Analysis of N-glycans from Ribonuclease B

Significance of the topic

The analysis of N-linked glycans plays a crucial role in biopharmaceutical quality control, disease biomarker discovery, and glycomics research. Glycan structures influence protein function, stability, and immunogenicity, making reliable analytical methods essential.

Objective and study overview

This study demonstrates the separation, characterization, and detection of N-glycans released from ribonuclease B using hydrophilic interaction liquid chromatography coupled with fluorescence and mass spectrometric detection.

Methodology and instrumentation

N-glycans were labeled with 2-aminobenzamide to enhance fluorescence sensitivity. Separation was achieved on a Shim-pack GIST-HP Amide column under a gradient of acetonitrile and 50 mM ammonium formate. The mobile phase composition varied from 75 % to 20 % acetonitrile over 60 minutes at a flow rate of 0.25 mL/min. The column temperature was maintained at 45 °C. Fluorescence detection was performed at excitation 330 nm and emission 420 nm. Mass spectrometry was conducted in positive heated electrospray ionization mode over an m/z range of 700–2500.

Instrumentation

• UHPLC system: Nexera X2
• Column: Shim-pack GIST-HP Amide, 150 mm × 2.1 mm ID, 1.9 µm
• Mass spectrometer: LCMS-9030 with heated ESI source

Key results and discussion

Fluorescence chromatograms resolved five major mannose-containing glycans (Man5–Man9). MS spectra confirmed the identity of each peak based on expected m/z values. Peak shapes and retention times demonstrated the efficacy of HILIC for separating structurally similar glycans.

Benefits and practical applications

The combined HILIC-fluorescence-MS approach offers high sensitivity, selectivity, and throughput, making it suitable for glycan profiling in biopharmaceutical development, quality control, and basic research.

Future trends and potential applications

Advances may include integration of MS/MS for detailed structural elucidation, ion mobility separation, and automated data analysis workflows. Expansion into comprehensive glycomics studies and clinical biomarker discovery is anticipated.

Conclusion

This method provides a robust, user-friendly platform for routine N-glycan analysis, delivering reliable separation and identification of glycan species essential for research and quality assurance.