Chemical methods for O-GlcNAc detection and preparation of O-GlcNAc modified proteins

Chemistry, through the synthesis of a radioactive sugar-donor, played a critical role in the discovery of O-GlcNAc and has been contributing indispensable tools ever since. Here, we cover where chemical methods have contributed to both the discovery and characterization of O-GlcNAc modifications. We will first discuss metabolic labeling and chemoenzymatic modification as two techniques that leverage the power of bioorthogonal chemistry to visualize, enrich, and even quantify O-GlcNAc modifications from living systems. We will then describe how solid-phase peptide synthesis can be combined with protein ligation methods as the only current pathway to homogeneously O-GlcNAc-modified proteins for downstream biological experiments. In each section, we point out the positives and considerations for each approach and provide protocols for normalization of best practices across the field.