This project investigates the spatiotemporal dynamics of protein O-GlcNAcylation mediated by optogenetically controlled localization of O-GlcNAc transferase (OGT) to the plasma membrane. Cells expressing the OGT optogenetic system were subjected to varied light intensities (0, 0.2, and 1 mW/cm² for 10 s) or exposure durations (1 mW/cm² for 0, 1, and 10 s). Cell membranes were biochemically fractionated, enzymatically labeled with azido-containing N-acetylgalactosamine (GalNAz), and further conjugated with alkynyl biotin via the click chemistry. Biotinylated proteins were isolated with streptavidin-coated beads and subjected to on-beads trypsin digestion. The resulting peptides were then isotopically derivatized by dimethyl labeling of amino groups by reductive amination, combined and subjected to relative quantification via mass spectrometry. The study reveals how subcellular targeting of OGT rewires signaling networks and provides mechanistic insights into the compartmentalized regulation of O-GlcNAcylation and its crosstalk with other post-translational modifications.