O-glycan profiling using HPAE-PAD hyphenated with a high-resolution accurate mass (HRAM) mass spectrometer

Importance of O-Glycan Profiling

Glycosylation represents a critical post-translational modification affecting protein stability, function, and cell signaling. O-glycosylation, in particular, is highly heterogeneous and lacks a consensus attachment motif, complicating its analysis. Detailed O-glycan profiling is essential for understanding disease markers, therapeutic protein characterization, and quality control in biopharmaceuticals.

Objectives and Study Overview

This work presents the development and demonstration of a robust workflow combining high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and high-resolution accurate mass spectrometry (HRAM-MS) to profile O-glycans released from four model glycoproteins: porcine gastric mucin type III, bovine fetuin, bovine fibrinogen, and bovine thyroglobulin.

Methodology and Workflow

• Release: Reductive β-elimination under alkaline conditions using NaOH and sodium borohydride to generate glycan alditols and prevent peeling.
• Cleanup: Porous graphitized carbon SPE to remove salts and proteins.
• Separation: Dionex CarboPac PA300-4 µm column at 30 °C with a gradient of NaOH and sodium acetate eluents.
• Desalting: Inline Dionex ERD 500 device exchanging sodium for protons to protect the mass spectrometer.
• Detection: Pulsed amperometric detection on gold electrodes for quantitative profiling and Q Exactive HF Orbitrap MS in negative electrospray mode with HCD for structural elucidation.
• Data analysis: Chromeleon, Xcalibur, and SimGlycan software for compositional assignments and MS/MS confirmation.

Použitá instrumentace

• Dionex ICS-5000+ HPIC system with dual pump, conductivity and electrochemical detectors, CarboPac PA300-4 µm guard and analytical columns.
• Dionex ERD 500 inline desalting device.
• Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer with HESI-II probe.
• Dionex AS-AP autosampler with cooling tray, HyperSep Hypercarb SPE plates, and related consumables.

Key Results and Discussion

• Porcine gastric mucin type III produced over 20 distinct O-glycan structures, including neutral, sialylated, and sulfated species, all resolved with high chromatographic resolution and identified with <5 ppm mass accuracy.
• Bovine fetuin yielded both N- and O-glycans; O-glycans were predominantly sialylated core-1 and core-2 tri- and tetrasaccharides, confirmed by diagnostic HCD fragments.
• Bovine fibrinogen profiles were dominated by biantennary mono- and disialylated N-glycans with trace core-1 O-glycans, illustrating the method’s sensitivity for low-abundance species.
• Bovine thyroglobulin showed high-mannose and complex N-glycans and, for the first time experimentally, a monosialylated core-1 O-glycan, demonstrating discovery potential.

Benefits and Practical Applications

• A label-free, non-derivatized workflow preserves native glycan structures and reduces sample preparation steps.
• Dual PAD and HRAM-MS detection offer complementary quantitative and structural information.
• Applicable to diverse glycoprotein analyses in biomarker discovery, biopharmaceutical characterization, and QA/QC laboratories.

Future Trends and Applications

• Automation and microfluidic integration for higher throughput and reproducibility.
• Expanded spectral libraries and machine learning for improved glycan identification and linkage assignment.
• Integration with ion mobility spectrometry and data-independent acquisition for in-depth structural characterization.
• Extension to glycoproteomics workflows and single-cell glycan profiling to explore cellular heterogeneity.

Conclusion

The HPAE-PAD/HRAM-MS workflow featuring the CarboPac PA300 column and Orbitrap detection enables comprehensive, high-confidence O-glycan profiling across multiple glycoproteins. This approach facilitates the discovery of novel glycan structures and supports a wide range of research and industrial applications.

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