DNA-Alkyne-Chain Probes Enable N-Glycosite-Resolved GlycoBarcoding of Cell-Surface Proteins

Area of Science:

• Proteomics
• Glycomics
• Cell Biology

Background:

• Cell surface glycoproteins are crucial for cell communication and are key therapeutic targets.
• Profiling the 'surface glycome' is challenging due to low protein abundance and glycosylation complexity.

Purpose of the Study:

• To develop a site-specific glycoproteomics strategy for high-resolution profiling of cell surface glycoproteins.
• To enable precise mapping of N-glycosylation signatures at the site level on live cells.

Main Methods:

• GlycoCSP utilizes alkyne-functionalized DNA scaffolds for GlycoBarcoding of cell surface proteins.
• The platform employs extended DNA chains and a high-density alkyne array for specific labeling and capture.
• Orthogonal tandem release proteolysis integrates protein enrichment for site-level mapping.

Main Results:

• Identified 2,016 extracellular N-glycosylation sites on 1,420 proteins using GlycoCSP on live cells.
• Validated glycosylation sites using deamidation mass shifts and the N-X-S/T/C motif.
• Discovered that site-specific glycosylation occupancy offers a distinct layer of surfaceome heterogeneity in breast cancer cell lines, independent of protein abundance.

Conclusions:

• GlycoCSP provides a mass spectrometry-readable framework for decoding the cell surface glycome.
• This strategy facilitates unbiased discovery of glycosylation-dependent biomarkers and therapeutic targets.
• GlycoCSP overcomes limitations of conventional proteomics for surface glycoprotein analysis.