Exact Structure Identification of Isomeric N-Glycans by High‑Resolution Ion Mobility LC/Q‑TOF

Importance of the Topic

Glycosylation is the most complex post-translational modification of proteins, playing critical roles in biological processes and disease mechanisms. Distinguishing isomeric N-glycan structures at the molecular level is essential for understanding glycan function. High-resolution ion mobility LC/Q-TOF techniques leveraging unique arrival time distributions (ATDs) offer a route to unambiguous glycan identification.

Objectives and Study Overview

• Establish a workflow combining multiplexed ion mobility LC/Q-TOF with high-resolution demultiplexing to assign exact N-glycan structures.
• Demonstrate method performance using synthetic standards and N-glycans released from guinea pig erythrocytes.

Methodology and Instrumentation

• Glycan Preparation: Enzymatic release of N-glycans with Endo F2, purification by C18 and PGC solid phase extraction, and labeling with 2-aminobenzoic acid via reductive amination.
• Instrumentation: Agilent 1290 Infinity II LC with Hypercarb PGC column (100×2.1 mm, 3 μm) at 75 °C; Agilent 6560 IM LC/Q-TOF with Jet Stream source operating in negative mode, 4-bit multiplexing, 60 ms drift time.
• Data Processing: Demultiplexing via PNNL PreProcessor Beta 3.0, high-resolution demultiplexing and conformer distribution fingerprinting (CDF) using HRdm 2.0 software.

Results and Discussion

Extracted ion chromatograms of 2-AA labeled N-glycans from erythrocytes revealed multiple isomeric peaks. MS/MS alone could not resolve these isomers. High-resolution IM demultiplexing delivered distinct ATDs and collision cross section (CCS) values for each standard, with resolution up to Ω/ΔΩ = 267. Conformer distribution fingerprints matched biological sample features to reference standards, enabling rapid and unambiguous identification of isomeric N-glycans with varying poly-LacNAc chain lengths. Standards 6–8 displayed unique CDFs but were absent in the erythrocyte sample.

Benefits and Practical Applications

• Accelerated assignment of isomeric N-glycan structures in complex matrices without extensive MSn workflows.
• High-confidence identifications supported by a comprehensive CDF library.
• Compatibility with routine glycomics protocols in biopharma, clinical research, and quality control settings.

Future Trends and Opportunities

• Expansion of CDF libraries to include sialylated, fucosylated, and other glycan classes.
• Integration of AI-driven spectral matching and automated annotation tools.
• Application of IM profiling to other biomolecules such as glycolipids and glycosaminoglycans.

Conclusion

High-resolution ion mobility LC/Q-TOF with multiplexed acquisition and advanced demultiplexing presents a robust platform for exact isomeric N-glycan structure assignment. Conformer distribution fingerprinting streamlines glycan characterization, benefiting glycomics research and industry QA/QC.

References

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