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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
Mateusz Dobrowolski
Mateusz
Dobrowolski holds a Master’s degree in molecular biophysics and is currently
pursuing an [...]
Mateusz
Dobrowolski holds a Master’s degree in molecular biophysics and is currently
pursuing an implementation doctorate in medical sciences at the Medical
University of Łódź. He began his scientific career at the University of Warsaw,
where he was engaged in research in molecular biology and biotechnology. Since
2017, he has been working at Mabion S.A., a Polish biopharmaceutical company,
where he currently leads the Process Development Division. His professional
experience includes protein science, mass spectrometry-based analytics, and the
development of therapeutic biologics. His dual academic and industrial
perspective informs his work on the sequence confirmation methods presented in this
manuscript.
1,2
,
Małgorzata Urbaniak
Małgorzata Urbaniak 1,* and
Tadeusz Pietrucha
Tadeusz Pietrucha
Tadeusz
Pietrucha is a molecular biologist affiliated with the Medical University of
Łódź, he a [...]
Tadeusz
Pietrucha is a molecular biologist affiliated with the Medical University of
Łódź, where he has served as a researcher and academic teacher since the early
1990s. He holds a Ph.D. and a D.Sc. in medical biology and was awarded the
title of full professor in 2007. His research focuses on molecular mechanisms
of cardiovascular disease, placental pathologies, and personalized medicine. He
has authored numerous publications on gene-based biomarkers, coagulation
disorders, and translational applications of molecular diagnostics. In addition
to his academic work, Prof. Pietrucha is involved in biotechnology
entrepreneurship and innovation policy. He is the founder or co-founder of
several biotech companies and initiatives, including Bio-Tech Consulting, Mabion
S.A., and the BioForum Central European Forum of Biotechnology. His
professional background bridges academic research and practical implementation
in the life sciences sector.
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
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.
Share and Cite
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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