利用定量蛋白组学方法发现涂阴肺结核患者血清差异表达蛋白并验证FCN3

DISCOVERY DIFFERENTIAL EXPRESSION PROTEINS FOR SMEAR-NEGATIVE PULMONARY TUBERCULOSIS BY PROTEOME QUANTITATIVE ANALYSIS AND VERIFYING FCN3

目的:利用定量蛋白组学方法检测涂阴肺结核(SNP-TB)患者血清差异表达蛋白,并验证关键蛋白FCN3。方法:采用iTRAQ标记结合MALDI-TOF/MS筛选30例健康对照者和30例SNP-TB患者血清的差异表达蛋白,并进行生物信息学分析,对关键差异蛋白FCN3进一步ELISA验证,并绘制ROC曲线。结果:3次独立重复实验一共筛选和鉴定到344种蛋白质,其中置信度在95%以上有283种蛋白质;以正常人血清为对照,SNP-TB患者血清中呈显著性差异表达的蛋白有26种(P<0.05);生物信息学分析发现差异蛋白主要定位在细胞外,参与酶抑制剂活性和应激反应过程;蛋白相互作用发现FCN3等9种蛋白处于相互作用网络关键节点;ELISA验证SNP-TB组血清FCN3蛋白表达水平显著低于正常对照组和涂阳肺结核组(P<0.001),与蛋白组学结果一致;ROC曲线下的面积为0.929,灵敏度和特异度分别是81.24%和83.31%。结论:iTRAQ标记结合MALDI-TOF/MS技术能够有效的筛选和鉴定血清差异表达蛋白;FCN3是值得进一步研究的肺结核候选血清标志物。

Objective：Differential expression proteins in smear-negative pulmonary tuberculosis（SNP-TB）serum were screened by quantitative proteomics approach,and the key protein FCN3 was validated.Methods：30cases of normal healthy controls and 30 cases of smear-negative pulmonary tuberculosis patients were recruited in this study,and serum protein expression profiles were detected by iTRAQ labeling in combination with MALDI-TOF/MS,then analyzed by bioinformatics software.FCN3 was validated by ELISA.Then ROC curve was built.Results：Within 344 identified proteins,there were 283 proteins with the confidential levels of above 95%.There were 26 significant differentially expressed proteins in smearnegative pulmonary tuberculosis patient serum when compared to the normal healthy controls（P 〈0.05）.Bioinformatics analysis showed that most proteins were primarily located outside of the cell,and mainly involved in activation of enzyme inhibitor and taking response to stimulus.The result of STRING showed that 9proteins such as FCN3 appeared at the central position of the functional network.FCN3 was found to be significantly decreased in smear-negative pulmonary tuberculosis patients when compared with the normal controls and smear-positive pulmonary tuberculosis patients by ELISA（P 〈0.001）,which was consistent with the iTRAQ result.Examining serum FCN3 by ELISA achieved an area under the ROC was 0.929,the sensitivity was 81.24% and specificity was 83.31%in diagnosis of smear-negative pulmonary tuberculosis.Conclusion：ITRAQ-MALDI-TOF/MS is a compelling way for discovery of differential expression proteins.FCN3 is a potential serum biomarker for pulmonary tuberculosis patients.