Chemoenzymatic Labeling Method for Detection of O-GlcNAcylated α-Synuclein Proteins by Western Blot

α-Synuclein (α-syn) aggregation has emerged as a key pathogenetic feature in several neurodegenerative disorders. The α-syn protein has various conformational strains, each with unique structural features that influence their cytotoxicity, propagation, and neuroinflammation. A post-translational modification known as O-GlcNAcylation has been found to influence the toxicity of α-syn and its propensity to aggregate. Difficulties in detecting and quantifying this modification are a major challenge to understanding its roles among the conformational forms of α-syn. We now describe a protocol for detecting O-GlcNAcylated α-syn that combines a click chemistry labeling approach and western blotting. This chemoenzymatic method involves the transfer of azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAcylated proteins, enabling their further functionalization with alkyne-containing polyethylene glycol of defined molecular weight. This protocol facilitates the determination of the glycosylation status of varying conformations of α-syn and their stoichiometric ratios. Key features • The method outlines a chemoenzymatic mass-tagging approach for the identification and quantification of O-GlcNAcylated α-syn. • The process involves using GalT 289L for GalNAz transfer, followed by PEG mass tag conjugation, and then subjected to western blotting. • Visualizable modified α-syn with a noticeable molecular weight shift, enabling estimation of its relative stoichiometry. • This protocol provides a rapid and simple strategy that can be completed within two or three days.