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.

  • Article
  • Published:

Global identification of peptidase specificity by multiplex substrate profiling

Nature Methods volume 9, pages 1095–1100 (2012)Cite this article

Subjects

Abstract

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography–tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

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: Design of a physiochemically diverse peptide library and development of a multiplex substrate assay.
Figure 2: Validation of the multiplex substrate-profiling technique using the aspartyl peptidase cathepsin E.
Figure 3: Substrate profiling of an exopeptidase PRCP.
Figure 4: Specificity constants of individual substrates can be calculated from the MSP-MS assay.
Figure 5: Class-specific peptidase inhibitors can dissect the proteolytic signatures of biological samples.

Similar content being viewed by others

References

  1. Puente, X.S., Gutiérrez-Fernández, A., Velasco, G. & López-Otín, C. A genomic view of the complexity of mammalian proteolytic systems. Biochem. Soc. Trans. 33, 331–334 (2005).

    Article  CAS  Google Scholar 

  2. Van Damme, P., Vandekerckhove, J. & Gevaert, K. Disentanglement of protease substrate repertoires. Biol. Chem. 389, 371–381 (2008).

    Article  CAS  Google Scholar 

  3. Matthews, D.J. & Wells, J.A. Substrate phage: selection of protease substrates by monovalent phage display. Science 260, 1113–1117 (1993).

    Article  CAS  Google Scholar 

  4. Boulware, K.T. & Daugherty, P.S. Protease specificity determination by using cellular libraries of peptide substrates (CLiPS). Proc. Natl. Acad. Sci. USA 103, 7583–7588 (2006).

    Article  CAS  Google Scholar 

  5. Turk, B.E., Huang, L.L., Piro, E.T. & Cantley, L.C. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat. Biotechnol. 19, 661–667 (2001).

    Article  CAS  Google Scholar 

  6. Harris, J.L. et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. USA 97, 7754–7759 (2000).

    Article  CAS  Google Scholar 

  7. Choe, Y. et al. Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J. Biol. Chem. 281, 12824–12832 (2006).

    Article  CAS  Google Scholar 

  8. auf dem Keller, U. & Schilling, O. Proteomic techniques and activity-based probes for the system-wide study of proteolysis. Biochimie 92, 1705–1714 (2010).

    Article  CAS  Google Scholar 

  9. Mahrus, S. et al. Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134, 866–876 (2008).

    Article  CAS  Google Scholar 

  10. Staes, A. et al. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC). Proteomics 8, 1362–1370 (2008).

    Article  CAS  Google Scholar 

  11. Impens, F. et al. A quantitative proteomics design for systematic identification of protease cleavage events. Mol. Cell. Proteomics 9, 2327–2333 (2010).

    Article  CAS  Google Scholar 

  12. Colaert, N., Helsens, K., Martens, L., Vandekerckhove, J. & Gevaert, K. Improved visualization of protein consensus sequences by iceLogo. Nat. Methods 6, 786–787 (2009).

    Article  CAS  Google Scholar 

  13. Chajkowski, S.M. et al. Highly selective hydrolysis of kinins by recombinant prolylcarboxypeptidase. Biochem. Biophys. Res. Commun. 405, 338–343 (2011).

    Article  CAS  Google Scholar 

  14. Sun, J. et al. Importance of the P4′ residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop. J. Biol. Chem. 276, 15177–15184 (2001).

    Article  CAS  Google Scholar 

  15. Cordingley, M.G., Callahan, P.L., Sardana, V.V., Garsky, V.M. & Colonno, R.J. Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro. J. Biol. Chem. 265, 9062–9065 (1990).

    CAS  PubMed  Google Scholar 

  16. Sojka, D. et al. Characterization of gut-associated cathepsin D hemoglobinase from tick Ixodes ricinus (IrCD1). J. Biol. Chem. 287, 21152–21163 (2012).

    Article  CAS  Google Scholar 

  17. Ingram, J.R. et al. Investigation of the proteolytic functions of an expanded cercarial elastase gene family in Schistosoma mansoni. PLoS Negl. Trop. Dis. 6, e1589 (2012).

