In-depth Peptide Mapping with Iterative MS/MS Acquisition on the Agilent 6545XT AdvanceBio LC/Q‑TOF

Significance of the Topic

Therapeutic monoclonal antibodies (mAbs) are among the fastest growing classes of biopharmaceuticals. Their structural heterogeneity, including sequence variants and post-translational modifications (PTMs) such as deamidation, oxidation and glycosylation, demands comprehensive analytical characterization. Peptide mapping via liquid chromatography–mass spectrometry (LC–MS) provides critical insights into primary sequence confirmation and PTM profiling, underpinning quality control and regulatory submissions.

Objectives and Study Overview

This application demonstrates an end-to-end workflow for in-depth peptide mapping of a human IgG1 mAb, integrating automated sample preparation, high-resolution LC–MS/MS analysis and advanced data processing. A novel Iterative MS/MS acquisition method on the Agilent 6545XT AdvanceBio LC/Q-TOF is evaluated for its ability to enhance sequence coverage and detect low-abundance PTMs.

Instrumentation

• Agilent AssayMAP Bravo liquid-handling workstation
• Agilent 1290 Infinity II LC system with AdvanceBio Peptide Mapping column (2.1×150 mm, 2.7 μm)
• Agilent 6545XT AdvanceBio Q-TOF with Dual Jet Stream ESI
• Agilent MassHunter BioConfirm B.09 software

Methodology

CHO-derived human IgG1 mAb was reduced, alkylated and trypsin-digested using an automated AssayMAP Bravo protocol, followed by desalting. Peptides were separated using either a 15- or 30-minute gradient at 0.4 mL/min, and injected at 0.6 µg or 2 µg. Data were acquired via conventional Auto MS/MS and Iterative MS/MS, which excludes previously fragmented precursors across multiple injections. MS settings included 110–1700 m/z mass range, 8 spectra/s survey scans, and top10 MS/MS selection. Data were processed with BioConfirm B.09, using semitryptic search, up to two missed cleavages, common PTMs, 10 ppm MS1 tolerance, 30 ppm MS2 tolerance and 0.1% FDR.

Main Results and Discussion

• Sequence coverage ≥96.2% for heavy and ≥98.6% for light chains with 15-minute gradient and 0.6 µg injection; full coverage achieved with additional runs.
• Iterative MS/MS enabled identification of low-abundance glycopeptide TKPREEQYNSTYR only in the second injection, raising heavy chain coverage to 99.12%.
• Comprehensive PTM profiling of peptide NTAYLQMNSLR revealed native (94.09%), M7-oxidation (2.79%), N8-deamidation (1.5% and 0.44%), N1-deamidation (1.03%) and double deamidation Q6N8 (0.16%).

Benefits and Practical Applications

This integrated, automated workflow reduces sample preparation variability and increases throughput. Iterative MS/MS enhances detection of trace peptides and PTMs, supporting rigorous comparability studies, biosimilarity assessment and quality control in biotherapeutic development.

Future Trends and Applications

• Integration with higher-throughput and higher-resolution mass analyzers for accelerated mapping.
• Implementation of real-time adaptive acquisition and machine learning for optimized precursor selection.
• Expansion to regulatory-compliant, GMP-like workflows for continuous biomanufacturing monitoring.

Conclusion

The combined use of automated sample prep, Agilent 6545XT AdvanceBio LC/Q-TOF with Iterative MS/MS and MassHunter BioConfirm B.09 yields high sequence coverage and detailed PTM characterization of monoclonal antibodies. Iterative MS/MS notably improves low-abundance peptide identification, enhancing the robustness of peptide mapping workflows in biopharmaceutical analysis.

References

1. Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform, Agilent Technologies, publication number 5991-2957EN.
2. An Integrated Workflow for Peptide Mapping of Monoclonal Antibodies, Agilent Technologies, publication number 5991-7811EN.
3. Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform, Agilent Technologies, publication number 5991-4872EN.
4. Yang H, Zubarev RA. Mass spectrometric analysis of asparagine deamidation and aspartate isomerization in polypeptides. Electrophoresis. 2010;31(11):1764–1772.