Abstract
Protease specificity profiling using proteome-derived, database-searchable peptide libraries is a novel approach to define the active site specificity of proteolytic enzymes we call PICS (Proteomic Identification of protease Cleavage Sites). Proteome-derived peptide libraries are generated by trypsin, GluC, or chymotrypsin digestion of biologically relevant proteomes, such as cytosolic lysates, to generate three separate libraries that each differ from the others in their C-terminal amino acid residues according to the protease specificity. Primary amines of all peptides are then chemically protected so that after incubation with a test protease, the neo-N-termini of the prime-side cleavage products with exposed α-amines can be specifically biotinylated, enriched, and identified by liquid chromatography-tandem mass spectrometry. The corresponding nonprime-side sequences are derived bioinformatically. Suited for all protease classes except carboxyproteases and those aminoproteases and dipeptidases requiring a free α-amine for cleavage, PICS simultaneously profiles the specificity of prime and nonprime positions and directly determines scissile peptide bonds of up to hundreds of cleavage site sequences in a single experiment. This wealth of sequence specificity information also allows for the investigation of subsite cooperativity. Herein we describe a simplified procedure to produce PICS peptide libraries, the methods to perform a PICS assay, and a new method of data analysis.
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Acknowledgments
The authors thank Bettina Mayer for technical assistance in establishing the simplified purification protocol. O.S. acknowledges support from the Deutsche Forschungsgemeinschaft (DFG) (grants SCHI 871/1-1 and 871/2-1) and the Michael Smith Foundation for Health Research (MSFHR). U.a.d.K. was supported by a DFG research fellowship. C.M.O. is supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) and from a program project grant in Breast Cancer Metastases from the Canadian Breast Cancer Research Alliance (CBCRA) with funds from the Canadian Breast Cancer Foundation and the Cancer Research Society as well as with an Infrastructure Grant from MSHFR.
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Schilling, O., auf dem Keller, U., Overall, C.M. (2011). Protease Specificity Profiling by Tandem Mass Spectrometry Using Proteome-Derived Peptide Libraries. In: Gevaert, K., Vandekerckhove, J. (eds) Gel-Free Proteomics. Methods in Molecular Biology, vol 753. Humana Press. https://doi.org/10.1007/978-1-61779-148-2_17
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DOI: https://doi.org/10.1007/978-1-61779-148-2_17
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