    Article  CAS  Google Scholar 

  18. Knudsen, G.M., Medzihradszky, K.F., Lim, K.-C., Hansell, E. & McKerrow, J.H. Proteomic analysis of Schistosoma mansoni cercarial secretions. Mol. Cell. Proteomics 4, 1862–1875 (2005).

    Article  CAS  Google Scholar 

  19. Curwen, R.S., Ashton, P.D., Sundaralingam, S. & Wilson, R.A. Identification of novel proteases and immunomodulators in the secretions of schistosome cercariae that facilitate host entry. Mol. Cell. Proteomics 5, 835–844 (2006).

    Article  CAS  Google Scholar 

  20. Nolan-Stevaux, O. et al. GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation. Genes Dev. 23, 24–36 (2009).

    Article  CAS  Google Scholar 

  21. Fricker, L. Carboxypeptidase E. in Handbook of Proteolytic Enzymes 2nd edn. (eds. Barrett, A.J., Rawlings, N.D. & Woessner, J.F.) 840–844 (Academic, 2004).

  22. Caglic, D. et al. Murine and human cathepsin B exhibit similar properties: possible implications for drug discovery. Biol. Chem. 390, 175–179 (2009).

    Article  CAS  Google Scholar 

  23. Mason, R.W., Johnson, D.A., Barrett, A.J. & Chapman, H.A. Elastinolytic activity of human cathepsin L. Biochem. J. 233, 925–927 (1986).

    Article  CAS  Google Scholar 

  24. Zaidi, N., Herrmann, T., Voelter, W. & Kalbacher, H. Recombinant cathepsin E has no proteolytic activity at neutral pH. Biochem. Biophys. Res. Commun. 360, 51–55 (2007).

    Article  CAS  Google Scholar 

  25. Impens, F. et al. MS-driven protease substrate degradomics. Proteomics 10, 1284–1296 (2010).

    Article  CAS  Google Scholar 

  26. Rawlings, N.D., Barrett, A.J. & Bateman, A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 40, D343–D350 (2012).

    Article  CAS  Google Scholar 

  27. Ruggles, S.W., Fletterick, R.J. & Craik, C.S. Characterization of structural determinants of granzyme B reveals potent mediators of extended substrate specificity. J. Biol. Chem. 279, 30751–30759 (2004).

    Article  CAS  Google Scholar 

  28. Prudova, A., auf dem Keller, U., Butler, G.S. & Overall, C.M. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics. Mol. Cell. Proteomics 9, 894–911 (2010).

    Article  CAS  Google Scholar 

  29. Zhou, C. et al. Design and synthesis of prolylcarboxypeptidase (PrCP) inhibitors to validate PrCP as a potential target for obesity. J. Med. Chem. 53, 7251–7263 (2010).

    Article  CAS  Google Scholar 

  30. Cohen, F.E. et al. Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling. Biochemistry 30, 11221–11229 (1991).

    Article  CAS  Google Scholar 

  31. Cruz-Monserrate, Z. et al. Detection of pancreatic cancer tumours and precursor lesions by cathepsin E activity in mouse models. Gut 61, 1315–1322 (2012).

    Article  CAS  Google Scholar 

  32. Barkan, D.T. et al. Prediction of protease substrates using sequence and structure features. Bioinformatics 26, 1714–1722 (2010).

    Article  CAS  Google Scholar 

  33. Harris, J.L., Peterson, E.P., Hudig, D., Thornberry, N.A. & Craik, C.S. Definition and redesign of the extended substrate specificity of granzyme B. J. Biol. Chem. 273, 27364–27373 (1998).

    Article  CAS  Google Scholar 

  34. Salter, J.P. et al. Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes. J. Biol. Chem. 277, 24618–24624 (2002).

    Article  CAS  Google Scholar 

  35. Chalkley, R.J., Baker, P.R., Medzihradszky, K.F., Lynn, A.J. & Burlingame, A.L. In-depth analysis of tandem mass spectrometry data from disparate instrument types. Mol. Cell. Proteomics 7, 2386–2398 (2008).

    Article  CAS  Google Scholar 

  36. 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  Google Scholar 

  37. Liu, H., Sadygov, R.G. & Yates, J.R. III. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 76, 4193–4201 (2004).

    Article  CAS  Google Scholar 

  38. Choi, H., Fermin, D. & Nesvizhskii, A.I. Significance analysis of spectral count data in label-free shotgun proteomics. Mol. Cell. Proteomics 7, 2373–2385 (2008).

