GlycoID Proximity Labeling to Identify O-GlcNAcylated Protein Interactomes in Live Cells.
Curr Protoc
; 4(5): e1052, 2024 May.
Article
in En
| MEDLINE
| ID: mdl-38752278
ABSTRACT
Cells 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 cells Basic Protocol 2 GlycoID labeling in live HeLa cells for O-GlcNAc proteomic comparisons.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Acetylglucosamine
Limits:
Animals
/
Humans
Language:
En
Journal:
Curr Protoc
Year:
2024
Document type:
Article
Country of publication: