High-Throughput Human Serum and Immunoglobulin G N-Glycome Profiling with the Agilent AdvanceBio Gly-X InstantPC Technology for Biomarker Discovery

Importance of the Topic

Protein glycosylation modulates many physiological and pathological processes including immune response, cancer, and inflammatory diseases. Detailed N-glycome profiling of serum and immunoglobulin G (IgG) supports biomarker discovery and therapeutic development. Traditional fluorescent labeling methods involve lengthy protocols and high sample requirements, limiting throughput and sensitivity.

Objectives and Study Overview

This study evaluates the Agilent AdvanceBio Gly-X InstantPC kit for high-throughput N-glycan profiling of human serum and purified IgG. The aim is to establish a streamlined workflow that reduces sample preparation time, lowers sample volume, and enhances reproducibility and detection sensitivity for biomarker discovery applications.

Used Instrumentation

• Agilent 1260 Infinity II liquid chromatography system
• Agilent AdvanceBio Glycan Mapping column (120 Å, 2.1×150 mm, 2.7 µm)
• Agilent OpenLab ChemStation software
• 96-well plate format for sample preparation

Methodology

Sample deglycosylation and labeling occur in a 96-well plate format. Human serum (1 µL) or 40 µg IgG is diluted in HEPES buffer, denatured at 90 °C for 3 minutes, and enzymatically digested with N-glycanase at 50 °C for 5 minutes. Released glycosylamines are labeled with InstantPC dye via activated carbamate chemistry at 50 °C for 1 minute. Cleanup employs formic acid/acetonitrile washes in a vacuum manifold. Labeled glycans are eluted in ammonium formate/acetonitrile solution. HILIC-FLD analysis uses a quaternary pump, a 1 µL injection (equivalent to glycans from 0.01 µL serum or 0.4 µg IgG), and fluorescence detection at excitation 285 nm and emission 345 nm. A linear gradient of acetonitrile against ammonium formate separates glycan species over a 35-minute run at 60 °C column temperature.

Main Results and Discussion

The InstantPC workflow resolves major glycan peaks with high reproducibility. IgG profiling revealed predominant species FA2G1 (24.9%), FA2 (18.4%), FA2G2 (13.0%), and monosialylated FA2G2S1 (11.1%). Human serum analysis showed diantennary disialylated A2G2S2 as the dominant peak (41.9%), followed by trisialylated A3G3S3 (7.6%), mono-sialylated A2G2S1 (6.1%), and FA2G1 (5.4%). The method consistently separates fucosylated, bisected, galactosylated, and sialylated N-glycans. Peak assignment was calibrated using an InstantPC-labeled maltodextrin ladder and glucose unit (GU) values.

Benefits and Practical Applications

• Rapid sample preparation in under ten minutes
• Minimal sample input (1 µL serum, 40 µg IgG)
• High sensitivity and reproducibility for quantitative glycomics
• Automation-friendly 96-well format increases throughput
• Potential integration into biomarker discovery pipelines and QA/QC workflows

Future Trends and Potential Applications

The InstantPC workflow could be extended to other glycoprotein classes and integrated with mass spectrometry for structural characterization. Combining high-throughput glycomics with bioinformatics and multi-omics platforms may accelerate biomarker validation and personalized medicine. Advances in labeling chemistries and microfluidic automation are expected to further reduce sample requirements and analysis time.

Conclusion

The Agilent AdvanceBio Gly-X InstantPC technology provides a rapid, sensitive, and reproducible platform for N-glycan profiling of human serum and IgG. Its low sample requirement and high-throughput format support large-scale biomarker studies and biopharmaceutical development, representing a significant advancement over conventional glycan analysis methods.

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