Cornwell, O, Bond, NJ, Radford, SE orcid.org/0000-0002-3079-8039 et al. (1 more author) (2019) Long-Range Conformational Changes in Monoclonal Antibodies Revealed Using FPOP-LC-MS/MS. Analytical Chemistry, 91 (23). pp. 15163-15170. ISSN 0003-2700
Abstract
Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochemical Oxidation of Proteins (FPOP) followed by proteolysis and LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labelling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal down-stream analysis. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity determining regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-association at high concentrations and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labelling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labelling patterns at the interfaces of the CL–CH1 and CH1–CH2 domains, with the non-aggregating variant undergoing up to four times more labelling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL – CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidised peptides. Some oxidised amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an analysis as an aid to MS/MS in assigning oxidation sites.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 American Chemical Society. This is an open access article under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) (https://creativecommons.org/licenses/by/4.0/) |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Biomolecular Mass Spectroscopy (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds) |
Funding Information: | Funder Grant number BBSRC BB/K000659/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 07 Nov 2019 11:55 |
Last Modified: | 25 Jun 2023 22:03 |
Status: | Published |
Publisher: | American Chemical Society |
Identification Number: | 10.1021/acs.analchem.9b03958 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:153205 |