    Article  CAS  Google Scholar 

  39. O'Donoghue, A.J. et al. Inhibition of a secreted glutamic peptidase prevents growth of the fungus Talaromyces emersonii. J. Biol. Chem. 283, 29186–29195 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank F.A. Lewis at the US National Institutes of Health–National Institute of Allergy and Infectious Disease Schistosomiasis Resource Center for providing S. mansoni–infected Biomphalaria glabrata and K.C. Lim of the Sandler Center for Drug Discovery at UCSF for maintenance of the S. mansoni life cycle. We thank C. Tajon, C. Brown, G. Lee and S. Clarke from UCSF, M. Tuohy from National University of Ireland, Galway, and W. Geissler from Merck & Co. for providing purified peptidases and D. Hanahan from UCSF for the mouse PDAC cell line. This project was supported by grants from the US National Institutes of Health: P50 GM082250 (C.S.C.), RO1 CA128765 (C.S.C.), P41 RR001614 (A.L.B.), P41 GM103481 (A.L.B.) and K12 GM081266 (A.A.E.-R.) and the Sandler Center (C.S.C.).

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco (UCSF), San Francisco, California, USA

    Anthony J O'Donoghue, A Alegra Eroy-Reveles, Giselle M Knudsen, Min Zhou, Jacob B Statnekov, Daniel R Hostetter, David A Maltby, Alma L Burlingame & Charles S Craik

  2. Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA

    A Alegra Eroy-Reveles & Marc O Anderson

  3. Sandler Center for Drug Discovery, UCSF, San Francisco, California, USA

    Jessica Ingram & James H McKerrow

  4. Howard Hughes Medical Institute, UCSF, San Francisco, California, USA

    Alexander L Greninger & Joseph L DeRisi

  5. Department of Biochemistry and Biophysics, UCSF, San Francisco, California, USA

    Alexander L Greninger & Joseph L DeRisi

  6. Department of Electrical and Computer Engineering, University of Maryland, College Park, Maryland, USA

    Gang Qu

Authors
  1. Anthony J O'Donoghue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A Alegra Eroy-Reveles
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Giselle M Knudsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jessica Ingram
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Min Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jacob B Statnekov
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexander L Greninger
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Daniel R Hostetter
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gang Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. David A Maltby
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Marc O Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Joseph L DeRisi
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. James H McKerrow
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Alma L Burlingame
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Charles S Craik
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.J.O., M.Z. and C.S.C. conceptualized the MSP-MS library and assay. C.S.C. directed and coordinated the project. M.O.A. and G.Q. wrote the pair-fitting script and J.B.S. wrote the MSP-MS extractor script. A.J.O., A.A.E.-R. and M.Z. synthesized and purified the peptides. A.J.O., A.A.E.-R. and G.M.K. performed the MSP-MS assays and analyzed the data. G.M.K., D.A.M. and A.L.B. developed the mass spectrometry protocol. J.I. and J.H.M. generated S. mansoni–infected snail samples. A.J.O. and D.R.H. generated conditioned PDAC medium. A.L.G. and J.L.D. provided viral peptidases. A.J.O., G.M.K., A.A.E.-R. and C.S.C. wrote the manuscript, and all authors participated in editing it.

Corresponding author

Correspondence to Charles S Craik.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–5 (PDF 785 kb)

Supplementary Data

P4–P4' cleavage sequences generated by multiplex substrate profiling by mass spectrometry for cathepsin E, prolylcarboxypeptidase, cruzain, MMP2, matriptase, DPP-IV, eqolisin, aspergillopepsin, HIV-1 and HIV-2 proteases, granzyme B, pancreatic ductal adenocarcinoma–conditioned medium and Shistosoma mansoni–infected snail sheds. (XLSX 130 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

O'Donoghue, A., Eroy-Reveles, A., Knudsen, G. et al. Global identification of peptidase specificity by multiplex substrate profiling. Nat Methods 9, 1095–1100 (2012). https://doi.org/10.1038/nmeth.2182

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2182

This article is cited by

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