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Article

Peptide Mapping for Sequence Confirmation of Therapeutic Proteins and Recombinant Vaccine Antigens by High-Resolution Mass Spectrometry: Software Limitations, Pitfalls, and Lessons Learned

by
Mateusz Dobrowolski
1,2,
Małgorzata Urbaniak
1,* and
Tadeusz Pietrucha
2
1
Research and Development Department, Mabion S.A., Langiewicza 60, 95-050 Konstantynów Łódzki, Poland
2
Department of Medical Biotechnology, Medical University of Lodz, Żeligowskiego 7/9, 90-752 Łódź, Poland
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(20), 9962; https://doi.org/10.3390/ijms26209962 (registering DOI)
Submission received: 31 July 2025 / Revised: 1 October 2025 / Accepted: 3 October 2025 / Published: 13 October 2025
(This article belongs to the Section Biochemistry)

Abstract

Peptide mapping is a well-established method for confirming the identity of therapeutic proteins as part of batch release testing and product characterization for regulatory filings. Traditionally based on enzymatic digestion followed by reversed-phase liquid chromatography and UV detection, the method has evolved with technological advancements to incorporate mass spectrometry (MS), enabling more detailed structural insights. Residue-level confirmation of amino acid sequences requires MS/MS fragmentation, which produces large amounts of data that must be processed using specialized software. In regulated environments, the use of academic algorithms is often limited by validation requirements, making it necessary to rely on commercially approved tools, although their built-in scoring systems have limitations that can affect sequence assignment accuracy. Here, we present representative examples of incorrect peptide assignments generated by commercial software. In antibody sequence analysis, misidentifications resulted from isobaric and near-isobaric dipeptides (e.g., SA vs. GT). Additional examples from the analysis of SARS-CoV-2 spike protein variants revealed software-induced artifacts, including artificial succinylation of aspartic acid residues to compensate for sequence mismatches, and incorrect deamidation site assignments due to misinterpretation of isotopic peaks. These findings underscore the necessity for expert manual review of MS/MS data, even when using validated commercial platforms, and highlight the molecular challenges in distinguishing true sequence variants from software-driven artifacts.
Keywords: antibody; peptide mapping; SARS-CoV-2; electrospray ionization mass spectrometry; sequence confirmation; false positives; orbitrap; biosimilar antibody; peptide mapping; SARS-CoV-2; electrospray ionization mass spectrometry; sequence confirmation; false positives; orbitrap; biosimilar

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MDPI and ACS Style

Dobrowolski, M.; Urbaniak, M.; Pietrucha, T. Peptide Mapping for Sequence Confirmation of Therapeutic Proteins and Recombinant Vaccine Antigens by High-Resolution Mass Spectrometry: Software Limitations, Pitfalls, and Lessons Learned. Int. J. Mol. Sci. 2025, 26, 9962. https://doi.org/10.3390/ijms26209962

AMA Style

Dobrowolski M, Urbaniak M, Pietrucha T. Peptide Mapping for Sequence Confirmation of Therapeutic Proteins and Recombinant Vaccine Antigens by High-Resolution Mass Spectrometry: Software Limitations, Pitfalls, and Lessons Learned. International Journal of Molecular Sciences. 2025; 26(20):9962. https://doi.org/10.3390/ijms26209962

Chicago/Turabian Style

Dobrowolski, Mateusz, Małgorzata Urbaniak, and Tadeusz Pietrucha. 2025. "Peptide Mapping for Sequence Confirmation of Therapeutic Proteins and Recombinant Vaccine Antigens by High-Resolution Mass Spectrometry: Software Limitations, Pitfalls, and Lessons Learned" International Journal of Molecular Sciences 26, no. 20: 9962. https://doi.org/10.3390/ijms26209962

APA Style

Dobrowolski, M., Urbaniak, M., & Pietrucha, T. (2025). Peptide Mapping for Sequence Confirmation of Therapeutic Proteins and Recombinant Vaccine Antigens by High-Resolution Mass Spectrometry: Software Limitations, Pitfalls, and Lessons Learned. International Journal of Molecular Sciences, 26(20), 9962. https://doi.org/10.3390/ijms26209962

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