Abstract
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.
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Acknowledgment
This work was supported by the SFB877 “Proteolysis as a Regulatory Event in Pathophysiology” (project Z2) and the Cluster of Excellence “Inflammation at Interfaces” funded by the DFG (Deutsche Forschungsgemeinschaft).
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Tucher, J., Tholey, A. (2017). Multiplexed Protease Specificity Profiling Using Isobaric Labeling. In: Schilling, O. (eds) Protein Terminal Profiling. Methods in Molecular Biology, vol 1574. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6850-3_12
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DOI: https://doi.org/10.1007/978-1-4939-6850-3_12
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