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.

  • Protocol
  • Published:

Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS

Nature Protocols volume 8, pages 1155–1168 (2013)Cite this article

Subjects

Abstract

Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography–mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2–3 d, and approximately 8–10 d are needed to complete the entire protocol.

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: Proteasome ABPP.
Figure 2: Results of fluorescence-based quantitative proteasome ABPP.
Figure 3: Schematic overview of the quantitative activity-based proteasome profiling experiments.
Figure 4: Direct and two-step activity-based affinity purification of the proteasome β-subunits with streptavidin beads from EL4 lysate.
Figure 5: Active site identification of the murine β2 subunit by one-step and two-step ABPP with LC-MS analysis.
Figure 6: Competitive ABPP assay in EL4 cells.

Similar content being viewed by others

References

  1. Rock, K.L. & Goldberg, A.L. Degradation of cell proteins and the generation of MHC class I-presented peptides. Annu. Rev. Immunol. 17, 739–779 (1999).

    Article  CAS  Google Scholar 

  2. Kloetzel, P.M. & Ossendorp, F. Proteasome and peptidase function in MHC-class-I-mediated antigen presentation. Curr. Opin. Immunol. 16, 76–81 (2004).

    Article  CAS  Google Scholar 

  3. Kane, R.C., Bross, P.F., Farrell, A.T. & Pazdur, R. Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist 8, 508–513 (2003).

    Article  Google Scholar 

  4. Jung, T., Catalgol, B. & Grune, T. The proteasomal system. Mol. Aspects Med. 30, 191–296 (2009).

    Article  CAS  Google Scholar 

  5. Marques, A.J., Palanimurugan, R., Matias, A.C., Ramos, P.C. & Dohmen, R.J. Catalytic mechanism and assembly of the proteasome. Chem. Rev. 109, 1509–1536 (2009).

    Article  CAS  Google Scholar 

  6. Borissenko, L. & Groll, M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem. Rev. 107, 687–717 (2007).

    Article  CAS  Google Scholar 

  7. Murata, S. et al. Regulation of CD8+ T cell development by thymus-specific proteasomes. Science 316, 1349–1353 (2007).

    Article  CAS  Google Scholar 

  8. Verdoes, M., Florea, B.I., van der Marel, G.A. & Overkleeft, H.S. Chemical tools to study the proteasome. Eur. J. Org. Chem. 20, 3301–3313 (2009).

    Article  Google Scholar 

  9. Kisselev, A.F. & Goldberg, A.L. Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates. Methods Enzymol. 398, 364–378 (2005).

    Article  CAS  Google Scholar 

  10. Li, N., Overkleeft, H.S. & Florea, B.I. Activity-based protein profiling: an enabling technology in chemical biology research. Curr. Opin. Chem. Biol. 16, 227–233 (2012).

    Article  CAS  Google Scholar 

  11. Fenteany, G. et al. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268, 726–731 (1995).

    Article  CAS  Google Scholar 

  12. Meng, L. et al. Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc. Natl. Acad. Sci. USA 96, 10403–10411 (1999).

    Article  CAS  Google Scholar 

  13. Bogyo, M. et al. Covalent modification of the active site threonine of proteasomal β-subunits and the Escherichia coli homolog HslV by a new class of inhibitors. Proc. Natl. Acad. Sci. USA 94, 6629–6634 (1997).

    Article  CAS  Google Scholar 

  14. Kessler, B.M. et al. Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic β-subunits. Chem. Biol. 8, 913–929 (2001).

    Article  CAS  Google Scholar 

  15. Berkers, C.R. et al. Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib. Nat. Methods 2, 357–362 (2005).

    Article  CAS  Google Scholar 

  16. Verdoes, M. et al. A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo. Chem. Biol. 13, 1217–1226 (2006).

    Article  CAS  Google Scholar 

  17. Florea, B.I. et al. Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t. Chem. Biol. 17, 795–801 (2010).

    Article  CAS  Google Scholar 

  18. Verdoes, M. et al. A panel of subunit-selective activity-based proteasome probes. Org. Biomol. Chem. 8, 2719–2727 (2010).

    Article  CAS  Google Scholar 

  19. Kraus, M. et al. Activity patterns of proteasome subunits reflect bortezomib sensitivity of hematologic malignancies and are variable in primary human leukemia cells. Leukemia 21, 84–92 (2007).

    Article  CAS  Google Scholar 

  20. Ruckrich, T. et al. Characterization of the ubiquitin-proteasome system in bortezomib-adapted cells. Leukemia 23, 1098–1105 (2009).

    Article  CAS  Google Scholar 

  21. Saxon, E. & Bertozzi, C. Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2017 (2000).

    Article  CAS  Google Scholar 

  22. Ovaa, H. et al. Chemistry in living cells: detection of active proteasomes by a two-step labeling strategy. Angew. Chem. Int. Ed. 42, 3626–3629 (2003).

