Quantification and structural characterization of glycans and glycopeptides by TQMS: the energy-resolved oxonium ion monitoring (Erexim) platform

Significance of the Topic

Glycans and glycopeptides play essential roles in biological recognition events and protein stability. Accurate analysis of glycan composition and structure supports the development of biopharmaceuticals and the discovery of disease biomarkers. A combined strategy for quantitation and structural resolution addresses challenges in distinguishing isobaric glycoforms and monitoring sample heterogeneity.

Objectives and Study Overview

This work introduces the energy resolved oxonium ion monitoring Erexim platform based on a triple quadrupole mass spectrometer. The aim is to achieve rapid quantitative profiling and detailed structural differentiation of glycans and glycopeptides. A dedicated software suite and a reference database of fragmentation profiles enable identification of glycan structures by pattern matching.

Methodology and Instrumentation

• Sample preparation involved 2 aminopyridine labeled N glycan standards covering 45 structures and tryptic digestion of human IgG with Fc glycopeptide enrichment by solid phase extraction
• Chromatographic separation used an Aeris Peptide column 1.7 µm 150×2.1 mm at 40 °C with a gradient of 2 to 30 percent acetonitrile in 0.1 percent formic acid at 0.3 mL per minute
• Mass spectrometry was performed on a Shimadzu LCMS 8050 triple quadrupole instrument offering ultrafast scanning and polarity switching
• Multiple reaction monitoring focused on m/z 138 as a universal oxonium ion, with sequential collision energy steps to generate energy resolved fragmentation profiles
• Custom software automated method file creation and enabled data acquisition and profile based glycan identification against a structural database

Main Results and Discussion

• The platform delivered linear quantitation of glycopeptides from 2 to 1000 ng of IgG with r squared values above 0.999
• Relative glycoform abundances were highly reproducible with coefficients of variation below 5 percent at the lowest injection levels
• Energy resolved fragmentation profiles distinguished isobaric sialylated glycans by differences in the collision energy dependence of key oxonium ions
• Distinct behaviors of m/z 366 and 657 signals reflected variations in branch linkage stability consistent with theoretical expectations

Benefits and Practical Applications of the Method

The Erexim platform integrates high sensitivity quantitation with in depth structural insight in a high throughput format. Its compatibility with labeled glycans and intact glycopeptides streamlines workflows for biopharmaceutical quality control and glycan biomarker screening. Rapid turnaround times and robust software support facilitate routine application in research and industrial laboratories.

Future Trends and Possibilities

• Expansion of the fragmentation profile database to encompass a broader range of glycan subclasses and linkages
• Integration with bioinformatics workflows for fully automated glycomics interpretation
• Application to complex biological matrices and clinical specimens to accelerate biomarker discovery
• Combination with complementary analytical platforms to achieve deeper structural resolution

Conclusion

The energy resolved oxonium ion monitoring Erexim platform provides a versatile solution for comprehensive glycan and glycopeptide analysis on a triple quadrupole mass spectrometer. It achieves both accurate quantification and differentiation of isobaric structures through collision energy dependent oxonium ion profiling. Continued development of the reference database will extend its utility for identifying unknown glycoforms in complex samples.

Reference

• Toyama Atsuhiko Shuuichi Nakaya Shinji Funatsu Koji Ueda Yoshihiro Hayakawa Ichiro Hirano Quantification and structural characterization of glycans and glycopeptides by TQMS the energy resolved oxonium ion monitoring Erexim platform ASMS 2015 MP686