Protocol abstract
Nature Protocols 2, - 2930 - 2944 (2007)
Published online: 15 November 2007 | doi:10.1038/nprot.2007.422
Subject Categories: Biochemistry and protein analysis | Chemical modification | Synthetic chemistry
Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation
Scott T Laughlin1 & Carolyn R Bertozzi1,2,3,4
Abstract
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.
- Department of Chemistry, University of California, Berkeley, California 94720, USA.
- Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
- Howard Hughes Medical Institute, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
- The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Correspondence to: Carolyn R Bertozzi1,2,3,4 e-mail: crb@berkeley.edu
nature-products
A-Z product listing
- 0.5 M EDTA solution(Invitrogen)
- 1-Butanol(Sigma)
- 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(Sigma-Aldrich)
- 1-Hydroxybenzotriazole(Fluka)
- 1-Methyl-2-aminoterephthalate(Aldrich)
- 1-Methyl-2-pyrrolidinone(Sigma-Aldrich)
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