Intracellular Imaging of Protein-Specific Glycosylation

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, Ac₄GlcNCyoc, into the cellular glycome via metabolic engineering. Ac₄GlcNCyoc 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, Förster 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 Ac₄GlcNCyoc, immunoprecipitation of EGFP-fusion proteins to examine the Ac₄GlcNCyoc 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.