Improved Recovery and Peak Shape of Sialylated O-glycopeptides of Erythropoietin (EPO) with ACQUITY Premier CSH Column

Significance of the Topic

Peptide mapping via liquid chromatography mass spectrometry serves a critical role in protein characterization and quality control in the biopharmaceutical sector. Among various analytes the class of sialylated O glycopeptides presents unique challenges due to non specific adsorption to metal surfaces leading to analyte loss and poor peak shape. Effective separation and recovery of these species is essential for accurate post translational modification analysis and relative quantitation in biologics development.

Study Objectives and Overview

This application brief examines the performance of a novel ACQUITY Premier CSH column featuring MaxPeak High Performance Surfaces compared with a conventional stainless steel column. The focus is on recovery and chromatographic peak shape of sialylated O glycopeptides derived from erythropoietin, a clinically relevant glycoprotein with one O glycosylation site and three N glycosylation sites.

Methodology and Instrumentation

The protein sample was denatured reduced alkylated and digested with trypsin. Peptide mixtures were analyzed on a BioAccord LC MS system using an ACQUITY UPLC I Class coupled to an ACQUITY RDa detector. Separations occurred at 60 °C on two columns of identical dimensions and chemistry except for surface treatment. Mobile phases used 0.1 percent formic acid in water and acetonitrile with a gradient from one to thirty five percent organic over fifty minutes. Mass spectrometry was performed in positive electrospray mode under data independent acquisition.

Key Results and Discussion

Extracted ion chromatograms for core and sialylated O glycopeptide forms derived from the tryptic peptide EAISPPDAASAAPLR showed a two and a half fold increase in peak area for all species on the Premier column. Doubly sialylated species in particular exhibited a fourfold gain in signal. Chromatographic asymmetry for the doubly sialylated glycopeptide improved sixfold, approaching Gaussian shape and enabling more precise integration. Relative quantitation matched closely with values obtained from intact de N glycosylated erythropoietin analysis, indicating mitigation of metal induced adsorption artifacts.

Benefits and Practical Applications

• Reduced non specific sample adsorption compared with conventional stainless steel hardware
• Enhanced analyte recovery for acidic and sialylated glycopeptides
• Improved peak symmetry and retention time reproducibility
• Better alignment with orthogonal intact protein quantitation for reliable profiling

Future Trends and Opportunities

The adoption of high performance surface technologies is expected to extend to other metal sensitive analytes including phosphorylated peptides and low abundance acidic species. Continued innovation in column surface design may further boost throughput and sensitivity in biologics characterization workflows. Integration with advanced mass spectrometry methods promises deeper structural elucidation of complex glycoforms.

Conclusion

The ACQUITY Premier CSH column featuring MaxPeak High Performance Surfaces offers a robust solution for the analysis of sialylated O glycopeptides. By reducing metal induced adsorption it delivers significant gains in sensitivity peak shape and quantitation accuracy. This advancement supports more reliable peptide mapping for biopharmaceutical research and quality control.

References

• Koshel B Simeone J Dao D Nguyen JM Rzewuski SC Lauber MA Birdsall RE Yu YQ Bypassing LC System Passivation Requirements Using ACQUITY Premier Column with MaxPeak HPS Technology for the Recovery of a Phosphorylated Peptide Waters Application Note 2020
• Birdsall RE Kellet J Ippoliti S Ranbaduge N Shion H Yu YQ Increasing Chromatographic Performance of Acidic Peptides in RPLC MS based Assays with ACQUITY Premier Column featuring MaxPeak HPS Technology Waters Application Note 2020
• Lauber MA Walter TH Gilar M DeLano M Boissel C Smith K Rainville P Belanger J Wyndam K Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces Waters White Paper 2020
• CarboMet Case Study Erythropoeitin EPO Professor Sabine Flitsch University of Manchester 2021