iTRAQ技术在筛选胆道肿瘤胆汁标志物中的应用  

Application of iTRAQ technique in screening bile biomarkers of biliary tract carcinoma

作　　者：郑晓 严斌 刘铁梅 ZHENG Xiao;YAN Bin;LIU Tiemei(Department of Endoscopy,Qingpu branch of Zhongshan Hospital,Fudan University,Shanghai 201700,China)

机构地区：[1]复旦大学附属中山医院青浦分院内镜科,上海201700

出　　处：《肿瘤》2024年第10期993-1002,共10页Tumor

基　　金：2022年度上海市"科技创新行动计划"自然科学基金项目(22ZR1412300)

摘　　要：目的:运用基于同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)的蛋白质组学相关技术,探究诊断胆道肿瘤的潜在生物标志物。方法:从复旦大学附属中山医院青浦分院中心实验室样本库中随机抽取经病理确诊的胆道肿瘤(biliarytractcarcinoma,BTC)患者和良性胆道疾病(benignbiliary disease,BBD)患者的胆汁标本各4例,组内两两混合后,采用iTRAQ标记技术结合二维液相色谱串联质谱分析(two-dimensional liquid chromatography-tandem mass spectrometry,2D LC-MS/MS),进行蛋白质组定量分析。然后,筛选出BTC组与BBD组之间的差异表达蛋白,并对其进行生物信息学分析,包括KEGG(Kyoto Encyclopedia of Genes and Genomes)通路富集分析、GO(Gene Ontology)富集分析和蛋白相互作用(protein-protein interaction,PPI)网络分析。从差异表达蛋白中选取表达丰度差异倍数最大的候选蛋白作为胆道肿瘤的潜在诊断生物标志物,并使用50例BTC患者和50例BBD患者的胆汁标本对该候选蛋白进行酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)验证以及受试者操作特征(receiver operating characteristic,ROC)曲线分析。结果:从4组BTC患者和BBD患者的胆汁标本中共筛选出12个差异表达的蛋白(FDR<1%),其中触珠蛋白(haptoglobin,Hp)和补体因子H(complementfactorH,CFH)作为蛋白互作网络中的枢纽蛋白,在BBD患者胆汁中的表达丰度明显上调。ELISA结果显示,Hp和CFH蛋白在各胆道肿瘤亚型患者的胆汁中均发生显著上调(P<0.01)。ROC曲线分析表明,胆汁Hp和CFH蛋白在胆道肿瘤诊断方面展现出显著的特异性和敏感度,且其诊断效果要明显优于血清蛋白CA19-9。结论:本研究采用iTRAQ标记技术联合2D LC-MS/MS,系统解析了BTC患者与BBD患者胆汁标本中蛋白质组的差异,鉴定出Hp和CFH蛋白作为核心差异表达蛋白,二者诊断胆道肿瘤的效能均显著优于血清蛋白CA19-9,可作为胆道Objective:To explore potential biomarkers of biliary tract carcinoma(BTC)by proteomic techniques based on isobaric tags for relative and absolute quantitation(iTRAQ).Methods:Bile samples from 4 patients with BTC and 4 patients with benign biliary tract disease(BBD)were randomly selected from sample library of central laboratory,Qingpu branch of Zhongshan Hospital,Fudan University.After mixing samples within each group,iTRAQ labeling technology combined with two-dimensional liquid chromatography-tandem mass spectrometry(2D-LC-MS/MS)was used for quantitative proteomic analysis.Then,differentially expressed proteins between the BTC and BBD groups were screened and subjected to bioinformatic analysis,including KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis,GO(Gene Ontology)enrichment analysis and protein-protein interaction(PPI)network analysis.The candidate protein with the largest fold change in expression abundance selected from the differentially expressed proteins as a potential diagnostic biomarker for BTC.Enzyme-linked immunosorbent assay(ELISA)validation and receiver operating characteristic(ROC)cure analysis were performed on this candidate protein using bile samples form 50 BTC patients and 50 BBD patients.Results:A total of 12 differential expressed proteins were screened from the bile samples of 4 BTC patients and 4 BBD patients(FDR<1%).Haptoglobin(Hp)and Complement Factor H(CFH),as hub proteins in the PPI network,were significantly upregulated in the bile of BBD patients.ELISA results showed that Hp and CFH were significantly upregulated in the bile of patients with various subtypes of BTC(P<0.01).ROC curve analysis indicated that Hp and CFH in bile exhibited significant specificity and sensitivity in the diagnosis of BTC,and their diagnostic performance was superior to that of serum protein CA19-9.Conclusion:In this study,iTRAQ labeling technology combined with 2D-LC-MS/MS was used to systematically analyze the proteomic differences between bile samples from BTC and BBD patient

关 键 词：胆道肿瘤 触珠蛋白 补体因子H 蛋白质组学 同位素相对和绝对定量 二维液相色谱串联质谱分析 

分 类 号：R735.8[医药卫生—肿瘤]