Robustness of RapiFluor-MS N-Glycan Sample Preparations and Glycan BEH Amide HILIC Chromatographic Separations

Significance of the topic

Protein N-glycosylation plays a vital role in biopharmaceutical quality control and disease biomarker research. Efficient, sensitive, and reproducible glycan analysis underpins therapeutic development and clinical diagnostics.

Study Objectives and Overview

This work evaluates the robustness of the GlycoWorks RapiFluor-MS N-Glycan Kit for rapid sample preparation and Waters Glycan BEH Amide HILIC columns for chromatographic separations. Performance is benchmarked against conventional 2-AB labeling and HPLC methods. Novel calibration standards are introduced to enhance retention time reproducibility.

Methodology and Instrumentation

The integrated 30-minute workflow comprises: heat-denaturation with RapiGest SF, rapid PNGase F digestion (10 min), RapiFluor-MS labeling (~5 min), and HILIC SPE cleanup. Labeled glycans are analyzed by HILIC-FLR and MS detection. Glucose unit (GU) calibration uses a dextran ladder derivatized with a RapiFluor-MS analog.

Instrumentation

• ACQUITY UPLC H-Class Bio System
• Glycan BEH Amide Columns (1.7 µm, 2.5 µm)
• ACQUITY QDa Mass Detector
• Xevo G2-XS QTof MS
• SYNAPT G2-Si HDMS

Key Results and Discussion

The kit achieves complete deglycosylation, >95% labeling efficiency, ~74% SPE recovery, and ~73% overall yield—over twice the ~35% yield of 2-AB workflows. Method performance is unaffected by extended heat treatment, reagent batch variation, or 300 consecutive UPLC runs. BEH Amide columns provide high peak capacities (Pc ~80–95) on UPLC and HPLC. GU calibration with the dextran ladder minimizes retention drift and improves data consistency.

Benefits and Practical Applications

• Sample prep in 30 min versus hours
• Enhanced MS/FLR sensitivity via RapiFluor-MS tag
• Quantitative, bias-minimized glycan recovery
• Seamless transfer between HPLC and UPLC platforms
• Standardized system suitability with dedicated test standards

Future Trends and Opportunities

Automation of rapid glycan workflows will support high-throughput biopharma analytics. Coupling with advanced MS and AI-driven data analysis can accelerate biomarker discovery. Expanded calibration libraries and multiplexed labeling chemistries will broaden glycomics applications.

Conclusion

The GlycoWorks RapiFluor-MS N-Glycan Kit combined with Glycan BEH Amide HILIC separation offers a robust, sensitive, and high-throughput solution for N-glycan analysis, enabling reliable glycoprofiling in research and quality control environments.

References

1. Ohtsubo K; Marth JD. Cell 2006,126(5):855–67
2. Mechref Y et al. Bioanalysis 2012,4(20):2457–69
3. Ruhaak LR et al. Mol Cell Proteomics 2013,12(4):846–55
4. Dalziel M et al. Science 2014,343(6166):1235681
5. Beck A et al. Anal Chem 2013,85(2):715–36
6. Mechref Y et al. Mol Cell Proteomics 2013,12(4):874–84
7. Ruhaak LR et al. Anal Bioanal Chem 2010,397(8):3457–81
8. Lauber MA et al. Waters Application Note 720005275EN 2015
9. Yu YQ et al. Rapid Commun Mass Spectrom 2005,19(16):2331–6
10. Koza SM et al. Waters Application Note 720005214EN 2015
11. Campbell MP et al. Bioinformatics 2008,24(9):1214–6