Intracellular Imaging of Protein-Specific Glycosylation

Abstract Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac 4 GlcNCyoc, into the cellular glycome via metabolic engineering. Ac 4 GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels–Alder chemistry with fluorophores inside living cells. Additionally, target proteins can be expressed as enhanced green fluorescent protein (EGFP)-fusion proteins. To assess the proximity of the donor EGFP and the glycan-anchored acceptor fluorophore, Forster resonance energy transfer (FRET) is employed and read out with high contrast by fluorescence lifetime imaging (FLIM) microscopy. In this chapter, we present a detailed description of methods required to perform protein-specific imaging of glycosylation inside living cells. These include the complete synthesis of Ac 4 GlcNCyoc, immunoprecipitation of EGFP-fusion proteins to examine the Ac 4 GlcNCyoc modification state, and a complete section on basics, performance, as well as data analysis for FLIM–FRET microscopy. We also provide useful notes necessary for reproducibility and point out strengths and limitations of the approach.