Updated project metadata. O-GlcNAcylation is a dynamic post-translational modification that diversifies the proteome with spatiotemporal precision in response to various stimuli. Its dysregulation is associated with many neurological disorders that impair cognitive function, and yet identification of phenotype-relevant protein substrates in specific brain regions remains unfeasible. By combining O-GlcNAc binding activity of Clostridium perfringens OGA (CpOGA) with TurboID proximity labeling, we developed an O-GlcNAcylation profiling tool that translates O-GlcNAc modification into biotin conjugation for tissue-specific substrates enrichment. We mapped the O-GlcNAcylated proteome in different brain regions of Drosophila.