Mologic, Ltd, Bedford Technology Park, Thurleigh, Bedfordshire, MK44 2YA, UK; School of Life & Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.
Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany; Center for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany; Institute of Marine Research, Spanish Council for Scientific Resesarch, (IIM-CSIC), Vigo, Spain.
Free Radic Biol Med. 2019 Nov 20;144:234-244. doi: 10.1016/j.freeradbiomed.2019.05.008. Epub 2019 May 7.
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 DPHECYAKVFDEFKPLV, 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.
脂质容易受到活性氧的损伤，并且从脂质氧化反应中可以形成许多短链脂质过氧化产物。4-羟基-2-壬烯醛（HNE）是最丰富和细胞毒性的脂质氧化产物之一，已知与蛋白质的亲核氨基酸形成共价加合物。HNE 修饰的蛋白质作为生物标志物具有价值，可以通过基于抗体的技术检测，但大多数市售的抗体是针对 HNE-钥孔贻贝血红蛋白（KLH）产生的。我们使用 HNE 处理的人血清白蛋白（HSA）来制备绵羊抗血清，并首次报告使用共价修饰的肽阵列来评估抗体（Abs）的表位结合。肽阵列覆盖 HSA 的序列，并在肽合成后用 HNE 进行处理，用于比较针对 HNE 处理的 KLH 产生的商业多克隆抗体（pAb）和内部抗 HNE 富集的 pAb 的不同结合模式。结果与通过高分辨率串联质谱分析 HNE 修饰的 HSA 进行了相关性分析。两种抗 HNE pAb 均被发现强烈结合到 HNE 处理的 HSA 膜上的八个常见肽上，这表明在两种情况下，HNE 加合物本身都诱导了免疫反应，尽管使用了不同的免疫原。两种抗体与肽 DPHECYAKVFDEFKPLV 的结合亲和力最高，该肽包含 K378，并且通过质谱分析也显示被修饰。总体而言，商业抗 HNE pAb 表现出更好的特异性，可识别 MS/MS 发现的 11 个加合物中的 9 个，而内部富集的 pAb 仅识别 6 个。然而，内部 pAb 识别了商业 pAb 未识别的特定肽，这表明存在针对 HSA 上 HNE 加合物的独特特异性的克隆。
