Cell-specific Bioorthogonal Tagging of Glycoproteins

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Abstract
Interactions between cells fundamentally impact biological processes. In cancer development, such interactions define key stages of disease that cannot be adequately recapitulated in cell monoculture. Complex co-culture studies have been key to unraveling the complexity of these processes, usually by sorting cells and transcriptome or bulk proteome analyses. However, these methods invariably lead to sample loss and do not capture aberrant glycosylation as an important corollary of cancer formation. Here, we report the development of Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped with a biosynthetic AND gate that uses bioorthogonally tagged sugars to generate glycosylation precursors. The cellular glycosylation machinery then introduces bioorthogonal tags into glycoproteins exclusively in cell lines expressing the enzymes of the biosynthetic AND gate. Modification with clickable reporter moieties allows for imaging or enrichment with mass spectrometry-proteomics in a cell-specific fashion. Making use of glycans as a property of most cell surface proteins, we use BOCTAG as an efficient means for cell-specific protein tracing. SUMMARY This work describes the development of a strategy to profile the glycoproteome with bioorthogonal tags in a cell-specific manner. We use a synthetic biology strategy in which an artificial biosynthetic AND gate leads to the cell-specific biosynthesis of a tagged UDP-sugar that, in turn, is used by the cellular glycosylation machinery. Comprehensive characterisation in mono- and co-culture experiments reveal a strong foundation for cell-specific glycoproteome analysis.
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