Profiling of Protease Cleavage Sites by Proteome-Derived Peptide Libraries and Quantitative Proteomics
| Autor: | Bettina Mayer, Chia-yi Chen, Oliver Schilling |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
chemistry.chemical_classification Proteases Protease medicine.medical_treatment Quantitative proteomics Peptide 03 medical and health sciences 030104 developmental biology 0302 clinical medicine chemistry Biochemistry Proteome medicine Peptide bond Peptide library Peptide sequence 030217 neurology & neurosurgery |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493968497 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|