Label-Free Analysis by UHPLC with Charged Aerosol Detection of Glycans Separated by Charge, Size, and Isomeric Structure

Significance of the topic

Glycoprotein function and therapeutic efficacy are critically influenced by attached oligosaccharides. Changes in glycan composition and linkages serve as disease biomarkers and impact biopharmaceutical quality. Conventional glycan analysis often requires derivatization to introduce a chromophore or fluorophore. A label-free approach using charged aerosol detection (CAD) streamlines workflows and reduces potential biases.

Study objectives and overview

This work aimed to establish a rapid, sensitive UHPLC method for direct quantification and profiling of native N-linked glycans released from glycoproteins. By combining a mixed-mode separation column with universal CAD, the method resolves glycans by charge, size, and isomeric structure without chemical labeling.

Methodology and Instrumentation

Glycans were enzymatically released from glycoproteins using PNGase F. Standards and protein samples (alpha-acid glycoprotein and fetuin) were prepared in ultrapure water and digested under controlled conditions. Separation was carried out on a GlycanPac AXR-1 column (1.9 µm, 2.1 × 100 mm) at 30 °C.

• Mobile phases: A = water; B = 100 mM ammonium formate, pH 4.4.
• Gradient: 4% B to 39% B over 35 min at 0.4 mL/min.
• Injection volume: 2 µL.
• CAD settings: evaporation temperature 50 °C, power function 1.00, data collection rate 10 Hz.

Main Results and Discussion

Key parameters were optimized: gradient slope (1 mM/min) for balance of resolution and runtime; purely aqueous mobile phase for robust signal-to-noise; and CAD evaporation temperature (50 °C) for sensitivity. The mixed-mode column separated neutral, mono-, di- and higher-charged glycans into distinct groups, with further isomer resolution via reversed-phase interactions. Calibration over 2–400 pmol injections yielded quadratic fits with R² > 0.995 and low-picomole detection limits. Precision was excellent, with retention time RSD < 0.1% and peak area RSD < 3%. Analysis of PNGase F digests showed reagent by-products well separated from glycan peaks, confirming method specificity in complex matrices.

Benefits and Practical Applications

• No labeling required, simplifying sample preparation and reducing cost.
• Universal CAD detection provides consistent response for all non-volatile glycans.
• High-resolution separation supports detailed glycan profiling and lot-to-lot quality control in biopharma.
• Low detection limits enable analysis of scarce samples.

Future Trends and Opportunities

Integration with automated glycan release and online UHPLC-CAD platforms could further accelerate workflows. Coupling label-free CAD with high-resolution MS may enhance structural characterization. Advances in column chemistry may improve isomer separation and shorten analysis times.

Conclusion

The presented UHPLC-CAD method delivers a robust, sensitive, and label-free platform for direct analysis of native N-linked glycans. It achieves high resolution, excellent quantitative performance, and reliable separation of analytes from matrix interferences, making it ideal for research and industrial glycoprotein characterization.

References

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4. Udayanath A et al. Integrated LC/MS Workflow for the Analysis of Labeled and Native N-Glycans Using a Mixed-Mode Column. Thermo Scientific Applications Note 595;2014.
5. Aich U et al. Separation of 2AB-labeled N-linked glycans from bovine fetuin on a novel mixed-mode column. Thermo Scientific Applications Note 20908;2014.