Chapter title |
Profiling of Protease Cleavage Sites by Proteome-Derived Peptide Libraries and Quantitative Proteomics
|
---|---|
Chapter number | 14 |
Book title |
Protein Terminal Profiling
|
Published in |
Methods in molecular biology, March 2017
|
DOI | 10.1007/978-1-4939-6850-3_14 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6849-7, 978-1-4939-6850-3
|
Authors |
Chia-yi Chen, Bettina Mayer, Oliver Schilling |
Editors |
Oliver Schilling |
Abstract |
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. |
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