Towards a More Complete Glycome: Advances in Ion Chromatography-Mass Spectrometry (IC-MS) for Improved Separation and Analysis of Carbohydrates

Importance of the Topic

Glycosylation is a highly diverse and functionally critical post-translational modification affecting cell signaling, adhesion, trafficking and immune recognition. Detailed profiling of native glycans poses challenges due to their structural heterogeneity, non-template biosynthesis and lack of universal enzymatic release methods, especially for O-linked glycans. High-resolution chromatographic separation coupled with accurate mass analysis is essential to resolve isomeric species and preserve labile moieties without derivatization artifacts.

Objectives and Overview of the Study

This work introduces an integrated ion chromatography–mass spectrometry (IC-MS) approach combining high-performance anion exchange (HPAE) using a novel Dionex CarboPac PA300 column with a Q Exactive Orbitrap mass spectrometer. The aim is to achieve comprehensive separation and structural characterization of both neutral and charged glycans in a single workflow, expanding analytical capabilities for biologics, food and clinical samples.

Methods and Instrumentation

Sample Preparation

• O-glycan release by reductive β-elimination in 50 mM NaOH/1 M NaBH₄ at 50 °C for 16 h
• Neutralization with formic acid, solvent evaporation and cleanup on graphitic carbon cartridges
• Final elution in 40 % acetonitrile with 0.1 % TFA

Chromatography and Detection

• Thermo Scientific Dionex ICS-6000 HPIC system with AS-AP autosampler
• Dionex CarboPac PA300 analytical column (2 × 250 mm) with guard (2 × 50 mm) at 0.25 mL/min
• Effluent directed to PAD or via an ERD 500 desalter into the mass spectrometer

Mass Spectrometry

• Q Exactive Hybrid Quadrupole-Orbitrap operated in negative ESI mode
• Full scan m/z 400–2000 at 60 000 resolving power, AGC target 3×10⁶, max IT 120 ms
• Data-dependent MS² (Top 10) with structural annotation via UniCarb-DB

Main Results and Discussion

The Dionex CarboPac PA300 column achieved enhanced resolution of early-eluting neutral glycans compared with PA200, while retaining separation of charged sialylated and sulfated species. Base peak chromatograms of porcine gastric mucin O-glycans show distinct peaks across a wide elution window. Structural isomers such as Hex₂Fuc(HexNAc)₃ vs. (Hex)₃(Fuc)₂(HexNAc)₄-S were baseline-separated, and MS² spectra provided diagnostic fragments for confident annotation. Analysis of commercial lactose-free milk revealed clean separation of neutral oligosaccharides from sialylated forms, demonstrating applicability to food glycomics. Mass accuracies for identified glycan peaks were within 5 ppm.

Benefits and Practical Applications

• Simultaneous profiling of neutral and acidic glycans without derivatization
• High isomeric resolution supports detailed glycome mapping in biopharmaceutical quality control
• Orthogonal data complementing RPLC-MS workflows enhances coverage of prebiotic and dietary glycans
• Accurate mass and MS² fragmentation ensure reliable structural assignments

Future Trends and Potential Uses

Further column innovations may push resolution limits for complex glycan pools. Integration with automated sample prep and bioinformatics pipelines will accelerate glycomics research. Expanding IC-MS to clinical biomarker discovery, microbial glycan profiling and real-time process monitoring in biomanufacturing represents promising directions.

Conclusion

The combination of Dionex CarboPac PA300 HPAE chromatography with Orbitrap MS delivers a robust IC-MS platform for comprehensive glycan separation and characterization. This workflow enables enhanced resolution of isomeric species, preserves native glycan structures and provides high-confidence structural data, addressing key challenges in glycomics.

References

1. Anal Bioanal Chem. 2017;409(12):3089–3101.
2. Mol Cell Proteomics. 2017;16(5):743–758.