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Probing the efficiency of proteolytic events by positional proteomics

Kim Plasman, Petra Van Damme UGent, Dion Kaiserman, Francis Impens UGent, Kimberly Demeyer UGent, Kenny Helsens UGent, Marc Goethals, Phillip I Bird, Joël Vandekerckhove and Kris Gevaert UGent (2011) MOLECULAR & CELLULAR PROTEOMICS. 10(2).
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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
CLEAVAGE, SPECIFICITIES, DIAGONAL CHROMATOGRAPHY, IDENTIFICATION, INDUCED APOPTOSIS, N-TERMINAL PEPTIDES, HUMAN GRANZYME-B, PATHWAY, EXPRESSION, BNIP-2
journal title
MOLECULAR & CELLULAR PROTEOMICS
Mol. Cell. Proteomics
volume
10
issue
2
pages
10 pages
Web of Science type
Article
Web of Science id
000287846500019
JCR category
BIOCHEMICAL RESEARCH METHODS
JCR impact factor
7.398 (2011)
JCR rank
5/72 (2011)
JCR quartile
1 (2011)
ISSN
1535-9476
DOI
10.1074/mcp.M110.003301
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1197697
handle
http://hdl.handle.net/1854/LU-1197697
date created
2011-03-28 10:50:19
date last changed
2016-12-19 15:45:12
@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},
}

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.