title: Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S.
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 (http://proteomecentral.proteomexchange.org/dataset/PXD001536; http://proteomecentral.proteomexchange.org/dataset/PXD001553).
authors: Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.|Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia.|International Postgraduate School Jozef Stefan, Ljubljana, Slovenia.
authors: Matej
authors: M
authors: Vizovišek
authors: Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.|International Postgraduate School Jozef Stefan, Ljubljana, Slovenia.
authors: Robert
authors: R
authors: Vidmar
authors: Department of Biochemistry, Ghent University, Ghent, Belgium.|Department of Medical Protein Research, Ghent, Belgium.
authors: Emmy
authors: E
authors: Van Quickelberghe
authors: Department of Biochemistry, Ghent University, Ghent, Belgium.|Department of Medical Protein Research, Ghent, Belgium.|Unité des Interactions Bactéries-Cellules, Institut Pasteur, Paris, France.
authors: Francis
authors: F
authors: Impens
authors: Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Belgrade, Serbia.
authors: Uroš
authors: U
authors: Andjelković
authors: Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.|International Postgraduate School Jozef Stefan, Ljubljana, Slovenia.
authors: Barbara
authors: B
authors: Sobotič
authors: Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
authors: Veronika
authors: V
authors: Stoka
authors: Department of Biochemistry, Ghent University, Ghent, Belgium.|Department of Medical Protein Research, Ghent, Belgium.
authors: Kris
authors: K
authors: Gevaert
authors: Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.|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.
authors: Boris
authors: B
authors: Turk
authors: Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia.|Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia.
authors: Marko
authors: M
authors: Fonović
mesh_terms: D000595:Amino Acid Sequence; D056657:Cathepsin K / Q000737:chemistry / Q000378:metabolism*; D056668:Cathepsin L / Q000737:chemistry / Q000378:metabolism*; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010455:Peptides; D002403:Cathepsins; D056668:Cathepsin L; C028217:cathepsin S; D056657:Cathepsin K
keywords: Cathepsin protease specificity; Intact protein-based cleavage site discovery; N-terminomics; Technology