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Probing the efficiency of proteolytic events by positional proteomics

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Abstract
Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with ( potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography ( 1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.
Keywords
CLEAVAGE, SPECIFICITIES, DIAGONAL CHROMATOGRAPHY, IDENTIFICATION, INDUCED APOPTOSIS, N-TERMINAL PEPTIDES, HUMAN GRANZYME-B, PATHWAY, EXPRESSION, BNIP-2

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Chicago
Plasman, Kim, Petra Van Damme, Dion Kaiserman, Francis Impens, Kimberly Demeyer, Kenny Helsens, Marc Goethals, Phillip I Bird, Joël Vandekerckhove, and Kris Gevaert. 2011. “Probing the Efficiency of Proteolytic Events by Positional Proteomics.” Molecular & Cellular Proteomics 10 (2).
APA
Plasman, K., Van Damme, P., Kaiserman, D., Impens, F., Demeyer, K., Helsens, K., Goethals, M., et al. (2011). Probing the efficiency of proteolytic events by positional proteomics. MOLECULAR & CELLULAR PROTEOMICS, 10(2).
Vancouver
1.
Plasman K, Van Damme P, Kaiserman D, Impens F, Demeyer K, Helsens K, et al. Probing the efficiency of proteolytic events by positional proteomics. MOLECULAR & CELLULAR PROTEOMICS. 2011;10(2).
MLA
Plasman, Kim, Petra Van Damme, Dion Kaiserman, et al. “Probing the Efficiency of Proteolytic Events by Positional Proteomics.” MOLECULAR & CELLULAR PROTEOMICS 10.2 (2011): n. pag. Print.
@article{1197697,
  abstract     = {Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with ( potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography ( 1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.},
  author       = {Plasman, Kim and Van Damme, Petra and Kaiserman, Dion and Impens, Francis and Demeyer, Kimberly and Helsens, Kenny and Goethals, Marc and Bird, Phillip I and Vandekerckhove, Jo{\"e}l and Gevaert, Kris},
  issn         = {1535-9476},
  journal      = {MOLECULAR \& CELLULAR PROTEOMICS},
  keyword      = {CLEAVAGE,SPECIFICITIES,DIAGONAL CHROMATOGRAPHY,IDENTIFICATION,INDUCED APOPTOSIS,N-TERMINAL PEPTIDES,HUMAN GRANZYME-B,PATHWAY,EXPRESSION,BNIP-2},
  language     = {eng},
  number       = {2},
  pages        = {10},
  title        = {Probing the efficiency of proteolytic events by positional proteomics},
  url          = {http://dx.doi.org/10.1074/mcp.M110.003301},
  volume       = {10},
  year         = {2011},
}

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