Structure · dataset · 2026
Middle-Down Mass Spectrometry Characterization of Monoclonal Antibody Enabled by Electron-Activated Dissociation on a Time-of-Flight Platform: A Case Study of Methionine Oxidation PTM Identification and Quantification
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Characterizing post-translational modifications (PTMs) in monoclonal antibody products is critical for ensuring product quality attributes understanding and monitoring.
Description
While the bottom-up approach is widely adopted, it suffers from the loss of molecular connectivity and is susceptible to artifact generation. Conversely, top-down approaches face limitations due to the high complexity and poor gas-phase fragmentation of large intact molecules to get site-specific post-translational information profiling.
Middle-down MS methods offer a complementary middle-ground approach to both bottom-up and top-down, with broader sequence coverage, and facilitate the simultaneous identification of multiple attributes and proteoforms. In this study, we optimized the electron-activated dissociation (EAD) approach for middle-down sequencing of NISTmAb subunits and present a potential MS and MS/MS strategy to rapidly characterize PTMs on antibodies, using methionine oxidation as an example.
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Peroxide-stressed and control mAb samples were digested and reduced into subunits using the IdeS enzyme and analyzed via LC-MS, with the optimized MRM<sup>HR</sup> EAD method. The resulting data enabled high-resolution subunit mass analysis, sequence confirmation, and potential for site-specific oxidation localization. Methionine oxidation localization in NISTmAb was achieved in light chain (M4), Fd’ (M34, M101), and Fc/2 (M16, M122, M192), with these locations confirmed with bottom-up peptide mapping data.
Quantification using the Fc/2 subunit enabled detection of oxidation levels up to 1% relative, with strong correlation (<i>R</i><sup>2</sup> > 0.99) between expected and observed values using MS1 data. This middle-down electron-activated dissociation (EAD) approach provides a potential method for site-specific characterization of PTMs in antibody products, offering valuable complementary mapping to support formulation development and production.
Links
Where it is published
- DOI doi.org/10.1021/acs.analchem.6c03432.s001 ↗
DOI / persistent id · from zivahub uct ac za
Catalogue records · 1
- OAI-PMH record api.figshare.com/v2/oai?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Af… ↗
metadata API · from zivahub uct ac za
Topics
- From keywords
- Bioinformatics and computational biology · Chemistry · Earth & Environmental Science · Infectious diseases · Inorganic chemistry · Life Sciences · Medicine & Health · Social Science · Sociology
- Inferred from text
- Mass spectrometry 75% · Sequencing 75%
Provenance · 1 source records, 16 field assertions
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|---|---|---|---|
| ZivaHub | oai:figshare.com:article/34018734 | 13 d ago | JSON v1 |
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