Complete de novo sequencing of a human immunoglobulin G using multiple enzyme digests and LC-ESI-QTOF-MS/MS analysis

Importance of the topic

Mass spectrometry de novo sequencing of monoclonal antibodies addresses critical needs when genetic information is incomplete or unavailable, enabling precise characterization of therapeutic and diagnostic antibodies.

Objectives and study overview

This work demonstrates a comprehensive workflow to determine the complete amino acid sequence of a human monoclonal IgG1 from scratch. By applying six distinct proteolytic digests and high-resolution LC-ESI-QTOF-MS/MS, the aim was to achieve full sequence coverage including all six complementarity-determining regions.

Methodology and Instrumentation

The antibody sample was deglycosylated with PNGase F, reduced and alkylated, then divided into six aliquots for digestion with trypsin, chymotrypsin, elastase, pepsin, AspN/LysC and GluC/LysC under optimized conditions. Digests were quenched with formic acid and analyzed individually by reversed-phase UHPLC coupled to an ESI-QTOF mass spectrometer. Data were processed in PEAKS AB software with mass tolerances of 10 ppm and 0.05 Da, fixed carbamidomethylation, and variable modifications including oxidation and deamidation. Manual inspection verified peptide assignments and resolved isobaric residues.

Instrumentation used

• Bruker impact II QTOF mass spectrometer with ESI source
• Agilent 1290 Infinity UHPLC system
• Waters ACQUITY UPLC Peptide CSH C18 column (150×2.1 mm, 1.7 μm, 130 Å)
• PEAKS AB 2.0 software for de novo sequencing and assembly

Main results and discussion

The combined datasets from six protease digestions enabled 100 % sequence coverage of both heavy and light chains. All six CDRs were identified correctly, and an N-terminal pyro-glutamine modification on the light chain was characterized. Ambiguities in leucine/isoleucine assignments were resolved by homology statistics and PSM counts. The workflow required approximately 4–5 days for wet-lab experiments and 3 days for data processing and manual curation.

Benefits and practical applications

• Facilitates sequence determination of originator antibodies lacking genetic data
• Supports biosimilar development, quality control and regulatory submissions
• Establishes a robust protocol for routine antibody de novo sequencing

Future trends and opportunities

Ongoing advances in AI-driven spectral interpretation, integration with expansive antibody sequence databases, and automated resolution of sequence ambiguities will shorten timelines and minimize manual intervention. Emerging high-throughput and cloud-based platforms will enable scalable, rapid de novo sequencing services.

Conclusion

The described multi-enzyme LC-ESI-QTOF-MS/MS approach combined with PEAKS AB software delivers a reliable, high-confidence solution for complete de novo sequencing of monoclonal antibodies, achieving full coverage and accurate identification of sequence variants.

References

• Bandeira N, Tang H, Shen Z, et al. Automated de novo protein sequencing of monoclonal antibodies. Nat Biotechnol. 2008;26(12):1336–1338.
• Tran NH, Qiao R, Xin L, et al. Complete de novo assembly of monoclonal antibody sequences. Sci Rep. 2016;6:31730.