Differentiation of α2,3 and α2,6 Sialic Acid Linked Glycan Isomers using SelexION® Differential Mobility Spectrometry Technology

Significance of Topic

Protein glycosylation represents one of the most prevalent and structurally diverse post-translational modifications. Terminal sialic acids in glycan chains play critical roles in immunity, pathogen recognition and disease progression. Distinct linkage isomers (α2,3 versus α2,6 to galactose or GalNAc) can profoundly affect biological function, including cancer metastasis and the efficacy of biopharmaceuticals. Reliable differentiation of these linkages is therefore essential for both basic research and quality control in therapeutic development.

Objectives and Study Overview

This study evaluated the selectivity of differential mobility spectrometry (DMS) using SelexION® technology coupled to a QTRAP® mass spectrometer for resolving isobaric glycan isomers bearing α2,3- and α2,6-linked sialic acids. Both small trisaccharide standards and larger disialylated biantennary glycans were examined to demonstrate method robustness and diagnostic value.

Methodology

Glycan standards were prepared in acetonitrile/water (20/80, v/v) with 10 mM ammonium bicarbonate at concentrations of 0.5–25 µg/mL. Continuous infusion of each sample at 5 µL/min was introduced into the SelexION DMS cell, positioned between the ion source orifice and the IonDrive™ Turbo V assembly. The DMS cell was maintained at 150 °C with nitrogen curtain gas at 30 psi. Methanol vapor (1.5 % in the curtain gas) served as a chemical modifier. Separation voltage (SV) and compensation voltage (CoV) were scanned while monitoring either MRM transitions or linear ion trap MS/MS spectra at each CoV increment.

Instrumentation Used

• SelexION® planar differential mobility spectrometry module (SCIEX)
• SCIEX QTRAP® 6500 and 6500+ systems
• IonDrive™ Turbo V atmospheric pressure ion source
• Methanol chemical modifier introduced via curtain gas

Main Results and Discussion

SelexION DMS achieved clear separation of α2,3- and α2,6-linked sialic acid trisaccharides and disialylated biantennary glycans. The α2,3 isomers consistently required a more negative CoV than their α2,6 counterparts, indicating stronger interactions with methanol vapor. Post-DMS MS/MS spectra revealed linkage-specific fragments (e.g., m/z 306 and m/z 655), facilitating unambiguous assignment. Compared to alternative ion mobility techniques, SelexION DMS uniquely resolved larger fucosylated structures that previously resisted separation.

Benefits and Practical Applications

• Orthogonal separation prior to MS enhances confidence in structural assignments.
• Rapid differentiation of sialic acid linkages supports glycoprotein profiling in biopharma R&D and QA/QC.
• Flexible tuning of DMS parameters and modifiers allows adaptation to diverse glycan classes.

Future Trends and Applications

Further work may explore additional chemical modifiers and coupling SelexION DMS with high-throughput workflows or tandem ion mobility platforms. Extending this approach to other glycoforms (e.g., O-linked glycans, Neu5Gc variants) and integrating software-driven CoV prediction models will broaden its utility in glycomics and biomarker discovery.

Conclusion

SelexION® DMS combined with QTRAP® MS provides a powerful, reproducible method for separating and identifying α2,3- and α2,6-sialylated glycan isomers across a range of sizes. The technique offers significant advantages for structural glycan analysis and biopharmaceutical product characterization.

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