Evaluating Protein Glycosylation in Limited-Quantity Samples by HPAE-PAD

Significance of the Topic

Changes in protein glycosylation are critical in cancer research as they can reveal biomarkers for disease detection and progression. Prostate‐specific antigen (PSA) glycosylation patterns differ between normal and cancerous states, making detailed glycan analysis essential. High‐performance anion exchange chromatography with pulsed amperometric detection (HPAE‐PAD) offers a direct, label-free approach capable of analyzing N- and O-linked glycans in limited protein samples (<10 µg), addressing challenges of low sample availability.

Objectives and Study Overview

This study aimed to develop HPAE-PAD methods to profile N- and O-glycosylation of PSA and transferrin in microgram‐scale samples. Two complementary strategies were validated:

• PNGase F digestion to release N-linked glycans and assess sialylation and fucosylation patterns.
• Acid hydrolysis of intact proteins or released glycans to quantify diagnostic monosaccharides (glucosamine, galactosamine) as indicators of O-glycosylation or sample contamination.

Methodology and Instrumentation

Samples (0.5–10 µg protein per injection) underwent:

• Denaturation and PNGase F digestion to release N-glycans.
• Ultrafiltration (10 kDa and 30 kDa MWCO) to separate glycans from proteins and salts.
• Optional enzymatic digests with neuraminidase (α2-3 and α2-6) and α-L-fucosidase to determine linkage and core fucosylation.
• Acid hydrolysis (6 N HCl, 100 °C, 4 h) for monosaccharide analysis.
• HPAE-PAD separation on CarboPac PA200 for oligosaccharide profiling (hydroxide and acetate gradients) and on CarboPac PA20/AminoTrap for monosaccharides (KOH eluent).

Used Instrumentation

• Thermo Scientific Dionex ICS-3000/5000 HPAE-PAD system with eluent generator and pulsed amperometric detector.
• CarboPac PA200 columns (3×250 mm analytical, 3×50 mm guard) for glycan profiling.
• CarboPac PA20 (3×150 mm) and AminoTrap (3×30 mm) for monosaccharide separations.
• Thermo Scientific NanoDrop 2000c for protein concentration checks.
• Amicon Ultra centrifugal filters (10 kDa, 30 kDa) for sample cleanup.

Key Results and Discussion

Monosaccharide hydrolysis distinguished O-glycan contributions: transferrin lacked galactosamine, while mucin contained both glucosamine and galactosamine; transferrin spiked with mucin showed both signals.
PSA hydrolysates before and after PNGase F displayed similar amino sugar ratios (~0.07 GalNAc/GlcNAc), indicating minimal O-glycosylation.
Oligosaccharide profiles revealed predominant mono- and disialylated biantennary species (A1F, A2F) with core fucosylation. Neuraminidase digests quantified ~6 mol Neu5Ac/mol PSA (3 mol α2-3, 2.8 mol α2-6), confirming linkage distribution. Fucosidase treatment removed fucosylated peaks, verifying core fucosylation of charged glycans.

Benefits and Practical Applications

• Direct, derivatization-free detection of glycans preserves labile sialic acid linkages.
• High sensitivity enables analysis of <10 µg total protein, suitable for limited clinical samples.
• Complementary monosaccharide and oligosaccharide workflows distinguish N- vs. O-glycosylation and identify linkage features.

Future Trends and Applications

Integration of HPAE-PAD with high-resolution MS could further elucidate structural microheterogeneity. Automated sample preparation and miniaturized formats may extend analysis to single‐cell glycomics or multiplexed biomarker screening in clinical diagnostics.

Conclusion

This work demonstrates robust HPAE-PAD methods for detailed glycan mapping of PSA and transferrin in low-quantity samples. The combination of enzymatic and acid treatments with optimized chromatography provides insights into glycan composition, sialic acid linkages, and core fucosylation, offering valuable tools for cancer biomarker research.

Reference

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[3] Dionex Application Notes and Technical Notes (TN 71, AU 141, AU 176, AN 215).
[4] Dionex TN 40: Glycoprotein Monosaccharide Analysis. 2012.
[5] Spik G. et al. FEBS Lett. 1975, 50, 296–299.