Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer
Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer
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DOI：
10.1038/sj.bjc.6601882
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发表时间：
2004-07-05
影响因子：
8.8
通讯作者：
Rice, GE
中科院分区：
文献类型：
--
作者：
Ahmed, N;Barker, G;Rice, GE
由于卵巢癌的无症状性和生存率低，筛查早期检测卵巢癌的特异性生物标志物是一个主要的健康优先事项。我们利用二维凝胶电泳（2DE）来鉴定卵巢癌患者血清中差异表达的蛋白质，这些蛋白质可能用作这种疾病的生物标志物。在这项研究中，38名不同病理分级的卵巢癌患者（1级（n = 6），2级（n = 8）和3级（n = 24））与对照组8名健康女性进行了比较。血清样品用Affigel-Blue和蛋白A（5：1）的混合物处理1小时以去除高丰度蛋白（例如，G.免疫球蛋白和白蛋白），并使用11 cm，pH 4-7等电聚焦条带进行第一维显示，使用10%丙烯酰胺凝胶电泳进行第二维显示。通过SYPRO-Ruby染色使蛋白质斑点可视化，通过FX-成像仪成像，并通过PDQuest软件进行比较和分析。在卵巢癌1级、2级和3级患者中，差异表达的蛋白质分别为24个、31个和25个（P <0.05）。在所有卵巢癌患者组中显著上调的6个蛋白质点通过纳米电喷雾四极杆四极杆飞行时间质谱（n-ESIQ（q）TOFMS）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOFMS）鉴定为结合珠蛋白-1前体（HAP 1）的同种型，HAP 1是存在于人血清中的肝糖蛋白。通过使用针对HAP 1内所含触珠蛋白表位的单克隆抗体的Western印迹法进一步鉴定不同病理等级的斑点。HAP 1样活性的免疫组织化学定位存在于恶性卵巢上皮和间质中，但强免疫染色存在于血管、粘液瘤间质和血管间隙中。在正常卵巢表面上皮中没有观察到HAP 1样免疫反应性的组织定位。这些数据强调了评估卵巢癌患者血清中HAP 1循环浓度的必要性，并评估其作为卵巢癌早期诊断生物标志物的潜力。
Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n = 6), grade 2 (n = 8) and grade 3 (n = 24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A ( 5 : 1) for 1 h to remove high abundance protein ( e. g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P < 0.05), 31 in grade 2 (P < 0.05) and 25 in grade 3 (P < 0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.