Global identification of peptidase specificity by multiplex substrate profiling
| Autor: | O'Donoghue, Anthony J, Eroy-Reveles, A Alegra, Knudsen, Giselle M, Ingram, Jessica, Zhou, Min, Statnekov, Jacob B, Greninger, Alexander L, Hostetter, Daniel R, Qu, Gang, Maltby, David A, Anderson, Marc O, Derisi, Joseph L, McKerrow, James H, Burlingame, Alma L, Craik, Charles S |
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| Rok vydání: | 2012 |
| Předmět: |
Technology
Carboxypeptidases Cathepsin E Medical and Health Sciences Granzymes Cell Line Substrate Specificity Mice Viral Proteins Peptide Library Tandem Mass Spectrometry Exopeptidases Animals Humans Chromatography Liquid Tumor Pancreatic Elastase Carcinoma 3C Viral Proteases Schistosoma mansoni Biological Sciences Cysteine Endopeptidases Pancreatic Ductal Peptides Peptide Hydrolases Developmental Biology |
| Zdroj: | Nature methods, vol 9, iss 11 |
| Popis: | We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors. |
| Databáze: | OpenAIRE |
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