Cheng Yongjing, Chen Yuling, Sun Xiaolin, Li Yuhui, Huang Cibo, Deng Haiteng, Li Zhanguo
Department of Rheumatology and Immunology, Beijing Hospital, 1 Dahua Road, 100730, Beijing, China.
Inflammation. 2014 Oct;37(5):1459-67. doi: 10.1007/s10753-014-9871-8.
The aim of this study was to find serum biomarkers of rheumatoid arthritis (RA) by high-resolution proteomic analysis. Low-abundance proteins from pooling serum sample of early RA patients and healthy controls were enriched using ProteoMiner™ enrichment kits. The enriched proteins were separated on SDS-PAGE, digested by trypsin, labeled with tandem mass tag (TMT) reagents, and desalted by C18 stage tip column. Then, the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with nano-LC combined with Orbitrap Q Exactive mass spectrometer, and experiments were carried out three times using different specimens, and differentially expressed proteins were screened by intensity ratios of identified peptides. Enzyme-linked immunosorbent assays (ELISAs) were performed to confirm differentially expressed proteins. Twenty-six proteins were found differentially expressed in RA serum by high-resolution proteomic analysis. Among them, levels of thrombospondin-1, ficolin-2, isoform 10 of fibronectin, and apolipoprotein E were higher in RA patients than in healthy controls (RA/healthy control (HC) ≥ 1.5, p<0.05), while levels of angiotensinogen, paraoxonase/arylesterase 1, isoform E of proteoglycan 4, and plasminogen were significantly lower (RA/HC ≤ 0.67, p<0.05). Further study by ELISA showed a higher level of ficolin-2 in the serum of RA patients compared to healthy controls; the level of ficolin-2 was found to be positively correlated with swollen joint counts (SJCs), disease activity score (DAS28), rheumatoid factor, and IgM in RA patients and with DAS28 and IgM in early RA patients through statistical analysis. The results of this study suggest that ficolin-2, as a newly screened biomarker by high-resolution quantitative proteomic analysis, offers the potentiality to become a diagnostic or disease evaluation tool in RA.
本研究旨在通过高分辨率蛋白质组学分析寻找类风湿关节炎（RA）的血清生物标志物。使用ProteoMiner™富集试剂盒对早期RA患者和健康对照的混合血清样本中的低丰度蛋白质进行富集。将富集的蛋白质在SDS-PAGE上分离，用胰蛋白酶消化，用串联质谱标签（TMT）试剂标记，并用C18反相柱进行脱盐。然后，用纳升液相色谱与Orbitrap Q Exactive质谱仪联用的液相色谱-串联质谱（LC-MS/MS）分析标记的肽段，使用不同样本进行3次实验，并通过鉴定肽段的强度比筛选差异表达的蛋白质。进行酶联免疫吸附测定（ELISA）以确认差异表达的蛋白质。通过高分辨率蛋白质组学分析发现26种蛋白质在RA血清中差异表达。其中，RA患者血清中血小板反应蛋白-1、纤维胶凝蛋白-2、纤连蛋白异构体10和载脂蛋白E的水平高于健康对照（RA/健康对照（HC）≥1.5，p<0.05），而血管紧张素原、对氧磷酶/芳基酯酶1、蛋白聚糖4异构体E和纤溶酶原的水平显著降低（RA/HC≤0.67，p<0.05）。ELISA进一步研究显示，RA患者血清中纤维胶凝蛋白-2的水平高于健康对照；通过统计分析发现，纤维胶凝蛋白-2的水平与RA患者的肿胀关节计数（SJC）、疾病活动评分（DAS28）、类风湿因子和IgM以及早期RA患者的DAS28和IgM呈正相关。本研究结果表明，纤维胶凝蛋白-2作为通过高分辨率定量蛋白质组学分析新筛选出的生物标志物，具有成为RA诊断或疾病评估工具的潜力。
