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

Significance of the topic

Glycosylation is the most prevalent post-translational modification influencing protein stability, folding, and biological activity. Aberrant O-linked glycosylation is implicated in cancer, autoimmune and cardiovascular diseases. Reliable profiling of O-glycans is therefore crucial for basic research, clinical diagnostics, and biopharmaceutical quality control.

Objectives and overview of the study

This work demonstrates an integrated workflow combining high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and high-resolution accurate-mass Orbitrap mass spectrometry to profile O-linked glycans released from four model glycoproteins: porcine gastric mucin type III, bovine fetuin, fibrinogen, and thyroglobulin. The goals are to achieve high-resolution separation, mass-accurate identification, and confident structural annotation of native O-glycans without derivatization.

Methodology and instrumentation

O-glycans were released by reductive β-elimination, purified on porous graphitized carbon, and separated on a Dionex CarboPac PA300-4 µm column under a gradient of NaOH and sodium acetate. The effluent was split post-column, with one branch directed to a pulsed amperometric detector and the other to a Q Exactive HF Orbitrap MS via an ERD 500 electrolytic desalter.

• Dionex ICS-5000+ system with CarboPac PA300 guard and analytical columns
• Gold working electrode pulsed amperometric detector (PAD)
• ERD 500 electrolytic desalter
• Thermo Scientific Q Exactive HF Quadrupole-Orbitrap MS with HESI-II source

Main results and discussion

Porcine gastric mucin yielded a diverse array of neutral, sialylated, and sulfated O-glycans. Bovine fetuin produced predominantly sialylated core-1 tri- and tetrasaccharides. Bovine fibrinogen displayed mainly biantennary mono-, di- and trisialylated O-glycans, with diagnostic MS2 fragments confirming linkage types. Bovine thyroglobulin analysis provided the first direct evidence of an O-glycan ([Neu5Ac]HexHexNAc) on this protein. All assignments were made at <5 ppm mass accuracy and verified by characteristic glycosidic and cross-ring fragments.

Benefits and practical applications

This label-free, high-resolution method obviates the need for derivatization, combines quantitative PAD responses with structural MS data, and is suited for glycoprotein characterization in pharmaceutical development, biomarker discovery, and clinical research.

Future trends and applications

Future developments may include integration with glycopeptide and N-glycan analyses, automation for high-throughput screening, deeper structural elucidation through MSn approaches, and application to complex biological and clinical sample sets for more comprehensive glycoproteomic studies.

Conclusion

The combined HPAE-PAD and high-resolution Orbitrap MS workflow using the CarboPac PA300 column enables comprehensive, sensitive, and accurate profiling of native O-linked glycans from diverse glycoproteins, supporting confident structural assignments and broad analytical applications.

Reference

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