    Article  CAS  Google Scholar 

  23. Willems, L.I. et al. Triple bioorthogonal ligation strategy for simultaneous labeling of multiple enzymatic activities. Angew. Chem. Int. Ed. 51, 4431–4434 (2012).

    Article  CAS  Google Scholar 

  24. Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009).

    Article  CAS  Google Scholar 

  25. Raijmakers, R. et al. Target profiling of a small library of phosphodiesterase 5 (PDE5) inhibitors using chemical proteomics. Chem.Med.Chem. 5, 1927–1936 (2010).

    Article  CAS  Google Scholar 

  26. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  27. Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).

    Article  CAS  Google Scholar 

  28. Kisselev, A.F., Songyang, Z. & Goldberg, A.L. Why does threonine, and not serine, function as the active site nucleophile in proteasomes? J. Biol. Chem. 275, 14831–14837 (2000).

    Article  CAS  Google Scholar 

  29. Singh, J., Petter, R.C., Baillie, T.A. & Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 10, 307–317 (2011).

    Article  CAS  Google Scholar 

  30. Muchamuel, T. et al. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat. Med. 15, 781–787 (2009).

    Article  CAS  Google Scholar 

  31. Kallemeijn, W.W. et al. Novel activity-based probes for broad-spectrum profiling of retaining β-exoglucosidases in situ and in vivo. Angew. Chem. Int. Ed. 51, 12529–12533 (2012).

    Article  CAS  Google Scholar 

  32. Chang, J.T. et al. Asymmetric proteasome segregation as a mechanism for unequal partitioning of the transcription factor T-bet during T lymphocyte division. Immunity 34, 492–504 (2011).

    Article  CAS  Google Scholar 

  33. Adibekian, A. et al. Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors. Nat. Chem. Biol. 7, 469–478 (2011).

    Article  CAS  Google Scholar 

  34. Weerapana, E., Speers, A.E. & Cravatt, B.F. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—a general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2, 1414–1425 (2007).

    Article  CAS  Google Scholar 

  35. Lin, G. et al. Inhibitors selective for mycobacterial versus human proteasomes. Nature 461, 621–626 (2009).

    Article  CAS  Google Scholar 

  36. Gu, C. et al. Proteasome activity profiling: a simple, robust and versatile method revealing subunit-selective inhibitors and cytoplasmic, defense-induced proteasome activities. Plant J. 62, 160–170 (2010).

    Article  CAS  Google Scholar 

  37. van der Linden, W.A. et al. Two-step bioorthogonal activity-based proteasome profiling using copper-free click reagents: a comparative study. Bioorg. Med. Chem. 20, 662–666 (2012).

    Article  CAS  Google Scholar 

  38. Wessel, D. & Flugge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).

    Article  CAS  Google Scholar 

  39. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  Google Scholar 

  40. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    Article  CAS  Google Scholar 

  41. Olsen, J.V. et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell Proteomics 4, 2010–2021 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to the Netherlands Genomics Initiative, the Netherlands Proteomics Center and the Netherlands Organization for Scientific Research (NWO) for funding this work.

Author information

Authors and Affiliations

  1. Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics Centre, Leiden, The Netherlands

    Nan Li, Chi-Lin Kuo, Guillem Paniagua, Hans van den Elst, Martijn Verdoes, Lianne I Willems, Wouter A van der Linden, Mark Ruben, Eric van Genderen, Jacob Gubbens, Gilles P van Wezel, Herman S Overkleeft & Bogdan I Florea

Authors
  1. Nan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chi-Lin Kuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guillem Paniagua
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hans van den Elst
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martijn Verdoes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lianne I Willems
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wouter A van der Linden
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mark Ruben
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Eric van Genderen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jacob Gubbens
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Gilles P van Wezel
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Herman S Overkleeft
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Bogdan I Florea
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.L. carried out the cell culture (treatment), affinity purification, SDS-PAGE imaging work and prepared the first draft of the manuscript and subsequent modifications. C.-L.K. and G.P. carried out the cell culture (treatment), affinity purification and SDS-PAGE imaging work. M.V., L.I.W., W.A.v.d.L. and M.R. refined the design and synthesis of the chemical tools used in this work. H.v.d.E., E.v.G. and J.G. performed LC-MS analysis and data processing. G.P.v.W., H.S.O. and B.I.F. supervised the work and were involved in manuscript preparation.

Corresponding author

Correspondence to Bogdan I Florea.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, N., Kuo, CL., Paniagua, G. et al. Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS. Nat Protoc 8, 1155–1168 (2013). https://doi.org/10.1038/nprot.2013.065

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.065

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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