GLYCOWORKS RapiFluor-MS QUICK START PROTOCOL - 24 sample (3 x 8 Format)

Significance of the Topic

The accurate profiling of N-linked glycans is essential in analytical characterization of therapeutic glycoproteins. Rapid and consistent workflows improve laboratory efficiency and ensure robust data for quality control and research applications.

Study Objectives and Overview

This protocol outlines a rapid deglycosylation and labeling workflow using GlycoWorks RapiFluor-MS reagents. The method accommodates 24 samples in a 3×8 format, focusing on disulfide-rich glycoproteins and achieving completion in under 20 minutes.

Methodology and Instrumentation

The three main steps include:

1. Deglycosylation
   Starting with 1.5 mg/mL glycoprotein, samples are denatured with RapiGest SF and DTT, heated at 90 °C, then treated with Rapid PNGase F at 50 °C for 5 minutes.
2. Labeling
   Deglycosylated glycosylamines are reacted with RapiFluor-MS reagent in DMF for 5 minutes at room temperature, employing a doubled reagent concentration in a reduced reaction volume.
3. HILIC Cleanup
   Labeled glycans are captured on a GlycoWorks HILIC µElution plate conditioned with water and acetonitrile, washed, and eluted in an acidic organic solvent for downstream LC-MS analysis.

Instrument Used

• Heat blocks set at 90 °C and 50 °C
• GlycoWorks HILIC µElution plate with shims and waste tray
• Standard laboratory pipettes and microcentrifuge tubes
• Reagents: RapiGest SF, DTT, Rapid PNGase F, RapiFluor-MS, acetonitrile, DMF, formic acid, water

Main Results and Discussion

By halving reaction volumes while doubling label concentration, the workflow achieves efficient glycan release and labeling with minimal hands-on time. Cleanup yields purified, fluorescently labeled glycans ready for high-sensitivity MS detection, demonstrating consistency across 24 samples.

Benefits and Practical Applications

• High throughput processing of glycoprotein samples
• Rapid turnaround suitable for QC and research
• Enhanced reproducibility with standardized reagents
• Seamless integration with LC-MS platforms

Future Trends and Possibilities

Automation of the workflow with robotic platforms, miniaturization to reduce reagent consumption, development of novel fluorescent labels, and coupling with high-resolution MS and informatics tools will further streamline glycan analysis.

Conclusion

The GlycoWorks RapiFluor-MS quick start protocol offers a fast, robust solution for glycan analysis of disulfide-rich glycoproteins, supporting high-throughput laboratories in achieving reproducible and high-quality glycan profiles.

Reference

Waters Corporation. GlycoWorks RapiFluor-MS Quick Start Protocol. Application Notes 720005506EN and 720007038EN. Care and Use Manual 715004903.