Molecular Medicine Research Center, Chang Gung University, Taiwan.
J Proteome Res. 2010 Nov 5;9(11):5803-15. doi: 10.1021/pr100576x. Epub 2010 Sep 17.
A urine sample preparation workflow for the iTRAQ (isobaric tag for relative and absolute quantitation) technique was established. The reproducibility of this platform was evaluated and applied to discover proteins with differential levels between pooled urine samples from nontumor controls and three bladder cancer patient subgroups with different grades/stages (a total of 14 controls and 23 cancer cases in two multiplex iTRAQ runs). Combining the results of two independent clinical sample sets, a total of 638 urine proteins were identified. Among them, 55 proteins consistently showed >2-fold differences in both sample sets. Western blot analyses of individual urine samples confirmed that the levels of apolipoprotein A-I (APOA1), apolipoprotein A-II, heparin cofactor 2 precursor and peroxiredoxin-2 were significantly elevated in bladder cancer urine specimens (n = 25-74). Finally, we quantified APOA1 in a number of urine samples using a commercial ELISA and confirmed again its potential value for diagnosis (n = 126, 94.6% sensitivity and 92.0% specificity at a cutoff value of 11.16 ng/mL) and early detection (n = 71, 83.8% sensitivity and 94.0% specificity). Collectively, our results provide the first iTRAQ-based quantitative profile of bladder cancer urine proteins and represent a valuable resource for the discovery of bladder cancer markers.
建立了一种用于 iTRAQ（相对和绝对定量的同位素标记）技术的尿样制备工作流程。评估了该平台的重现性，并将其应用于发现来自非肿瘤对照和三个具有不同分级/分期的膀胱癌患者亚组的混合尿样之间差异水平的蛋白质（两个多重 iTRAQ 运行中共有 14 个对照和 23 个癌症病例）。结合两个独立临床样本集的结果，共鉴定出 638 种尿蛋白。其中，55 种蛋白质在两个样本集中均表现出>2 倍的差异。对个体尿样的 Western blot 分析证实，载脂蛋白 A-I（APOA1）、载脂蛋白 A-II、肝素辅因子 2 前体和过氧化物酶-2 的水平在膀胱癌尿标本中显著升高（n=25-74）。最后，我们使用商业 ELISA 定量测定了一些尿样中的 APOA1，并再次证实了其在诊断（n=126，截断值为 11.16ng/ml 时的敏感性为 94.6%，特异性为 92.0%）和早期检测（n=71，敏感性为 83.8%，特异性为 94.0%）中的潜在价值。总的来说，我们的结果提供了基于 iTRAQ 的膀胱癌尿蛋白的定量图谱，为膀胱癌标志物的发现提供了有价值的资源。
