基于iTRAQ技术筛选儿童急性早幼粒细胞白血病血清差异蛋白

Screening Serum Differential Proteins in Children with Acute Promyelocytic Leukemia Based on iTRAQ Technique

目的:筛选并鉴定儿童急性早幼粒细胞白血病(APL)血清差异表达蛋白,寻找潜在的APL生物标记物。方法:选取APL与健康儿童血清各20例,采用同位素标记的相对和绝对定量(i TRAQ)联合二维液相色谱串联质谱(2DLC-MS/MS)技术筛查差异蛋白,并进行生物信息学分析;对差异蛋白S100A8、LRG1及SPARC应用ELISA进一步验证;采用受试者工作特征(ROC)曲线分析法,评价差异蛋白对儿童APL的诊断效能。结果:APL患儿血清中检测出83种差异表达蛋白,其中33种蛋白表达上调,50种蛋白表达下调;生物信息学分析显示这些差异蛋白涉及的生物功能包括细胞运动、免疫细胞运输、血液系统发育与功能、细胞间信号传导和相互作用、组织发育等;差异蛋白涉及多条信号通路,其中受影响最显著的2条通路为LXR/RXR活化与急性期反应信号通路。ELISA验证结果显示,S100A8与LRG1蛋白浓度高于正常对照组、SPARC蛋白浓度低于正常对照组,与蛋白质组学筛选结果一致。ROC曲线分析显示,S100A8、LRG1及SPARC的ROC曲线下面积(AUC)分别为0.841、1.000、0.944。结论:血清中筛选出的差异表达蛋白S100A8、LRG1及SPARC是值得进一步研究的儿童APL候选血清标志物。

Objective:To screen the serum differentially expressed proteins of APL in children.Methods:Serum protein expression profiles from 20 cases of normal healthy controls,and 20 cases of APL patients were detected by iTRAQ(isobaric tag for relative and absolute quantification)labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2 DLC-MS/MS),and analyzed by bio informatics software.S100 A8,LRG1 and SPARC were validated by ELISA.ROC was built by SPSS 20.0 software.Results;Analysis identified 83 differentially expressed proteins in APL serum compared with control according to our defined criteria,of which 33 proteins were up-regulated and 50 proteins were down-regulated(P<0.05).IPA analysis revealed that these differentially expressed proteins were related to the function of Cellular Movement,Immune Cell Trafficking,Hematological System Development and Function,Cell-To-Cell Signaling and Interaction,Tissue Development,and involved in a variety of signalling Pathways,the most representative pathways including LXR/RXR Activation and Acute Phase Response Signaling.S100 A8 and LRG1 were found to be elevated and SPARC was markedly down-regulated in serum of childhood APL when compared to the normal controls as examined by ELISA(P<0.05),which was consistent with the iTRAQ result.The overall predictive accuracy of each protein was reflected by the area under the ROC curve(AUC),S100 A8,LRG1 and SPARC with ROC areas of 0.841,1.000 and 0.944 respectively.Conclusion:S100 A8,LRG1 and SPARC may be serve as serum candidate biomarkers for pediatric APL.