基于iTRAQ标记结合2D nano HPLC-ESI-OrbiTrap MS/MS技术筛选卵巢癌血清标记物  

Search for Differentially Expressed Protein in Ovarian Cancer Serum Using iTRAQ and 2D Nano HPLC-ESI-OrbiTrap MS/MS

作　　者：李莉[1] 唐杰[1] 于春霞[1] 丁珊珊[1] 王茜[2] 

机构地区：[1]新疆医科大学附属肿瘤医院妇科,乌鲁木齐市830011 [2]新疆医科大学公共卫生学院

出　　处：《中国肿瘤临床》2012年第24期2075-2079,共5页Chinese Journal of Clinical Oncology

基　　金：新疆维吾尔自治区自然科学基金(编号:201 121 1A038)资助~~

摘　　要：目的:利用相对和绝对同位素标记(iTRAQ)和纳升级两维高效液相色谱-电喷雾-OrbiTrap质谱(2D nanoHPLC-ESI-OrbiTrap MS/MS)蛋白组学方法探寻卵巢癌血清标记物,以提高早期卵巢癌诊断率。方法:收集卵巢癌患者血清20例(卵巢癌组),CA125异常的卵巢良性囊肿16例(良性囊肿组),健康者20例(正常对照组),每组取10例组内等量混合后去除高丰度蛋白,iTRAQ试剂标记,进行强离子交换柱分离多肽、nanoLC分离并在线连接电喷雾串联OrbiTrap质谱分析,筛选出重要的差异蛋白。随后采用Western blot方法,将筛选出的重要差异蛋白进行56例临床血清样本逐例验证。结果:共鉴定出差异蛋白326个。其中重点筛选出了8个重要的差异蛋白:蛋白PARD3、SRP1-alpha、LAMA4、LRP16、IgSF2、NRAMP1、NF-E2-related factor 1和APOA4;验证了3个重要差异蛋白:PARD3、NRAMP1、APOA4。结论:应用iTRAQ标记的定量蛋白质组学技术筛选出了8个重要的差异蛋白,可能是潜在的肿瘤标记物。其中APOA4蛋白有可能成为区分恶性与良性卵巢疾病的生物标志物。Objective： The objective of this study is to detect ovarian cancer tumor markers using proteomics approaches of isobaric tags for relative and absolute quantification （iTRAQ） and two-dimensional nano high-performance liquid chromatography-electrospray-OrbiTrap mass spectrometry （2D nano HPLC-ESI-OrbiTrap MS/MS） to improve the early diagnosis rate. Methods： Serum samples from 10 patients with ovarian cancer, 10 with benign ovarian tumor, and 10 normal women subjects were pooled with equal volume. A high abundance of protein was depleted after the balanced mix of the samples. The samples were then detected and identified by the iTRAQ - coupled 2D nano HPLC-ESI-OrbiTrap MS/MS technology. The proteins with significant differences were checked using Western blot in the 56 serum samples. Results： A total of 326 proteins were identified. Among these proteins, eight important proteins were interpreted, including proteins PARD3, SRP1-alpha, LAMA4, LRP16, IgSF2, NRAMP1, NF-E2-related factor 1, and APOA4. Using Western blot technology, three important proteins with significant differences were validated, namely, proteins APOA4, PARD3, and NRAMP1. Conclusion： The eight important proteins discovered using iTRAQ may be the potential tumor makers of ovarian cancer. As a novel protein, APOA4 may become a biological marker to differentiate malignant from benign tumor in a serum that has an elevated CA-125.

关 键 词：卵巢癌 蛋白质组学 肿瘤标志物 ITRAQ 

分 类 号：R737.31[医药卫生—肿瘤]