Document Detail


Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation.
MedLine Citation:
PMID:  18007630     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic analysis are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approximately 1 week. A cell metabolic labeling experiment can be completed in approximately 4 d.
Authors:
Scott T Laughlin; Carolyn R Bertozzi
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Nature protocols     Volume:  2     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2007  
Date Detail:
Created Date:  2007-11-16     Completed Date:  2008-02-19     Revised Date:  2008-03-24    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  2930-44     Citation Subset:  IM    
Affiliation:
Department of Chemistry, University of California, Berkeley, California 94720, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Azides / chemical synthesis*,  chemistry,  metabolism
Blotting, Western
Carbohydrates / chemistry*
Cells, Cultured
Flow Cytometry
Fluorescent Dyes
Glycoproteins / analysis*,  chemistry
Molecular Probe Techniques*
Molecular Probes / chemical synthesis*,  chemistry,  metabolism
Phosphines / chemistry
Polysaccharides / analysis*,  biosynthesis,  chemistry
Vertebrates / metabolism
Chemical
Reg. No./Substance:
0/Azides; 0/Carbohydrates; 0/Fluorescent Dyes; 0/Glycoproteins; 0/Molecular Probes; 0/Phosphines; 0/Polysaccharides; 7803-51-2/phosphine

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