A Strategy for Functional Proteomic Analysis of Glycosidase Activity from Cell Lysates

A multipurpose flag: The inactivator 1 covalently labels the catalytic nucleophiles of retaining β‐glycosidases to form species 2. The small azide group allows labeling of enzymes with sterically congested active sites. Staudinger ligation of 2 with phosphine–FLAG yields adduct 3, which can be used...

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Bibliographic Details
Published in:Angewandte Chemie (International ed.) 2004-10, Vol.43 (40), p.5338-5342
Main Authors: Vocadlo, David J., Bertozzi, Carolyn R.
Format: Article
Language:English
Subjects:
Animals
beta-Glucosidase - genetics
beta-Glucosidase - metabolism
carbohydrates
Cell Extracts - analysis
Cells, Cultured
Electrophoresis, Gel, Two-Dimensional
Escherichia coli - enzymology
glycosidases
Glycoside Hydrolases - genetics
Glycoside Hydrolases - metabolism
Humans
mechanism-based inactivators
Protein Binding
proteomics
Proteomics - methods
Staudinger ligation
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Summary:A multipurpose flag: The inactivator 1 covalently labels the catalytic nucleophiles of retaining β‐glycosidases to form species 2. The small azide group allows labeling of enzymes with sterically congested active sites. Staudinger ligation of 2 with phosphine–FLAG yields adduct 3, which can be used to detect and profile retaining β‐glycosidase activities in complex mixtures.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.200454235