In-depth Peptide Mapping of Monoclonal Antibody (mAb) by A de novo Peptide Sequencing Method on Q-TOF Mass Spectrometer with Data-independent Acquisition

Significance of the Topic

Peptide mapping is essential for detailed primary structure characterization of monoclonal antibodies (mAbs), enabling detection of single amino acid changes and post-translational modifications (PTMs). A robust, unbiased workflow improves the accuracy and reproducibility of biopharmaceutical quality control.

Study Objectives and Overview

This study demonstrates an integrated data-independent acquisition (DIA) approach on the Shimadzu LCMS-9030 Q-TOF mass spectrometer for de novo peptide sequencing of a bevacizumab biosimilar, aiming to verify the complete primary sequence and identify any structural alterations without relying solely on comparative reference maps.

Methodology and Instrumentation

Sample Preparation:

• Denaturation and reduction: bevacizumab digested in Tris-HCl buffer with ProteaseMAX and DTT.
• Alkylation: iodoacetamide treatment.
• Enzymatic digestion: overnight trypsin incubation.
• Quenching: trifluoroacetic acid addition.

Instrumental Setup:

• Liquid chromatography: Shimadzu Nexera X2 UHPLC with Shim-pack GISS-HP (3 μm, 150 × 3.0 mm) column at 40 °C, gradient from 0% to 75% acetonitrile over 35 min.
• Mass spectrometry: Shimadzu LCMS-9030 Q-TOF with heated electrospray interface, full-scan MS (100–2000 m/z) and MS/MS DIA segments covering 210–1690 m/z in 40 m/z windows.
• Data processing: MS-DIAL for peak extraction, Skyline for theoretical y and b ion transition prediction.

Results and Discussion

The workflow extracted 11 852 mass features, leading to confident identification of 61 tryptic peptides (42 heavy-chain and 19 light-chain) covering 100% of the mAb sequence. The example peptide ALPAPIEK exhibited clear matching of extracted MS/MS fragments to predicted transitions, validating the de novo sequencing capability. Observations included PTMs such as N-glycosylation and C-terminal lysine processing.

Benefits and Practical Applications

• Unbiased fragmentation: DIA captures all fragment ions without precursor selection, enhancing sequence coverage.
• Comprehensive mapping: full amino acid coverage and detection of PTMs supports in-depth characterization.
• Improved reproducibility: standardized UHPLC-Q-TOF conditions reduce variability in comparative analyses.
• Regulatory compliance: robust primary structure confirmation aligns with biopharma quality requirements.

Future Trends and Opportunities

Advancements may include machine learning-driven spectral interpretation for automated de novo sequencing, integration of real-time PTM monitoring, expansion of DIA methods to other large biomolecules, and streamlined QC workflows incorporating high-throughput peptide mapping.

Conclusion

The described DIA strategy on the LCMS-9030 Q-TOF provides a straightforward, high-coverage approach for de novo peptide sequencing of mAbs, offering significant benefits for biopharmaceutical characterization and quality control.

References

1. Searle BC, Pino LK (2018) Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. Nat Commun 9:5128.
2. Riken MS-DIAL software for metabolomics and DIA processing.
3. MacLean B et al. Skyline: an open source document editor for targeted proteomics.
4. Shimadzu (2019) Peptide mapping of monoclonal antibody using LCMS-9030 Q-TOF. Application News AD-0212B.