Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan.
J Proteomics. 2011 May 1;74(5):744-57. doi: 10.1016/j.jprot.2011.02.023. Epub 2011 Mar 2.
Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is not easily diagnosed until advanced stages. To discover potential biomarkers for improving NPC diagnosis, we herein identified the aberrant plasma proteins in NPC patients. We first removed the top-seven abundant proteins from plasma samples of healthy controls and NPC patients, and then labeled the samples with different fluorescent cyanine dyes. The labeled samples were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Proteins showing altered levels in NPC patients were identified by in-gel tryptic digestion and LC-MS/MS. When the biological roles of the 45 identified proteins were assessed via MetaCore™ analysis, the blood coagulation pathway emerged as the most significantly altered pathway in NPC plasma. Plasma kallikrein (KLKB1) and thrombin-antithrombin III complex (TAT) were chosen for evaluation as the candidate NPC biomarkers because of their involvement in blood coagulation. ELISAs confirmed the elevation of their plasma levels in NPC patients versus healthy controls. Western blot and activity assays further showed that the KLKB1 active form was significantly increased in NPC plasma. Collectively, our results identified the significant alteration of blood coagulation pathway in NPC patients, and KLKB1 and TAT may represent the potential NPC biomarkers.
鼻咽癌（NPC）是东南亚地区最常见的癌症之一，直到晚期才容易被诊断出来。为了发现改善 NPC 诊断的潜在生物标志物，我们在此鉴定了 NPC 患者血浆中的异常蛋白。我们首先从健康对照和 NPC 患者的血浆样本中去除前 7 种丰富的蛋白质，然后用不同的荧光 Cy3 染料标记样本。标记的样本然后用离子交换色谱法进行等份分离，然后进行 SDS-PAGE。通过凝胶内胰蛋白酶消化和 LC-MS/MS 鉴定 NPC 患者中蛋白水平改变的蛋白。当通过 MetaCore™分析评估 45 种鉴定蛋白的生物学作用时，血液凝固途径成为 NPC 血浆中改变最显著的途径。由于其在血液凝固中的参与，血浆激肽释放酶（KLKB1）和凝血酶抗凝血酶 III 复合物（TAT）被选为候选 NPC 生物标志物进行评估。ELISA 证实 NPC 患者血浆中它们的水平升高与健康对照组相比。Western blot 和活性测定进一步表明，NPC 血浆中的 KLKB1 活性形式明显增加。总的来说，我们的结果确定了 NPC 患者血液凝固途径的显著改变，KLKB1 和 TAT 可能代表潜在的 NPC 生物标志物。
