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Title

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

AuthorsCampos-Pinto, Isabel; Méndez, Lucía ; Schouten, James; Wilkins, John; Fedorova, Maria; Pitt, Andrew R.; Davis, Paul; Spickett, Corinne M.
KeywordsLipid peroxidation
Protein lipoxidation
Mass spectrometry
Diagnostics
Antibodies
Peptide arrays
Epitope mapping
Issue Date2019
PublisherElsevier
CitationFree Radical Biology and Medicine 144: 234-244 (2019)
AbstractLipids 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 HNEtreated 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
Description11 pages, 7 figures, 1 table.-- This is an open access article under the CC BY-NC-ND license
Publisher version (URL)https://doi.org/10.1016/j.freeradbiomed.2019.05.008
URIhttp://hdl.handle.net/10261/196388
DOI10.1016/j.freeradbiomed.2019.05.008
ISSN0891-5849
E-ISSN1873-4596
Appears in Collections:(IIM) Artículos
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