2454–2460 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  PubMed  Google Scholar 

  29. Jordan, M.A. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr. Med. Chem. Anticancer Agents 2, 1–17 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. Wisniewski, J.R. et al. Constitutive and dynamic phosphorylation and acetylation sites on NUCKS, a hypermodified nuclear protein, studied by quantitative proteomics. Proteins 73, 710–718 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Gigant, B. et al. Structural basis for the regulation of tubulin by vinblastine. Nature 435, 519–522 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Ravelli, R.B. et al. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 428, 198–202 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Unk, I. et al. Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Proc. Natl. Acad. Sci. USA 103, 18107–18112 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Derrien, D. et al. Muramyl dipeptide bound to poly-L-lysine substituted with mannose and gluconoyl residues as macrophage activators. Glycoconj. J. 6, 241–255 (1989).

    Article  CAS  PubMed  Google Scholar 

  35. Kirkpatrick, D.S., Weldon, S.F., Tsaprailis, G., Liebler, D.C. & Gandolfi, A.J. Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin. Proteomics 5, 2104–2111 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    Article  CAS  PubMed  Google Scholar 

  38. de Godoy, L.M. et al. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome Biol. 7, R50 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Xu, G., Shin, S.B. & Jaffrey, S.R. Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini. Proc. Natl. Acad. Sci. USA 106, 19310–19315 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Elias, J.E. & Gygi, S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Silva, J.C. et al. Quantitative proteomic analysis by accurate mass retention time pairs. Anal. Chem. 77, 2187–2200 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Mortensen, P. et al. MSQuant, an open source platform for mass spectrometry-based quantitative proteomics. J. Proteome Res. 9, 393–403 (2010).

    Article  CAS  PubMed  Google Scholar 

  43. Thomas, P.D. et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 13, 2129–2141 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Dennis, G. Jr. et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, 3 (2003).

    Article  Google Scholar 

  45. Lu, Z. et al. Predicting subcellular localization of proteins using machine-learned classifiers. Bioinformatics 20, 547–556 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Rosner, J. Test of auditory analysis skills (TAAS) in helping children overcome learning difficulties: a step-by-step guide for parents and teachers (Academic Therapy, New York, 1979).

  47. Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).

    Article  CAS  PubMed  Google Scholar 

  48. Linding, R. et al. Protein disorder prediction: implications for structural proteomics. Structure 11, 1453–1459 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Hubbard, S.J., Campbell, S.F. & Thornton, J.M. Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors. J. Mol. Biol. 220, 507–530 (1991).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank T. Neubert and G. Zhang (New York University) for useful suggestions, P. Zhou (Weill Cornell Medical College, WCMC) for the His6-ubiquitin plasmid, U. Hengst, A. Deglincerti, R. Almeida and B. Derakhshan for the assistance during initial cell culturing, S. Gross and Y. Ma (WCMC Mass Spectrometry Core Facility) for helpful discussion in MS/MS analysis, F. Campagne, L. Skrabanek, J. Sun (WCMC Institute for Computational Biomedicine) for instructions and assistance in bioinformatic analysis. The mass spectrometry work was performed at the WCMC Mass Spectrometry Core Facility using instrumentation supported by US National Institutes of Health (NIH) RR19355 and RR22615. This work was supported by grants from Weill Cornell, NIH (MH086128) (S.R.J.), and a pharmacology cancer training grant from the National Cancer Institute (T32CA062948) (G.X. and J.S.P.).

Author information

Authors and Affiliations

  1. Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA

    Guoqiang Xu, Jeremy S Paige & Samie R Jaffrey

Authors
  1. Guoqiang Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeremy S Paige
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Samie R Jaffrey
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.R.J. and G.X. conceived and designed the study. G.X. and J.S.P. conducted the experiments, and G.X. and S.R.J. analyzed the data. S.R.J. and G.X. wrote the manuscript.

Corresponding author

Correspondence to Samie R Jaffrey.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1 and 2 and Supplementary Figs. 1–18 (PDF 12207 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xu, G., Paige, J. & Jaffrey, S. Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28, 868–873 (2010). https://doi.org/10.1038/nbt.1654

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1654

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research