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bioRxiv · 10.1101/2025.10.16.682811

Time-Resolved Tracking and Secretome Mapping of Intracellular, Cell Surface, and Extracellular Glycoconjugates with a Mini-Tetrazine N-Acetylglucosamine

Hillman, A. S.·Burns, M. W. N.·Janney, S. L.·Stark, A. M.·Cai, F.·Chaya, T.·Grimes, C. L.·Kohler, J. J.·Fox, J. M.
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Abstract

Glycosylation is instrumental in governing essential biological processes that dictate health outcomes. Herein we describe a mini-tetrazine N-acetylglucosamine (HTz-GlcNAc) that is incorporated into N-linked glycans and through rapid live-cell labeling with trans-cyclooctene (TCO) reagents enables real-time visualization and evaluation of glycoconjugate trafficking in living cells. We utilize cell-permeable and impermeable TCO-fluorophores to perform time-resolved, multicolor live labeling of cell-surface glycoconjugates, as well as intracellular, and internalized glycoconjugates. We observe intracellular glycoconjugate trafficking throughout the secretory pathway of live cells in real-time, as well as cell surface glycan turnover and extracellular vesicle secretion across three days. In the toolkit of probes for metabolic oligosaccharide engineering, HTz-GlcNAc brings the capability of precise interrogation of intracellular and extracellular glycoconjugate trafficking, and selective tagging of intra-and-extracellular vesicles, while providing a handle for the purification and enrichment of glycans for immunoblot and proteomic analyses. TOC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/682811v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@f65188org.highwire.dtl.DTLVardef@162c58corg.highwire.dtl.DTLVardef@1977b73org.highwire.dtl.DTLVardef@29e9ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Hillman, A. S., Burns, M. W. N., Janney, S. L., Stark, A. M., Cai, F., Chaya, T., Grimes, C. L., Kohler, J. J., Fox, J. M.. 2025-10-16. Time-Resolved Tracking and Secretome Mapping of Intracellular, Cell Surface, and Extracellular Glycoconjugates with a Mini-Tetrazine N-Acetylglucosamine. https://doi.org/10.1101/2025.10.16.682811

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