Multiplexed Protease Specificity Profiling Using Isobaric Labeling.
The determination of a protease's cleavage site specificity is one of the major goals in degradomics. In the last years, the use of proteome-derived peptide libraries and liquid chromatography-mass spectrometry (LC-MS) in a proteomic identification of protease cleavage sites (PICS) experiment became popular for that purpose.In this chapter, we offer a step-by-step protocol for the execution of a quantitative proteomic identification of protease cleavage sites (Q-PICS) experiment, which enables the relative quantification of proteolytic events by isobaric labeling, e.g., with tandem mass tags (TMT). In this way, the cleavage site specificity and activity of a protease can be compared under different reaction conditions (e.g., buffer, pH, temperature, inhibitor). Multiplexing can further be used to analyze replicate experiments in parallel, decreasing instrument times and work effort significantly, or to perform internal controls.
AG Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Campus UKSH-Kiel, Haus 1, Niemannsweg 11, 24105, Kiel, Germany. j.tucher@iem.uni-kiel.de.
Joanna
J
Tucher
AG Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, Campus UKSH-Kiel, Haus 1, Niemannsweg 11, 24105, Kiel, Germany. a.tholey@iem.uni-kiel.de.
Andreas
A
Tholey
D002853:Chromatography, Liquid; D010450:Endopeptidases / Q000378:metabolism*; D007553:Isotope Labeling; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D013223:Statistics as Topic / Q000379:methods; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D057188:Workflow
D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D020543:Proteome; D010450:Endopeptidases
Cleavage site specificity; Heat map; Isobaric labeling; LC-MS; Protease; Proteome-derived peptide library