基于串联质谱标签技术的甲状腺相关眼病眼外肌蛋白组学研究

Proteomic analysis of extraocular muscles from patients with thyroid-associated ophthalmopathy by tandem mass tag

目的使用串联质谱标签(TMT)方法筛选甲状腺相关眼病(TAO)继发限制性斜视患者和共同性斜视患者的眼外肌差异表达蛋白。方法收集2019年8月至2020年12月于北京大学人民医院眼科诊断为TAO继发限制性斜视并行斜视矫正术5例患者的眼外肌标本,收集同期5例共同性内斜视行斜视矫正术患者的眼外肌作为对照。采用基于TMT的蛋白质组学技术,对TAO组和对照组眼外肌的差异表达蛋白进行筛选及生物信息学分析。设定倍数变化≥1.2或≤0.83且P值<0.05作为筛选差异表达蛋白的阈值。利用UniProtGOA蛋白数据库和STRING蛋白网络互作数据库分析差异表达蛋白的基因本体(GO)注释、KEGG通路富集和蛋白-蛋白相互作用关系。结果TAO组与对照组眼外肌总差异表达蛋白数量为53个,其中表达上调蛋白34个,表达下调蛋白19个。通过GO注释发现,按其生物学过程分类,这些差异表达蛋白主要参与了对刺激的反应过程、多细胞生物过程、代谢过程、发育过程、细胞内信号转导、正向生物调控过程;KEGG通路富集分析发现差异表达蛋白主要参与了局部黏附、紧密连接、细胞骨架调控、凋亡等通路。蛋白-蛋白相互作用分析发现肌球蛋白重链2、肌球蛋白重链7、肌球蛋白调节轻链、α辅肌动蛋白2、纤维蛋白原α链和纤维蛋白原β链6个关键蛋白。结论TAO继发限制性斜视患者与共同性斜视患者的眼外肌中蛋白表达存在差异,肌球蛋白、辅肌动蛋白、细丝蛋白可能通过参与细胞骨架调控、局部黏附等参与TAO的发病机制。

Objective To identify the proteins differentially expressed in extraocular muscles between restrictive strabismus patients with thyroid-associated ophthalmopathy(TAO)and concomitant esotropia patients by proteomic analysis using tandem mass tag(TMT).Methods Extraocular muscles samples from 5 restrictive strabismus patients with TAO and 5 concomitant esotropia patients were collected at Peking University People's Hospital from August 2019 to December 2020.All the patients received strabismus surgery.Differentially expressed proteins(DEPs)in extraocular muscles samples were identified by quantitative proteomic analysis and bioinformatic analysis based on TMT.Fold change≥1.2 or≤0.83 and P value<0.05 was regarded as the threshold to screen DEPs.GO annotation,KEGG pathways enrichment analysis and protein-protein interaction(PPI)network of DEPs were conducted through UniProtGOA and STRING.This study protocol was approved by the Ethics Committee of Peking University People's Hospital(No.2021PHB058-001).Results A total of 53 DEPs were identified,34 of which were up-regulated and 19 were down-regulated.The biological processes DEPs mainly participated included response to stimulation,multicellular organismal process,metabolism,developmental process,intracellular signal transduction,and positive regulation of biological process.DEPs were involved in pathways including focal adhesion,tight junction,regulation of action cytoskeleton,and apoptosis.Six key proteins identified using PPI network were myosin heavy chain 2,myosin heavy chain 7,myosin regulatory light chain,α-actinin-2,fibrinogen alpha chain and fibrinogen beta chain.Conclusions There are DEPs in extraocular muscles between restrictive strabismus patients with TAO and concomitant esotropia patients.Myosin,actinin and filamin may be involved in the pathogenesis of TAO through regulation of actin cytoskeleton and focal adhesion.