An MSn platform for detailed characterisation of both the peptide and the glycan moieties and the peptide/glycan linkage in glycoproteins

Importance of the topic

The detailed analysis of glycoproteins is critical in biopharmaceutical research and quality control, particularly for biosimilars and follow-on biologics. Accurate mapping of glycosylation sites, glycan composition and isomeric structures ensures consistency with innovator drugs, supports regulatory approval and informs manufacturing process control.

Objectives and study overview

This study demonstrates an integrated MSn platform combining high-resolution MALDI-QIT-TOF mass spectrometry with advanced software tools for comprehensive glycoprotein characterization. The goals include pinpointing peptide–glycan linkage sites, sequencing peptide backbones and resolving glycan isomers to deliver a fully detailed molecular profile of human transferrin glycopeptides.

Methodology and instrumentation

Samples of reduced, alkylated and trypsin-digested human apo-transferrin underwent glycopeptide enrichment followed by multi-stage MSn analysis. In parallel, PNGase F–released glycans were purified, aminopyridine-labelled and examined by directed MS up to MS4.

• MALDI-QIT-TOF mass spectrometer (Axima Resonance, Shimadzu).
• SIMSE software for glycopeptide composition, sequence and linkage analysis (Shimadzu).
• Accurate Glycan Analyser (AGA) for isomeric glycan identification using an empirical MSn spectral database.

Key results and discussion

SIMSE detected characteristic HexNAc triplets in MS2 spectra, revealing glycosylation at Asn432 and Asn630. Mass differences established a composition of Hex5HexNAc4. AGA MS→MS4 workflows resolved the bi-antennary glycan isomer and confirmed sialylation states, showing native glycans bearing zero, one or two sialic acids.

Benefits and practical applications of the method

This platform enables:

• Precise glycosylation site mapping and peptide sequencing.
• Accurate glycan composition assignment and isomer discrimination.
• Regulatory-grade data supporting biosimilar comparability.

Future trends and potential applications

Advances in automated MSn data interpretation, expanded glycan spectral libraries and integration with LC-MS workflows will further improve throughput and structural coverage. Potential applications extend to biomarker discovery, bioprocess monitoring and synthetic glycoprotein design.

Conclusion

The combined MALDI-QIT-TOF/MSn approach with SIMSE and AGA software delivers a robust, end-to-end solution for in-depth glycoprotein characterization, meeting the stringent demands of modern biopharmaceutical development and quality control.