Software Platform for Glycan Quantification and Qualification by LCMS-8060/8050 - Erexim Application Suite

Importance of the Topic

Site-specific analysis of N-linked glycan heterogeneity is critical for understanding protein function, therapeutic efficacy and batch-to-batch consistency in biopharmaceuticals. Conventional approaches that release glycans from all glycoproteins yield averaged profiles, masking variations at individual glycosylation sites. The Erexim Application Suite addresses this need by combining quantitative and qualitative mass spectrometric workflows to reveal fine structural detail at targeted sites.

Objectives and Study Overview

The primary goal is to introduce a software platform that streamlines MRM-based quantitation and energy-resolved oxonium ion monitoring for site-specific glycan analysis. Key features include a customizable glycan structure database, automated MRM transition generation, and integrated data processing for both relative abundance and collision energy profiling.

Methodology and Instrumentation

Sample Preparation

• Proteolytic digestion of glycoproteins with trypsin
• SPE clean-up to enrich target glycopeptides

Chromatography and Mass Spectrometry

• LCMS platform: Shimadzu Triple Quadrupole LCMS-8060/8050
• Column: Aeris Peptide XB-C18 (2.1 × 150 mm)
• Mobile phases: 0.1% formic acid in water and 90% acetonitrile with 0.1% formic acid
• Gradient: 2–30% B over 10 minutes, ramp to 98% B, then re-equilibrate
• Flow rate: 0.3 mL/min; injection volume: 10 µL
• MRM acquisition: user-defined dwell time, pause time, collision energy

Software Components

• Profile Database Manager with 45 default glycan entries and user extension capability
• MRM Method Maker automating transition list generation based on composition, adduct type and peptide sequence
• Data Analyzer for peak integration, relative quantitation and Erexim profile plotting

Key Results and Discussion

In an example using a commercial monoclonal IgG, 24 glycan structures attached to the Fc region peptide were targeted. Relative abundance data showed two dominant species at 43% and 11% of total glycan area. Subsequent Erexim profiling of the major glycan at m/z corresponding to structure ID 45100 produced a collision energy–dependent oxonium ion pattern matching the reference profile, confirming structural assignment. In contrast, data for structure ID 44100 revealed an intermediate CE profile between two isomeric references, highlighting the ability to detect structural heterogeneity beyond mass alone.

Benefits and Practical Applications

The integrated suite significantly reduces manual workload by automating transition design and data processing. Combining quantitative MRM and qualitative Erexim profiling enables precise site-specific glycoform characterization. Applications include therapeutic antibody QC, biomarker discovery, glycoengineering optimization and regulatory compliance in biologics production.

Future Trends and Potential Use

Advances may include expansion of the glycan database with novel linkages, integration with laboratory information management systems, higher-throughput multiplexed analyses and real-time monitoring during bioprocessing. Machine learning approaches could further refine collision energy prediction and structural deconvolution for complex glycoproteomes.

Conclusion

The Erexim Application Suite for LCMS-8060/8050 provides a robust, user-friendly workflow for detailed glycan heterogeneity analysis at specific glycosylation sites. By uniting automated MRM quantitation with energy-resolved oxonium ion monitoring, researchers gain both relative abundance data and structural insights with minimal manual intervention.

Reference

Toyama A, et al. Anal. Chem. 2012, 84, 9655–9662