Tagging-via-substrate strategy for probing O-GlcNAc modified proteins

Area of Science:

• Proteomics
• Post-Translational Modifications
• Chemical Biology

Background:

• Identifying proteins with specific post-translational modifications is crucial for understanding their functions.
• Proteomic analysis of modified proteins is challenging due to complexity, wide dynamic range, and lack of efficient enrichment methods.

Purpose of the Study:

• To develop and validate a novel strategy for the detection, isolation, and profiling of O-linked N-acetylglucosamine (O-GlcNAc) modified proteins.
• To demonstrate the utility of the tagging-via-substrate (TAS) approach for O-GlcNAc proteomics.

Main Methods:

• Metabolic labeling of cells with peracetylated azido-GlcNAc, an unnatural GlcNAc analogue.
• Chemoselective conjugation of azido-GlcNAc modified proteins using Staudinger ligation with a biotinylated phosphine reagent.
• Detection and affinity purification of conjugated O-GlcNAc modified proteins, followed by nano-HPLC-MS/MS analysis.

Main Results:

• Successfully labeled and detected azido-GlcNAc modified proteins in cytosolic lysates from HeLa, 3T3, COS-1, and S2 cell lines.
• Isolated azido-GlcNAc modified proteins from S2 cell cytosolic extract.
• Identified 10 known and 41 putative O-GlcNAc modified proteins using nano-HPLC-MS/MS.

Conclusions:

• The tagging-via-substrate (TAS) strategy is effective for the specific detection and isolation of O-GlcNAc modified proteins.
• This approach provides a valuable tool for comprehensive proteomic analysis of O-GlcNAc modifications across diverse cell lines and species.