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Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation

Nature Protocols volume 2, pages 2930–2944 (2007)Cite this article

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.

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Figure 1: Analysis of glycans using the bioorthogonal chemical reporter strategy.
Figure 2: Synthesis of reagents for metabolic labeling of glycans and subsequent reaction with bioorthogonal chemistries.
Figure 3: Anticipated results from unnatural monosaccharide metabolic labeling experiments.

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Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, 94720, California, USA

    Scott T Laughlin & Carolyn R Bertozzi

  2. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Carolyn R Bertozzi

  3. Howard Hughes Medical Institute, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Carolyn R Bertozzi

  4. The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Carolyn R Bertozzi

Authors
  1. Scott T Laughlin
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  2. Carolyn R Bertozzi
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Correspondence to Carolyn R Bertozzi.

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Laughlin, S., Bertozzi, C. Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation. Nat Protoc 2, 2930–2944 (2007). https://doi.org/10.1038/nprot.2007.422

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