Profiling of Protease Cleavage Sites by Proteome-Derived Peptide Libraries and Quantitative Proteomics.
Biochemical profiling of active site specificity is a crucial step to characterize proteases, which play key roles in health and disease. Here, we present a protocol using proteome-derived peptide libraries in combination with quantitative proteomics to simultaneously identify cleavage motifs N- and C-terminal to the scissile peptide bond. First, bacterial or eukaryotic cell lysate is used to generate peptide libraries. Without further chemical modification, peptide libraries are then split into control and treated (incubate with active protease) aliquots. Control and treated libraries are stable isotope-labeled, mixed, and analyzed by liquid chromatography-tandem mass spectrometry. Enriched, semi-specific peptides represent the cleavage products of the test protease and the entire peptide sequence that encompasses the scissile peptide bond is reconstructed bioinformatically. The method is fast, cost-effective, and suited for proteases with narrow or loose specificity.
Institute of Molecular Medicine and Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, 79104, Freiburg, Germany.
Chia-Yi
CY
Chen
Institute of Molecular Medicine and Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, 79104, Freiburg, Germany.
Bettina
B
Mayer
Institute of Molecular Medicine and Cell Research, University of Freiburg, Stefan-Meier-Strasse 17, 79104, Freiburg, Germany. oliver.schilling@mol-med.uni-freiburg.de.|BIOSS Centre of Biological Signaling Studies, University of Freiburg, 79104, Freiburg, Germany. oliver.schilling@mol-med.uni-freiburg.de.|German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany. oliver.schilling@mol-med.uni-freiburg.de.
Oliver
O
Schilling
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D019295:Computational Biology / Q000379:methods; D007553:Isotope Labeling; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D013223:Statistics as Topic; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D057188:Workflow
D019151:Peptide Library; D010455:Peptides; D020543:Proteome; D010447:Peptide Hydrolases
Biochemical profiling; Peptide library; Protease specificity; Quantitative proteomics