Analysis of Isomeric Permethylated Lacto-oligosaccharides Using Electron Transfer Dissociation Combined with Ion Mobility MS

Significance of the Topic

Glycosylation plays a crucial role in protein function, stability, and intercellular communication. Structural variations in oligosaccharides are linked to biological processes and disease markers. Distinguishing isomeric glycans is analytically demanding due to branching, multiple linkage sites, and anomeric configurations. Advanced mass spectrometry techniques that combine electron transfer dissociation with ion mobility offer a route to resolve closely related oligosaccharide structures.

Objectives and Study Overview

This work examines two isomeric permethylated milk oligosaccharides—lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT)—using an integrated ETD-ion mobility mass spectrometry approach. The goals are to:

• Demonstrate how ETD fragmentation produces distinct product ions for isomeric glycans.
• Showcase ion mobility separation to disentangle overlapping mass spectral features.
• Establish reliable markers for differentiating LNT and LNnT.

Methods and Instrumentation

Permethylation was applied to both oligosaccharides to enhance stability and structural information. Samples at 3 μM concentration were complexed with magnesium in 50% methanol, generating doubly charged [M+Mg]2+ precursors. Analyses were conducted on a SYNAPT G2 High Definition MS system equipped with T-Wave triwave ion guides and an ion mobility cell.

• Ionization: Electrospray in positive mode for MS and glow discharge for ETD reagent.
• MS settings: Capillary voltage 3.0 kV, cone voltage 25 V, desolvation temperature 200 °C.
• ETD: Glow discharge current 100 μA, trap T-Wave wave velocity 300 m/s.
• Ion Mobility: Nitrogen drift gas at 3.5 mbar, wave velocity 650 m/s, amplitude 25 V.

Main Results and Discussion

ETD generated a rich pattern of cross-ring and glycosidic cleavages. Both LNT and LNnT yielded major singly charged fragments from GlcNAc–Gal bond cleavage (Y2 at m/z 463.2 and B2 at m/z 464.2). A distinct internal E2 ion (m/z 228) was more abundant for LNT. CID spectra further confirmed differences: LNnT showed a characteristic methanol loss from the E2 ion due to linkage proximity to the N‐acetyl group.
Ion mobility enabled temporal separation of isobaric or overlapping ions. In the ATD domain, Y2, B2, and the precursor appeared at distinct drift times, facilitating independent extraction of each mass spectrum. The more compact Y2 species drifted slower than the larger B2 fragment.

Benefits and Practical Applications

This combined ETD-IM-MS methodology offers:

• Unambiguous differentiation of linkage isomers without extensive prior separation.
• Improved spectral clarity by separating overlapping ions based on mobility.
• Enhanced structural detail from permethylation and ETD cross-ring fragments.

This approach can be applied to glycoprotein profiling, quality control in biopharmaceutical production, and biomarker discovery in clinical glycomics.

Future Trends and Applications

Emerging directions include:

• Integration with higher-resolution mobility devices to resolve complex glycan mixtures.
• Automation of mobility-guided spectral deconvolution for high-throughput glycomics.
• Extension to intact glycopeptide analysis, combining ETD sequencing and mobility separation to map site-specific glycosylation.

Conclusion

The synergy of ETD fragmentation and ion mobility separation on the SYNAPT G2 platform enables clear discrimination of isomeric permethylated oligosaccharides. This workflow improves confidence in structural assignments and offers a powerful tool for advanced glycan analysis in research and industry.

Used Instrumentation

• SYNAPT G2 High Definition MS System (Waters Corporation)
• T-Wave Triwave ion guides and mobility cell
• Electrospray ion source with positive‐mode ESI
• Glow discharge ETD reagent supply

References

1. Domon B., Costello C. E. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconjugate Journal. 1988;5:397–409.
2. Han L., Costello C. E. Electron transfer dissociation of milk oligosaccharides. Journal of the American Society for Mass Spectrometry. 2011;22(6):997–1013.