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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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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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.
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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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DOI: https://doi.org/10.1038/nmeth.1469
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