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Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S

(2015) PROTEOMICS. 15(14). p.2479-2490
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Organization
Abstract
Proteases are important effectors of numerous physiological and pathological processes. Reliable determination of a protease's specificity is crucial to understand protease function and to develop activity-based probes and inhibitors. During the last decade, various proteomic approaches for profiling protease substrate specificities were reported. Although most of these approaches can identify up to thousands of substrate cleavage events in a single experiment, they are often time consuming and methodologically challenging as some of these approaches require rather complex sample preparation procedures. For such reasons their application is often limited to those labs that initially introduced them. Here, we report on a fast and simple approach for proteomic profiling of protease specificities (fast profiling of protease specificity (FPPS)), which can be applied to complex protein mixtures. FPPS is based on trideutero-acetylation of novel N-termini generated by the action of proteases and subsequent peptide fractionation on Stage Tips containing ion-exchange and reverse phase chromatographic resins. FPPS can be performed in 2 days and does not require extensive fractionation steps. Using this approach, we have determined the specificity profiles of the cysteine cathepsins K, L and S. We further validated our method by comparing the results with the specificity profiles obtained by the N-terminal combined fractional diagonal chromatography method. This comparison pointed to almost identical substrate specificities for all three cathepsins and confirmed the reliability of the FPPS approach. All MS data have been deposited in the ProteomeXchange with identifiers PXD001536 and PXD001553 (; ).
Keywords
N-terminomics, Technology, Intact protein-based cleavage site discovery, Cathepsin protease specificity, N-TERMINAL PEPTIDES, CYSTEINE CATHEPSINS, CLEAVAGE SITES, BOVINE SPLEEN, IDENTIFICATION, PROTEOME, CANCER, DATABASE, DISEASE, TARGETS

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MLA
Vizovišek, Matej et al. “Fast Profiling of Protease Specificity Reveals Similar Substrate Specificities for Cathepsins K, L and S.” PROTEOMICS 15.14 (2015): 2479–2490. Print.
APA
Vizovišek, M., Vidmar, R., Van Quickelberghe, E., Impens, F., Andjelković, U., Sobotič, B., Stoka, V., et al. (2015). Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S. PROTEOMICS, 15(14), 2479–2490.
Chicago author-date
Vizovišek, Matej, Robert Vidmar, Emmy Van Quickelberghe, Francis Impens, Uroš Andjelković, Barbara Sobotič, Veronika Stoka, Kris Gevaert, Boris Turk, and Marko Fonović. 2015. “Fast Profiling of Protease Specificity Reveals Similar Substrate Specificities for Cathepsins K, L and S.” Proteomics 15 (14): 2479–2490.
Chicago author-date (all authors)
Vizovišek, Matej, Robert Vidmar, Emmy Van Quickelberghe, Francis Impens, Uroš Andjelković, Barbara Sobotič, Veronika Stoka, Kris Gevaert, Boris Turk, and Marko Fonović. 2015. “Fast Profiling of Protease Specificity Reveals Similar Substrate Specificities for Cathepsins K, L and S.” Proteomics 15 (14): 2479–2490.
Vancouver
1.
Vizovišek M, Vidmar R, Van Quickelberghe E, Impens F, Andjelković U, Sobotič B, et al. Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S. PROTEOMICS. 2015;15(14):2479–90.
IEEE
[1]
M. Vizovišek et al., “Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S,” PROTEOMICS, vol. 15, no. 14, pp. 2479–2490, 2015.
@article{6981379,
  abstract     = {Proteases are important effectors of numerous physiological and pathological processes. Reliable determination of a protease's specificity is crucial to understand protease function and to develop activity-based probes and inhibitors. During the last decade, various proteomic approaches for profiling protease substrate specificities were reported. Although most of these approaches can identify up to thousands of substrate cleavage events in a single experiment, they are often time consuming and methodologically challenging as some of these approaches require rather complex sample preparation procedures. For such reasons their application is often limited to those labs that initially introduced them. Here, we report on a fast and simple approach for proteomic profiling of protease specificities (fast profiling of protease specificity (FPPS)), which can be applied to complex protein mixtures. FPPS is based on trideutero-acetylation of novel N-termini generated by the action of proteases and subsequent peptide fractionation on Stage Tips containing ion-exchange and reverse phase chromatographic resins. FPPS can be performed in 2 days and does not require extensive fractionation steps. Using this approach, we have determined the specificity profiles of the cysteine cathepsins K, L and S. We further validated our method by comparing the results with the specificity profiles obtained by the N-terminal combined fractional diagonal chromatography method. This comparison pointed to almost identical substrate specificities for all three cathepsins and confirmed the reliability of the FPPS approach. All MS data have been deposited in the ProteomeXchange with identifiers PXD001536 and PXD001553 (; ).},
  author       = {Vizovišek, Matej and Vidmar, Robert and Van Quickelberghe, Emmy and Impens, Francis and Andjelković, Uroš and Sobotič, Barbara and Stoka, Veronika and Gevaert, Kris and Turk, Boris and Fonović, Marko},
  issn         = {1615-9853},
  journal      = {PROTEOMICS},
  keywords     = {N-terminomics,Technology,Intact protein-based cleavage site discovery,Cathepsin protease specificity,N-TERMINAL PEPTIDES,CYSTEINE CATHEPSINS,CLEAVAGE SITES,BOVINE SPLEEN,IDENTIFICATION,PROTEOME,CANCER,DATABASE,DISEASE,TARGETS},
  language     = {eng},
  number       = {14},
  pages        = {2479--2490},
  title        = {Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S},
  url          = {http://dx.doi.org/10.1002/pmic.201400460},
  volume       = {15},
  year         = {2015},
}

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