A Chemical Glycoproteomics Platform Reveals O-GlcNAcylation of Mitochondrial Voltage-Dependent Anion Channel 2

Protein modification by O-linked β-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2−/− cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts. [Display omitted] •Glyco-DIGE allows comparison of sample-dependent changes in O-GlcNAcylated proteins•Glyco-DIGE identifies VDAC2 as a mitochondrial glycoprotein•Cellular responses to perturbed global O-GlcNAc require VDAC2 Protein modification by O-linked β-N-acetylglucosamine (O-GlcNAc) is a critical signaling modality, but identifying signal-specific O-GlcNAcylation events is a considerable challenge. Here, Bertozzi, Boyce, and colleagues describe “glyco-DIGE,” a method for analyzing changes in O-GlcNAcylated proteins. The authors show that voltage-dependent anion channel 2 (VDAC2) is O-GlcNAcylated and that VDAC2 ablation protects cells from the effects of O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc and mitochondrial physiology. Glyco-DIGE should facilitate the discovery of signal-specific O-GlcNAcylation events in a variety of contexts.