RAPID PREPARATION OF RELEASED N-GLYCANS FOR HILIC ANALYSIS USING A NOVEL FLUORESCENCE AND MS-ACTIVE LABELING REAGENT

Significance of the Topic

The detailed profiling of N-linked glycans is essential in biopharmaceutical development, quality control of therapeutic glycoproteins, and fundamental glycomics research. Traditional workflows for releasing, labeling, and analyzing N-glycans by HILIC-FLR-MS often involve lengthy deglycosylation steps, time-consuming labeling chemistries, and multiple sample clean-up procedures that compromise throughput and sensitivity.

Objectives and Study Overview

This work introduces an integrated workflow based on a novel labeling reagent, RapiFluor-MS, coupled with a rapid PNGase F formulation and HILIC μElution SPE clean-up. The study aims to demonstrate accelerated sample preparation (<30 minutes), enhanced fluorescence and mass spectrometric sensitivity, and robust recovery of released N-glycans from glycoprotein standards.

Methodology and Used Instrumentation

The streamlined protocol comprises three main steps: deglycosylation, labeling, and SPE clean-up. Key instrumentation and materials include:

• LC system: ACQUITY UPLC H-Class Bio with Glycan BEH Amide 130 Å, 1.7 μm, 2.1 × 50 mm column.
• Fluorescence detection: Ex 265 nm/Em 425 nm for RapiFluor-MS.
• Mass spectrometer: Synapt G2-S HDMS operating in ESI+, TOF resolution mode, managed with MassLynx V4.1.
• Reagents: RapiFluor-MS labeling kit, Rapid PNGase F with RapiGest SF surfactant, HILIC μElution SPE plate.

Main Results and Discussion

Benchmarking against Instant AB™ and conventional 2-AB labels revealed:

• RapiFluor-MS yields equivalent glycan profiles in HILIC-FLR and base peak intensity MS chromatograms with no bias.
• Fluorescence signal enhancement: ~2× compared to Instant AB; MS signal boost: ~780× over Instant AB.
• Rapid PNGase F with RapiGest SF achieves complete deglycosylation in ~5 minutes at 50 °C, confirmed by SDS-PAGE gel shift assays.
• HILIC μElution SPE ensures quantitative recovery of neutral to highly sialylated N-glycans without evaporation steps.

Benefits and Practical Applications

The integrated workflow enables preparation of FLR- and MS-active N-glycan samples from glycoproteins to analysis-ready material in under 30 minutes. This approach improves throughput in biopharmaceutical glycan profiling, enhances sensitivity for trace glycoforms, and simplifies routine QA/QC assays in pharmaceutical and academic laboratories.

Future Trends and Opportunities

Further development could include:

• Automation of the deglycosylation and clean-up steps in microplate formats to support high-throughput screening.
• Extension of labeling chemistry to O-glycans and glycolipids.
• Integration with ion mobility and MS/MS workflows for deeper structural characterization.

Conclusion

The RapiFluor-MS N-Glycan Kit offers a rapid, high-sensitivity workflow for released N-glycan analysis by HILIC-FLR-MS. By combining fast labeling, accelerated enzymatic release, and efficient SPE clean-up, it addresses common bottlenecks in glycomics and supports robust profiling of therapeutic glycoproteins.

References

• Mechref Y.; Hu Y.; Desantos‐Garcia J. L.; Hussein A.; Tang H. Quantitative glycomics strategies. Mol. Cell Proteomics 2013, 12(4), 874–884.
• Cook K. S.; Bullock K.; Sullivan T. Development and qualification of an antibody rapid deglycosylation method. Biologicals 2012, 40(2), 109–117.
• Klapoetke S.; Zhang J.; Becht S.; Gu X.; Ding X. Profiling and identification of N-linked glycans with a procainamide tag by HPLC with fluorescent and mass spectrometric detection. J. Pharm. Biomed. Anal. 2010, 53(3), 315–324.