Systematic Evaluation of Affinity Enrichment Methods for O-GlcNAc Proteomics

Area of Science:

• Proteomics
• Post-translational modifications
• Biochemistry

Background:

• O-Linked β-N-acetylglucosamine (O-GlcNAc) modification regulates numerous biological processes.
• Large-scale analysis of protein O-GlcNAcylation remains challenging.
• Various enrichment methods exist, but their comparative performance is unclear.

Purpose of the Study:

• To conduct a head-to-head comparison of three affinity enrichment methods for site-specific O-GlcNAc proteomics.
• To evaluate the performance of antibody, lectin AANL6, and OGA mutant enrichment materials.
• To assess the impact of different data analysis packages on O-GlcNAc proteomics outcomes.

Main Methods:

• Utilized PANC-1 cell lysates for enrichment experiments.
• Employed antibody, lectin AANL6, and OGA mutant affinity enrichment strategies.
• Analyzed enriched peptides using HCD product-dependent EThcD mass spectrometry.
• Processed data with Sequest HT, Byonic, and FragPipe analysis packages.

Main Results:

• Each enrichment method identified a distinct subpopulation of O-GlcNAc modified proteins.
• Complementarity was observed between different data analysis tools.
• No single method provided exhaustive O-GlcNAc protein coverage.

Conclusions:

• Individual O-GlcNAc enrichment methods capture only a fraction of the total O-GlcNAc proteome.
• Combining diverse enrichment strategies and data analysis tools is crucial for comprehensive O-GlcNAc site-specific proteomics.
• This comparative study provides insights for optimizing O-GlcNAc proteomics workflows.