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:

A proteomics approach to understanding protein ubiquitination

Abstract

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Strategy for identifying the precise site of ubiquitination by MS/MS.
Figure 2: Isolation and sequence analysis of yeast ubiquitin conjugates.
Figure 3: (a) Comparison among yeast proteome (n = 6,139), identified proteins in the candidate ubiquitin-conjugate fraction (n = 1,075) and proteins whose ubiquitination sites in the molecular environment is known (n = 72).

Similar content being viewed by others

References

  1. Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378 (2001).

    Article  CAS  Google Scholar 

  2. Oda, Y., Nagasu, T. & Chait, B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379–382 (2001).

    Article  CAS  Google Scholar 

  3. Ficarro, S.B. et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 (2002).

    Article  CAS  Google Scholar 

  4. Weissman, A.M. Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell. Biol. 2, 169–178 (2001).

    Article  CAS  Google Scholar 

  5. Glickman, M.H. & Ciechanover, A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82, 373–428 (2002).

    Article  CAS  Google Scholar 

  6. Layfield, R., Alban, A., Mayer, R.J. & Lowe, J. The ubiquitin protein catabolic disorders. Neuropathol. Appl. Neurobiol. 27, 171–179 (2001).

    Article  CAS  Google Scholar 

  7. Spence, J. et al. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 102, 67–76 (2000).

    Article  CAS  Google Scholar 

  8. Finley, D. et al. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant. Mol. Cell Biol. 14, 5501–5509 (1994).

    Article  CAS  Google Scholar 

  9. Gygi, M.P., Licklider, L.J., Peng, J. & Gygi, S.P. Combining two-dimensional chromatography and mass spectrometry for the separation of complex peptide mixtures. in Protein analysis: A Laboratory Manual (ed. Simpson, R.) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2003), in the press.

    Google Scholar 

  10. Peng, J. & Gygi, S.P. Proteomics: the move to mixtures. J. Mass Spectrom. 36, 1083–1091 (2001).

    Article  CAS  Google Scholar 

  11. Eng, J., McCormack, A.L. & Yates, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    Article  CAS  Google Scholar 

  12. Rotin, D., Staub, O. & Haguenauer-Tsapis, R. Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J. Membr. Biol. 176, 1–17 (2000).

    Article  CAS  Google Scholar 

  13. Hicke, L. Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels. Trends Cell Biol. 9, 107–112 (1999).

    Article  CAS  Google Scholar 

  14. Pickart, C.M. Ubiquitin in chains. Trends Biochem. Sci. 25, 544–548 (2000).

    Article  CAS  Google Scholar 

  15. Mastrandrea, L.D., You, J., Niles, E.G. & Pickart, C.M. E2/E3-mediated assembly of lysine 29-linked polyubiquitin chains. J. Biol. Chem. 274, 27299–27306 (1999).

    Article  CAS  Google Scholar 

  16. Spence, J., Sadis, S., Haas, A.L. & Finley, D. A ubiquitin mutant with specific defects in DNA repair and multiubiquitination. Mol. Cell Biol. 15, 1265–1273 (1995).

    Article  CAS  Google Scholar 

  17. Baboshina, O.V. & Haas, A.L. Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5. J. Biol. Chem. 271, 2823–2831 (1996).

    Article  CAS  Google Scholar 

  18. Finley, D. Signal transduction. An alternative to destruction. Nature 412, 283, 285–286 (2001).

    Article  CAS  Google Scholar 

  19. Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    Article  CAS  Google Scholar 

  20. Ciechanover, A., Orian, A. & Schwartz, A.L. Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 22, 442–451 (2000).

    Article  CAS  Google Scholar 

  21. Sloper-Mould, K.E., Jemc, J.C., Pickart, C.M. & Hicke, L. Distinct functional surface regions on ubiquitin. J. Biol. Chem. 276, 30483–30489 (2001).

    Article  CAS  Google Scholar 

  22. Cook, W.J., Jeffrey, L.C., Kasperek, E. & Pickart, C.M. Structure of tetraubiquitin shows how multiubiquitin chains can be formed. J. Mol. Biol. 236, 601–609 (1994).

    Article  CAS  Google Scholar 

  23. Goldknopf, I.L. et al. Isolation and characterization of protein A24, a “histone-like” non-histone chromosomal protein. J. Biol. Chem. 250, 7182–7187 (1975).

    CAS  PubMed  Google Scholar 

  24. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  25. Washburn, M.P., Wolters, D. & Yates, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).

    Article  CAS  Google Scholar 

  26. Link, A.J. et al. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682 (1999).

    Article  CAS  Google Scholar 

  27. Licklider, L.J., Thoreen, C.C., Peng, J. & Gygi, S.P. Automation of nanoscale microcapillary liquid chromatography–tandem mass spectrometry with a vented column. Anal. Chem. 74, 3076–3083 (2002).

    Article  CAS  Google Scholar 

  28. Gygi, S.P., Rist, B., Griffin, T.J., Eng, J. & Aebersold, R. Proteome analysis of low abundance proteins using multidimensional chromatography and isotope coded affinity tags. J. Proteome Res. 1, 47–54 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Yao and R. Cohen for sharing ubiquitin proteins and J. Rush and M. Comb for phosphopeptide synthesis. We also thank A. Goldberg, R. Cohen, D. Moazed, S. Gerber and L. Licklider for encouraging discussions. This work was supported in part by National Institutes of Health grants HG00041 (S.P.G.), GM67945 (S.P.G.), GM43601 (D.F.), Giovani-Armenise Harvard Foundation (S.P.G.), Jane Coffin Childs Memorial Fund for Medical Research (J.P.) and a European Molecular Biology Organization long-term fellowship (J.R.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Steven P Gygi.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Peng, J., Schwartz, D., Elias, J. et al. A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21, 921–926 (2003). https://doi.org/10.1038/nbt849

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt849

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing