Structural characterization of mucin O-linked glycans using a novel anion exchange column in HPAE-PAD-MS

Importance of the topic

The analysis of O-linked glycans is critical for understanding protein function in health and disease. Mucin glycoproteins exhibit dense clusters of O-glycans that modulate cell signaling, adhesion, and immune response. Alterations in mucin O-glycosylation patterns correlate with cancer and other pathologies, making detailed structural profiling essential for biomarker discovery and therapeutic design.

Aims and Study Overview

This work presents a combined high-performance anion-exchange chromatography with pulsed amperometric detection and high-resolution mass spectrometry (HPAE-PAD-MS) workflow. The goal is to demonstrate the efficient release, cleanup, chromatographic separation, and structural characterization of native O-glycans from porcine gastric mucin. A newly introduced Dionex CarboPac PA300-4µm column is evaluated for its ability to resolve neutral, sialylated, and sulfated glycan isomers without derivatization.

Methodology and Instrumentation

Sample preparation involves reductive β-elimination to liberate glycan alditols, followed by porous graphitized carbon cleanup. The HPAE-PAD-MS platform uses a Thermo Scientific Dionex ICS-6000 system configured for sequential PAD and Q Exactive Orbitrap MS detection. Key components include:

• Dionex CarboPac PA300-4µm guard (2×50 mm) and analytical (2×250 mm) columns
• PdH solid-state reference electrode in the electrochemical cell
• Dionex ERD 500 electrolytically regenerated desalter for salt removal
• Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer operating in negative ion HCD mode

Main Results and Discussion

Neutral O-glycans eluted early in the gradient, followed by sialylated and sulfated species, achieving high resolution and reproducibility. PAD detection provided sensitive profiles, while Orbitrap MS delivered accurate mass (<5 ppm) and detailed MS/MS fragmentation for structural elucidation. The PA300 column improved neutral glycan separation compared to PA200, while still resolving charged glycans effectively. Diagnostic fragment ions enabled assignment of core types, linkage positions, and blood-group epitopes. The ERD 500 incurred minor peak broadening but maintained acceptable resolution for downstream MS analysis.

Benefits and Practical Applications

• Label-free workflow preserves native glycan structures and avoids derivatization artifacts.
• Simultaneous PAD and high-resolution MS/MS offers comprehensive detection and structural annotation.
• The PA300 column expands the glycoanalytical toolkit, allowing tailored separation strategies for diverse glycoproteins.
• Applicable to biologics QA/QC, biomarker discovery, and fundamental glycoscience research.

Future Trends and Potential Uses

Advances in column chemistries and ion chromatography will further enhance isomer resolution. Integration with online glycan databases and automated annotation software will streamline data interpretation. Coupling with ion mobility or chemical derivatization strategies could deepen structural insight. The platform may be extended to complex biological samples, high-throughput screening, and real-time monitoring of glycosylation dynamics.

Conclusion

This study establishes a robust HPAE-PAD-MS method for detailed structural analysis of mucin O-linked glycans. The Dionex CarboPac PA300-4µm column, PdH reference electrode, and ERD 500 desalter form a powerful platform that delivers sensitive detection, high resolution, and rich MS/MS data without derivatization. This workflow supports accurate glycan profiling for research and industrial applications.

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