Protease cleavage site fingerprinting by label-free in-gel degradomics reveals pH-dependent specificity switch of legumain.

Autor: Vidmar R; Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia.; International Postgraduate School Jožef Stefan, Ljubljana, Slovenia., Vizovišek M; Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia., Turk D; Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia.; International Postgraduate School Jožef Stefan, Ljubljana, Slovenia.; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia., Turk B; Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia boris.turk@ijs.si marko.fonovic@ijs.si.; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia.; Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia., Fonović M; Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia boris.turk@ijs.si marko.fonovic@ijs.si.; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia.
Jazyk: angličtina
Zdroj: The EMBO journal [EMBO J] 2017 Aug 15; Vol. 36 (16), pp. 2455-2465. Date of Electronic Publication: 2017 Jul 21.
DOI: 10.15252/embj.201796750
Abstrakt: Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel-based label-free proteomic approach (DIPPS-direct in-gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in-gel digestion of the gel-separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length-limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase-7, and legumain) to broadly specific (matrix-metalloproteinase-3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH-dependent change in the specificity of this protease, further supporting its broad applicability.
(© 2017 The Authors.)
Databáze: MEDLINE
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