GLYCOWORKS RapiFluor-MS QUICK START PROTOCOL

Significance of the topic

The GlycoWorks RapiFluor-MS Quick Start Protocol streamlines N-glycan analysis for high-throughput glycoprotein characterization. Rapid deglycosylation, fluorescent labeling of released glycans, and efficient HILIC-based cleanup reduce sample preparation time while maintaining sensitivity and reproducibility, supporting biopharmaceutical quality control and glycomics research.

Objectives and protocol overview

This workflow is designed to process up to 96 glycoprotein samples (4×24 format) using a concise three-step procedure. It integrates rapid enzymatic deglycosylation, chemical labeling, and solid-phase extraction to prepare glycans for LC-MS profiling.

Methodology and instrumentation

Key reagents and steps:

• Denaturation: RapiGest SF surfactant (3% w/v) with heat at 90 °C.
• Deglycosylation: Rapid PNGase F enzyme at 50 °C for 5 minutes.
• Labeling: RapiFluor-MS reagent in anhydrous DMF, room temperature for 5 minutes.
• Cleanup: Waters GlycoWorks HILIC µElution Plate with sequential conditioning, equilibration, loading, washing, and elution using ACN and formic acid solutions.

Instrumentation includes a dual-temperature heat block, multichannel pipettes, a vacuum manifold for SPE, and standard LC-MS equipment for downstream analysis.

Main results and discussion

The streamlined protocol achieves complete deglycosylation and labeling in under 15 minutes of hands-on time. HILIC cleanup demonstrates high glycan recovery and purity, yielding consistent MS signal intensities across replicates. The workflow’s reproducibility and speed make it suitable for routine analysis of therapeutic antibodies and other glycoproteins.

Benefits and practical applications

• High throughput: processes 96 samples with minimal manual interventions.
• Enhanced sensitivity: RapiFluor-MS labeling improves fluorescence and mass spectrometric detection.
• Efficient cleanup: µElution SPE reduces solvent consumption and sample loss.
• Versatile use: ideal for biopharmaceutical QA/QC, glycoform profiling, and academic glycomics studies.

Future trends and potential applications

Future developments may include full automation on robotic liquid-handling platforms, integration with ion-mobility MS for isomer separation, and expansion to O-glycan analysis. Novel reagents may further accelerate labeling or enable multiplexed workflows for comparative glycan profiling.

Conclusion

The GlycoWorks RapiFluor-MS Quick Start Protocol provides a rapid, reproducible, and scalable approach to glycan sample preparation. Its streamlined design and high throughput capacity address the growing needs of biopharmaceutical and glycoscience laboratories for reliable glycan analysis.

Reference

• Waters Application Note 715004793EN: GlycoWorks Care and Use Manual.
• Waters Application Note 720005506EN: Glycan Sample Preparation Guide.
• Waters Application Note 720007038EN: Rapid Labeling and Cleanup Strategies.