Label-Free Profiling of O-linked Glycans by HPLC with Charged Aerosol Detection

Importance of the Topic

O-linked glycans play critical roles in protein structure, stability and function across biopharmaceutical, biomedical and quality control applications. Accurate, sensitive profiling of these glycans is essential for monitoring batch-to-batch consistency, detecting glycosylation changes linked to disease, and guiding downstream characterization by mass spectrometry.

Study Objectives and Overview

This work aimed to establish a label-free, quantitative UHPLC assay for native O-linked glycans released by reductive β-elimination. By leveraging charged aerosol detection (CAD) integrated with a mixed-mode UHPLC column, the method targets low-nanogram sensitivity, broad dynamic range and high precision without fluorescent derivatization.

Instrumental Setup

• UHPLC System: Thermo Scientific Vanquish UHPLC
• Detector: Vanquish Charged Aerosol Detector with spray chamber, evaporation tube (50 °C) and Corona charger
• Analytical Column: Thermo Scientific GlycanPac AXH-1 (1.9 µm, 2.1 × 100 mm), combining weak anion exchange and HILIC
• Data System: Thermo Scientific Dionex Chromeleon CDS 7.2

Methodology

O-linked glycans were liberated from glycoproteins (e.g., fetuin, mucin, IgG) by overnight reductive β-elimination in NaOH/NaBH4, followed by neutralization, desalting and spin-filtration. Separation was achieved using a binary gradient of acetonitrile/water (95:5) and 50 mM ammonium formate (pH 4.4) at 0.5 mL/min and 30 °C. CAD detected native glycan alditols directly, eliminating the need for fluorescent tags.

Results and Discussion

Native O-glycan pools from various proteins were baseline-resolved according to charge states (neutral, mono-, di-sialylated, etc.) and further separated by HILIC. Key figures of merit included:

• Sensitivity: low ng on-column detection (pmol range)
• Dynamic Range: over two orders of magnitude (2–400 ng on column)
• Retention Time Precision: <0.1 % RSD
• Peak Area Precision: ~3 % RSD

Comparative tests showed minimal interference from residual reagents or reaction by-products. Mixed-mode separation proved MS-compatible and offered robust glycan resolution.

Benefits and Practical Applications

• Label-free quantification simplifies workflow and reduces sample manipulation.
• Uniform CAD response affords accurate relative quantitation without primary standards.
• MS-compatible mobile phase enables downstream structural analysis.
• Applicable for QC labs, batch monitoring and glycan biomarker research.

Future Trends and Potential Applications

Advancements will focus on:

• Extending calibration with authentic O-glycan core standards to refine limits of detection and linearity.
• Integrating CAD with high-resolution mass spectrometry for simultaneous quantitation and identification.
• Automating O-glycan release and cleanup to further streamline workflows in regulated environments.

Conclusion

The developed UHPLC-CAD method delivers a straightforward, precise and sensitive platform for label-free profiling of native O-linked glycans. Its mixed-mode separation and universal detection support robust quantitation, minimal sample preparation and compatibility with MS, making it a valuable tool for biopharmaceutical and analytical laboratories.

References

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