Proteomics Research Center, National Yang-Ming University, Taipei 112, Taiwan.
Division of Allergy, Immunology, and Rheumatology, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan; Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
J Proteomics. 2018 Jan 6;170:141-150. doi: 10.1016/j.jprot.2017.08.012. Epub 2017 Sep 1.
This study identified and validated four differentially expressed novel malondialdehyde (MDA)-modified peptide adducts and evaluated autoantibodies against native and MDA-modified peptides among Taiwanese women patients with rheumatoid arthritis (RA), osteoarthritis (OA) and healthy controls (HCs). Ig kappa chain C region, alpha-1-antitrypsin, alpha-2-macroglobulin, and apolipoprotein B-100 exhibiting 2-fold differences in relative modification ratios were identified by concanavalin A (Con A) affinity chromatography, 1D SDS-PAGE, in-gel digestion, nano-LC/MS/MS and nano-LC/MS using pooled serum-derived Con A-captured proteins from 9 RA and 9 age-matched HCs. Furthermore, the levels of proteins, serum MDA, and MDA-modified protein adducts were further validated against individual serum from 20 RA and 20 HCs, and autoantibodies against native and their MDA-modified peptides used 45 RA, 30 OA and 45 HCs. Levels of serum MDA and MDA-modified protein adducts were significantly higher in RA than HCs but protein levels were not significantly different. Serum Igs G and M against MDA-modified peptides showed better diagnostic performance in differentiating among patients with RA, OA and HCs, with an area under the receiver operating characteristic curve of 0.96-0.98, sensitivity of 88.9%-97.8%, and specificity of 88.9%-100%. Autoantibodies against MDA-modified epitopes become useful clinical biomarkers for RA.
By using a label-free relative quantitative proteomic analysis of concanavalin A (Con A)-bound serum samples, the current study discovered and validated malondialdehyde (MDA)-modified peptide adducts as novel biomarkers for differentiating between rheumatoid arthritis (RA) patients and healthy controls (HCs). In addition, the serum levels of MDA, proteins, and MDA-modified protein adducts as well as the MDA modification of proteins were determined. Isotypes of autoantibodies against MDA-modified peptide adducts can be used as serological biomarkers for further discriminating among RA patients, osteoarthritis patients and HCs. This strategy can become the basis for identifying potential diagnostic and pathological biomarkers for RA.
本研究旨在鉴定和验证四种差异表达的新型丙二醛（MDA）修饰肽加合物，并评估台湾类风湿关节炎（RA）、骨关节炎（OA）和健康对照（HC）患者的天然和 MDA 修饰肽的自身抗体。通过 ConA 亲和层析、1D SDS-PAGE、胶内消化、纳升 LC/MS/MS 和纳升 LC/MS，使用来自 9 名 RA 和 9 名年龄匹配的 HC 的 Pooled 血清衍生的 ConA 捕获蛋白，鉴定出 Ig kappa 链 C 区、α-1-抗胰蛋白酶、α-2-巨球蛋白和载脂蛋白 B-100，这些蛋白的相对修饰比值存在 2 倍差异。此外，还针对来自 20 名 RA 和 20 名 HC 的个体血清进一步验证了蛋白、血清 MDA 和 MDA 修饰蛋白加合物的水平，并使用 45 名 RA、30 名 OA 和 45 名 HC 验证了针对天然和 MDA 修饰肽的自身抗体。RA 患者的血清 MDA 和 MDA 修饰蛋白加合物水平明显高于 HC，但蛋白水平无明显差异。MDA 修饰肽的血清 Ig G 和 M 自身抗体在区分 RA、OA 和 HC 患者方面具有更好的诊断性能，ROC 曲线下面积为 0.96-0.98，灵敏度为 88.9%-97.8%，特异性为 88.9%-100%。MDA 修饰表位的自身抗体成为 RA 的有用临床生物标志物。
通过使用 ConA 结合血清样本的无标记相对定量蛋白质组学分析，本研究发现并验证了丙二醛（MDA）修饰肽加合物作为区分类风湿关节炎（RA）患者和健康对照（HC）的新型生物标志物。此外，还测定了 MDA、蛋白、MDA 修饰蛋白加合物以及蛋白 MDA 修饰的水平。MDA 修饰肽加合物自身抗体的同种型可作为进一步区分 RA 患者、OA 患者和 HC 的血清生物标志物。该策略可以成为识别 RA 潜在诊断和病理生物标志物的基础。
