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Complementary positional proteomics for screening substrates of endo- and exoproteases

Nature Methods volume 7, pages 512–515 (2010)Cite this article

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Abstract

We describe a positional proteomics approach to simultaneously analyze N- and C-terminal peptides and used it to screen for human protein substrates of granzyme B and carboxypeptidase A4 in human cell lysates. This approach allowed comprehensive proteome studies, and we report the identification of 965 database-annotated protein C termini, 334 neo–C termini resulting from granzyme B processing and 16 neo–C termini resulting from carboxypeptidase A4 processing.

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Figure 1: Outline of the COFRADIC-based complementary positional proteomics procedure.
Figure 2: N- and C-terminal peptides provide complementary data.
Figure 3: Sequence analysis of identified hGrB cleavage sites.

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Acknowledgements

P. Bird (Monash University) kindly provided recombinant hGrB. P.V.D. was supported by a postdoctoral fellow grant of the Research Foundation, Flanders (FWO-Vlaanderen). K.H. is supported by a PhD grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). This work was supported by research grants from the Fund for Scientific Research, Flanders (Belgium) (project numbers G.0156.05, G.0077.06 and G.0042.07), the Concerted Research Actions (project BOF07/GOA/012) from Ghent University and the Interuniversity Attraction Poles (IUAP06). F.X.A. and S.B. acknowledge support from grants LSH6-2006-018830-CAMP (EC) and BIO2007-68046 (Ministerio de Ciencia y Innovación, Spain).

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Authors and Affiliations

  1. Department of Medical Protein Research, Vlaams Instituut voor Biotechnologie, Ghent, Belgium

    Petra Van Damme, An Staes, Kenny Helsens, Niklaas Colaert, Evy Timmerman, Joël Vandekerckhove & Kris Gevaert

  2. Department of Biochemistry, Ghent University, Ghent, Belgium

    Petra Van Damme, An Staes, Kenny Helsens, Niklaas Colaert, Evy Timmerman, Joël Vandekerckhove & Kris Gevaert

  3. Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain

    Silvia Bronsoms & Francesc X Aviles

Authors
  1. Petra Van Damme
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  2. An Staes
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  3. Silvia Bronsoms
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  4. Kenny Helsens
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  5. Niklaas Colaert
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  8. Joël Vandekerckhove
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  9. Kris Gevaert
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Contributions

P.V.D., S.B., F.X.A., J.V. and K.G. designed the experiments. P.V.D., A.S. and E.T. generated the data. P.V.D., K.H. and N.C. analyzed the data. P.V.D., F.X.A., J.V. and K.G. wrote the manuscript.

Corresponding authors

Correspondence to Petra Van Damme or Kris Gevaert.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Tables 2–3 (PDF 467 kb)

Supplementary Table 1

General overview of peptide and protein identifications. (XLS 1432 kb)

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Van Damme, P., Staes, A., Bronsoms, S. et al. Complementary positional proteomics for screening substrates of endo- and exoproteases. Nat Methods 7, 512–515 (2010). https://doi.org/10.1038/nmeth.1469

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