Boyce, MichaelCarrico, Isaac SGanguli, Anjali SYu, Seok-HoHangauer, Matthew JHubbard, Sarah CKohler, Jennifer JBertozzi, Carolyn R2020-01-012020-01-012011-02-070027-84241091-6490https://hdl.handle.net/10161/19698Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked β-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.Cell LineHumansAcetylgalactosamineAcetylglucosamineAffinity LabelsChromatography, AffinityMethodsReceptor Cross-TalkProtein Processing, Post-TranslationalGlycosylationMetabolic Networks and PathwaysJournal article2020-01-01