Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation.
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.
Laughlin
Scott T
ST
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Bertozzi
Carolyn R
CR
D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D002241:Carbohydrates / Q000737:chemistry*; D002478:Cells, Cultured; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D010720:Phosphines / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D014714:Vertebrates / Q000378:metabolism
D001386:Azides; D002241:Carbohydrates; D005456:Fluorescent Dyes; D006023:Glycoproteins; D015335:Molecular Probes; D010720:Phosphines; D011134:Polysaccharides; C044646:phosphine