Spin labelling via metabolic glycoengineering for studying post-translational protein modification by electron paramagnetic resonance spectroscopy

Post-translational modifications are involved in many cellular processes. The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification essential for regulatory processes in mammalian cells. Here, we demonstrate specific spin labelling of post-translational modifications, namely glycosylation modifications, via metabolic glycoengineering and subsequent copper-catalyzed azide-alkyne cycloaddition. Electron paramagnetic resonance spectroscopy and subsequent quantitative spectral simulations allow for detection of the labelled post-translational modifications and for monitoring the levels of O-GlcNAcylation in presence of different inhibitors relevant for metabolic glycoengineering.