Chemically Cleavable Enrichment Enables Site-Specific Profiling of Cell-Surface N-Glycan Microheterogeneity Dynamics

Area of Science:

• Biochemistry
• Glycobiology
• Chemical Biology

Background:

• Cell-surface glycans are diverse but their dynamic behavior is poorly understood.
• Existing analytical methods lack the resolution to study site-specific glycan dynamics.

Purpose of the Study:

• To develop a chemical enrichment strategy for profiling temporal dynamics of intact cell-surface sialylated N-glycopeptides.
• To achieve site-specific and single-glycoform resolution in analyzing glycan dynamics.
• To provide a robust platform for investigating cell-surface glycan dynamics.

Main Methods:

• Metabolic labeling of sialylated glycans with azide reporters.
• Copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) with a photocleavable biotin probe.
• Affinity enrichment of intact glycopeptides coupled with pulse-chase analysis.

Main Results:

• Distinct turnover kinetics observed among glycoforms at the same glycosylation site.
• Glycan-level dynamics can differ significantly from protein-level stability.
• Quantitative tracking of glycan turnover across glycoproteins, glycosylation sites, glycans, and site-specific glycoforms.

Conclusions:

• The developed chemical enrichment strategy enables high-resolution profiling of cell-surface glycan temporal dynamics.
• This method provides unprecedented insights into the dynamic behavior of cell-surface glycans.
• The platform facilitates deeper understanding of glycan involvement in cellular functions and disease.