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

• 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

SummaryProtein 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.

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