GlycoID Proximity Labeling to Identify O‐GlcNAcylated Protein Interactomes in Live Cells

Abstract

AbstractCells continuously remodel their intracellular proteins with the monosaccharide O‐linked N‐acetylglucosamine (O‐GlcNAc) to regulate metabolism, signaling, and stress. This protocol describes the use of GlycoID tools to capture O‐GlcNAc dynamics in live cells. GlycoID constructs contain an O‐GlcNAc binding domain linked to a proximity labeling domain and a subcellular localization sequence. When expressed in mammalian cells, GlycoID tracks changes in O‐GlcNAc‐modified proteins and their interactomes in response to chemical induction with biotin over time. Pairing the subcellular localization of GlycoID with the chemical induction of activity enables spatiotemporal studies of O‐GlcNAc biology during cellular events such as insulin signaling. However, optimizing intracellular labeling experiments requires attention to several variables. Here, we describe two protocols to adapt GlycoID methods to a cell line and biological process of interest. Next, we describe how to conduct a semiquantitative proteomic analysis of O‐GlcNAcylated proteins and their interactomes using insulin versus glucagon signaling as a sample application. This articles aims to establish baseline GlycoID protocols for new users and set the stage for widespread use over diverse cellular applications for the functional study of O‐GlcNAc glycobiology. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Expression of targeted GlycoID constructs to verify subcellular location and labeling activity in mammalian cellsBasic Protocol 2: GlycoID labeling in live HeLa cells for O‐GlcNAc proteomic comparisons