蛋白质组学技术筛选缺血性结肠炎与溃疡性结肠炎鉴别诊断的血清学标志物

Screening of serological markers for differential diagnosis ischemic colitis and ulcerative colitis by proteomic techniques

目的采用串联质谱标签(TMT)联合液相色谱-串联质谱(LC-MS/MS)筛选和鉴定用于缺血性结肠炎(IC)与UC鉴别诊断的血清蛋白质标志物。方法选择浙江大学医学院附属第一医院2018年1月至2019年1月收治的UC和IC患者各10例,以及健康对照者10名,分别纳入UC组、IC组和NC组。采集所有研究对象的空腹血清样本,经去除高丰度蛋白、蛋白酶解、肽段标记和分馏等处理后,通过质谱分析仪检测,获取在3组试验中均具有TMT数据且去除TMT值为0的蛋白质,构建蛋白质聚类热图,设定差异倍数>1.5或<0.67且差异有统计学意义的蛋白质为差异蛋白质。通过Reactome数据库对组间差异蛋白质所参与的通路进行聚类分析。统计学方法采用t检验、hypergeometry检验和BH多重检验校正。结果经蛋白质谱分析,共鉴定出357种血清蛋白质。IC组与NC组比较共有27个差异蛋白质,其中上调蛋白质6个,下调蛋白质21个;UC组与NC组比较共有228个差异蛋白质,其中上调蛋白质75个,下调蛋白质153个;UC组与IC组比较共有49个差异蛋白质,其中上调蛋白质22个,下调蛋白质27个。在NC组、IC组、UC组3组的差异蛋白质比较中,仅纤维凝胶蛋白质3表达量在3组间两两比较的差异均有统计学意义(其在UC组与NC组、UC组与IC组、IC组与NC组中的差异倍数分别为0.24、0.46和0.53,t=-5.089、-7.298、-3.919,P均<0.01)。聚类分析结果显示:IC组与NC组比较的差异蛋白质中,仅富集到血小板脱颗粒通路,有10个蛋白质参与了这条通路(P<0.01);UC组与NC组比较的差异蛋白质中,共富集得到58条通路,其中38个蛋白质参与血小板脱颗粒通路,16个蛋白质参与补体初始触发通路,13个蛋白质参与补体级联调节通路,11个蛋白质参与抗体介导的补体激活通路(P均<0.01);UC组与IC组比较的差异蛋白质中,共富集得到3条通路,其中9个蛋白质参与血小板脱颗粒通路,7个蛋白质参与补体初始触发通路,5个蛋白质参与补体级联调节通路(P均<0.01)。结论IC和UC患者的血清蛋白质组学存在差异,且差异蛋白质主要参与血小板活化和补体激活过程,这些差异蛋白质或可作为今后IC与UC鉴别诊断的生物标志物。

Objective To screen and identify serum protein biomarkers for the differential diagnosis between ischemic colitis(IC)and ulcerative colitis(UC)by tandem mass tag(TMT)combined with liquid chromatography/tandem mass spectrometry(LC-MS/MS).Methods From January 2018 to January 2019,at the First Affiliated Hospital of School of Medicine of Zhejiang University,patients with UC or IC,and health controls,each 10 cases,were enrolled into UC group,IC group and normal control(NC)group,respectively.Fasting serum samples of all the subjects were collected.After removal of high-abundance protein,followed by proteolysis,peptide labeling and fractionating,the samples were then processed by mass spectrometry.The protein with TMT data of three groups was obtained and protein with TMT value 0 were removed.Heat map of protein was constructed.The differential protein was defined as the protein fold change over 1.5 or less than 0.67.The Reactome database was used to cluster the pathways of differential proteins among groups.Statistical methods included t test,hypergeometry test and corrected by BH multiple test.Results A total of 357 serum proteins were identified by proteomic profiling.There were 27 differential proteins between the IC group and the NC group,including six up-regulated proteins and 21 down-regulated proteins.There were 228 differential proteins between the UC group and the NC group,including 75 up-regulated proteins and 153 down-regulated proteins.There were 49 differential proteins between UC group and IC group,including 22 up-regulated proteins and 27 down-regulated proteins.In the comparison of differential proteins between the NC group,IC group and UC group,only the expression of fibrin 3 was statistically significant(the fold change between UC and NC,between UC and IC,between IC and NC were 0.24,0.46 and 0.53,respectively;t=-5.089,-7.298 and-3.919,all P<0.01).The results of pathway cluster analysis showed that in the comparison of differential proteins between IC group and NC group,only the platelet degranulation pathway was enriched,and 10 proteins were involved in this pathway(P<0.01).In the comparison of differential proteins between UC group and NC group,there were 58 pathways enriched,of which 38 proteins were involved in the platelet degranulation pathway,16 proteins were involved in the initial complement trigger pathway,13 proteins were involved in the complement cascade pathway,and 11 proteins were involved in antibody-mediated complement activation pathway(all P<0.01).In the comparison of differential proteins between UC group and IC group,three different pathways were obtained.Among them,nine proteins were involved in the platelet degranulation pathway,seven proteins were involved in the initial complement trigger pathway,and five proteins were involved in the complement cascade pathway(all P<0.01).Conclusions The difference in serum proteome between IC patients and UC patients was significant,and the differential proteins are mainly involved in platelet activation and complement activation.The candidate proteins identified in this study may be used as biomarkers for the differential diagnosis of UC and IC in the future.