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Molecular & Cellular Proteomics 8:258-272, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Analysis of Protein Processing by N-terminal Proteomics Reveals Novel Species-specific Substrate Determinants of Granzyme B Orthologs ^*,S

Petra Van Damme^,, Sebastian Maurer-Stroh^¶,||, Kim Plasman^,, Joost Van Durme^¶,**, Niklaas Colaert^,, Evy Timmerman^,, Pieter-Jan De Bock^,, Marc Goethals^,, Frederic Rousseau^¶, Joost Schymkowitz^¶, Joël Vandekerckhove^, and Kris Gevaert^,,

From the Department of Medical Protein Research, Flanders Institute for Biotechnology (VIB), B-9000 Ghent, Belgium, Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium, and ^¶ Switch Laboratory, Flanders Interuniversity Institute of Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.

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