Epitope mapping and characterization of 4-hydroxy-2-nonenal modified-human serum albumin using two different polyclonal antibodies


Lipids are susceptible to damage by reactive oxygen species, and from lipid oxidation reactions many short chain lipid peroxidation products can be formed. 4-Hydroxy-2-nonenal (HNE) is one of the most abundant and cytotoxic lipid oxidation products and is known to form covalent adducts with nucleophilic amino acids of proteins. HNE-modified proteins have value as biomarkers and can be detected by antibody-based techniques, but most commercially available antibodies were raised against HNE-keyhole limpet hemocyanin. We used HNE-treated human serum albumin (HSA) to raise sheep antiserum and report for the first time the use of covalently modified peptide arrays to assess epitope binding of antibodies (Abs). Peptide arrays covering the sequence of HSA and treated post peptide synthesis with HNE were used to compare the different binding patterns of a commercial polyclonal antibody (pAb) raised against HNE-treated KLH and an in-house anti-HNE enriched pAb. The results were correlated with analysis of HNE-modified HSA by high-resolution tandem mass spectrometry. Both anti-HNE pAbs were found to bind strongly to eight common peptides on the HNE-treated HSA membranes, suggesting that HNE adducts per se induced an immune response in both cases even though different immunogens were used. Both antibodies bound with the highest affinity to the peptide 365DPHECYAKVFDEFKPLV381, which contains K378 and was also shown to be modified by the mass spectrometry analysis. Overall, the commercial anti-HNE pAb showed better specificity, recognizing nine out of the eleven adducts found by MS/MS, while the in-house enriched pAb only recognizes six. Nevertheless, the in-house pAb recognized specific peptides that were not recognized by the commercial pAb, which suggests the presence of clones uniquely specific to HNE adducts on HSA.

Publication DOI: https://doi.org/10.1016/j.freeradbiomed.2019.05.008
Divisions: College of Health & Life Sciences
College of Health & Life Sciences > Aston Pharmacy School
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
College of Business and Social Sciences > Aston Business School
College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > Chronic and Communicable Conditions
Additional Information: © 2019 The Authors. Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Funding: European Union’s Horizon 2020 research and innovation programme under the MASSTRPLAN Marie Sklowdowska-Curie grant agreement number 67513. The financial support from German Federal Ministry of Education and Research (BMBF); Xunta de Galicia postdoctoral scholarship.
Uncontrolled Keywords: Antibodies,Diagnostics,Epitope mapping,Lipid peroxidation,Mass spectrometry,Peptide arrays,Protein lipoxidation,Biochemistry,Physiology (medical)
Publication ISSN: 1873-4596
Last Modified: 17 Jun 2024 07:40
Date Deposited: 19 Aug 2019 09:40
Full Text Link:
Related URLs: https://linking ... 891584919303545 (Publisher URL)
http://www.scop ... tnerID=8YFLogxK (Scopus URL)
http://research ... ston.ac.uk/426/ (Related URL)
PURE Output Type: Article
Published Date: 2019-11-20
Published Online Date: 2019-05-07
Accepted Date: 2019-05-06
Authors: Campos-pinto, Isabel
Méndez, Lucía
Schouten, James
Wilkins, John
Fedorova, Maria
Pitt, Andrew (ORCID Profile 0000-0003-3619-6503)
Davis, Paul
Spickett, Corinne (ORCID Profile 0000-0003-4054-9279)

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