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:

Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry

Nature Protocols volume 4, pages 1167–1183 (2009)Cite this article

Abstract

Measuring enzymatic activities in biological fluids is a form of activity-based proteomics and may be utilized as a means of developing disease biomarkers. Activity-based assays allow amplification of output signals, thus potentially visualizing low-abundant enzymes on a virtually transparent whole-proteome background. The protocol presented here describes a semiquantitative in vitro assay of proteolytic activities in complex proteomes by monitoring breakdown of designer peptide substrates using robotic extraction and a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometric readout. Relative quantitation of the peptide metabolites is carried out by comparison with spiked internal standards, followed by statistical analysis of the resulting mini-peptidome. Partial automation provides reproducibility and throughput essential for comparing large sample sets. The approach may be used for diagnostic or predictive purposes and it enables profiling of 96 samples in 30 h. It could be tailored to many diagnostic and pharmaco-dynamic purposes as a readout of catalytic and metabolic activities in body fluids or tissues.

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: Exopeptidase Assay Operation.
Figure 2: Automation of sample processing for serum peptidome analysis.
Figure 3: C3f peptide degradation ladder.
Figure 4: Exopeptidase assay: C3f isotope-labeling design.
Figure 5: Assay performance experiment.
Figure 6: MALDI-TOF MS overlays of selected ion peaks corresponding to C3f_13C degradation products in sera from thyroid cancer patients and healthy controls.

Similar content being viewed by others

References

  1. Lopez-Otin, C. & Bond, J.S. Proteases: multifunctional enzymes in life and disease. J. Biol. Chem. 283, 30433–30437 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Overall, C.M. & Blobel, C.P. In search of partners: linking extracellular proteases to substrates. Nat. Rev. 8, 245–257 (2007).

    Article  CAS  Google Scholar 

  3. Palermo, C. & Joyce, J.A. Cysteine cathepsin proteases as pharmacological targets in cancer. Trends Pharmacol. Sci. 29, 22–28 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Lopez-Otin, C. & Matrisian, L.M. Emerging roles of proteases in tumour suppression. Nat. Rev. Cancer 7, 800–808 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Egeblad, M. & Werb, Z. New functions for the matrix metalloproteinases in cancer progression. Nat. Rev. Cancer 2, 161–174 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Leiting, B. et al. Catalytic properties and inhibition of proline-specific dipeptidyl peptidases II, IV and VII. Biochem. J. 371, 525–532 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Martinez, J.M. et al. Aminopeptidase activities in breast cancer tissue. Clin. Chem. 45, 1797–1802 (1999).

    CAS  PubMed  Google Scholar 

  8. Villanueva, J. et al. Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry. Anal. Chem. 76, 1560–1570 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Villanueva, J. et al. Differential exoprotease activities confer tumor-specific serum peptidome patterns. J. Clin. Invest. 116, 271–284 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Villanueva, J. et al. Serum peptidome patterns that distinguish metastatic thyroid carcinoma from cancer-free controls are unbiased by gender and age. Mol. Cell. Proteomics 5, 1840–1852 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Villanueva, J. et al. A sequence-specific exopeptidase activity test (SSEAT) for 'functional' biomarker discovery. Mol. Cell. Proteomics 7, 509–518 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Villanueva, J., Lawlor, K., Toledo-Crow, R. & Tempst, P. Automated serum peptide profiling. Nat. Protoc. 1, 880–891 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Villanueva, J., Philip, J., DeNoyer, L. & Tempst, P. Data analysis of assorted serum peptidome profiles. Nat. Protoc. 2, 588–602 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by US National Institutes of Health Grants R33 CA111942 and U24 CA126485. It is part of National Cancer Institute's Clinical Proteomic Technologies Initiative and Clinical Proteomic Technology Assessment for Cancer (CPTAC) consortium (Broad Institute of MIT and Harvard, Memorial Sloan-Kettering Cancer Center, Purdue University, University of California, San Francisco and Vanderbilt University School of Medicine).

Author information

Authors and Affiliations

  1. Protein Center, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Josep Villanueva, Arpi Nazarian, Kevin Lawlor & Paul Tempst

  2. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Josep Villanueva & Paul Tempst

Authors
  1. Josep Villanueva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Arpi Nazarian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kevin Lawlor
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paul Tempst
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul Tempst.

Supplementary information

Supplementary Fig. 1

Design diagram of the custom aliquoting rack. Custom aliquoting rack designed to hold sixteen rows of nine 0.5-mL eppendorf tubes for use with a liquid handler robot of variable pipette separation (Tecan Genesis Freedom). At the end of each row is a sample source vial kept in a separate rack. The rack is machined in aluminum and placed on a base with inserts that match the work surface of the liquid handler. The component parts of the rack: (a) 9 × 16 aliquot vial tray (yellow), (b) 16 source vial block (green), mounted on (c) the matching base for the Tecan handling robot (gray, red). (PDF 113 kb)

Supplementary Fig. 2

Design diagrams of the custom side-magnet rack. Panel A shows the exploded assembly view of the top part, magnets and bottom part of the rack (and a 96-well vial plate). Panel B shows the projected design views of the top and bottom parts of the rack. The machined material used is clear Lexan and all dimensions are in millimeters. (PDF 238 kb)

Supplementary Fig. 3

Design diagrams of the custom bottom-magnet rack. Panel A shows the exploded assembly view of the top part, magnets, and bottom part of the rack (and a 96-well vial plate). Panel B shows the orthogonal projected design views of the top and bottom parts of the rack. The machined material used is clear Lexan and all dimensions are in millimeters. (PDF 270 kb)

Supplementary Manual 1

Instructions for the use of the Mass Spectra Viewer (MSV). (PDF 894 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Villanueva, J., Nazarian, A., Lawlor, K. et al. Monitoring peptidase activities in complex proteomes by MALDI-TOF mass spectrometry. Nat Protoc 4, 1167–1183 (2009). https://doi.org/10.1038/nprot.2009.88

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.88

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