高度近视患者房水非标记定量蛋白质组学分析

Label-free quantitative proteomic analysis of aqueous humor in patients with high myopia

目的采用定量蛋白质组学技术分析高度近视患者房水中的蛋白质组谱。方法纳入2019年1—8月在天津医科大学眼科医院于白内障手术中用1 ml注射器采集伴或不伴高度近视的年龄相关性白内障患者房水标本各34例34眼(每例100μl),分别作为高度近视白内障组和单纯白内障组。各组分别选取16例16眼房水标本,采用BCA法进行蛋白定量并比较,采用非标记液相色谱串联质谱分析得到2个组的差异表达蛋白。进一步将生物大数据用基因本体功能富集和京都基因与基因组百科全书通路富集分析差异表达蛋白的功能和信号转导通路。各组分别选取18例18眼房水标本采用酶联免疫吸附实验(ELISA)进行质谱检测结果的扩大样本量验证。结果高度近视白内障组房水标本平均蛋白质量浓度为(1134.91±104.78)ng/L,明显高于单纯白内障组的(706.71±85.43)ng/L,差异有统计学意义(t=11.977,P<0.01)。2个组患者房水标本中共鉴定出463个可定量蛋白质,其中86个差异表达蛋白质,包括49个表达上调蛋白和37个表达下调蛋白。这些差异表达蛋白的分类主要包括蛋白结合活性调节因子、细胞外基质蛋白、载体蛋白、细胞间信号分子、蛋白质修饰酶等,分别占32.70%、14.50%、9.10%、9.10%和7.30%。生物信息学分析表明,86个差异表达蛋白主要富集在补体激活及其调节、急性炎症反应、细胞外基质组织重塑等生物学过程。其中21个差异表达蛋白富集于补体和凝血级联通路,15个差异表达蛋白富集于细胞外基质-受体相互作用通路,8个差异表达蛋白富集于PI3K-Akt信号通路。ELISA结果表明,随机选取的3个差异表达蛋白在2个组之间表达变化趋势均与非标记定量蛋白质组学分析结果一致。结论高度近视白内障与单纯白内障之间房水蛋白质表达谱有显著变化,高度近视与炎症和免疫相互作用以及细胞外基质的重塑密切相关。

Objective To characterize proteomic profile in aqueous humor of patients with high myopia using quantitative proteomic analysis.Methods Sixty-eight age-related cataract patients were divided into high myopic cataract group and simple cataract group according to that they had high myopia or not,with 34 patients(34 eyes)in each group.Aqueous humor samples(100μl/patient)were collected from each patient using a 1 ml tuberculin syringe during cataract surgery at Tianjin Medical University Eye Hospital from January 2019 to August 2019.Sixteen samples from each group were selected for protein quantification and comparison by BCA method.The differentially expressed proteins between the two groups were analyzed using label-free liquid chromatography tandem mass spectrometry.The function and signal transduction pathways of differentially expressed proteins were further analyzed by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes.Eighteen aqueous humor samples from each group were selected to verify the results of mass spectrometry by enzyme-linked immunosorbent assay(ELISA).This study protocol adhered to the Declaration of Helsinki.The use of human samples was approved by an Ethics Committee of Tianjin Medical University Eye Hospital(No.2020KY[L]-40).Written informed consent was obtained from each patient prior to surgery.Results The mean protein mass concentration of aqueous humor sample in the high myopic cataract group was(1134.91±104.78)ng/L,which was significantly higher than that in the simple cataract group(706.71±85.43)ng/L,showing statistically significant difference(t=11.977,P<0.01).A total of 463 proteins were identified and 86 proteins were found to be differentially expressed,including 49 up-regulated proteins and 37 down-regulated proteins in the two groups.These differentially expressed proteins were mainly protein-binding activity modulator,extracellular matrix protein,carrier protein,intercellular signal molecule,protein modifying enzyme and so on,accounting for 32.70%,14.50%,9.10%,9.10%and 7.30%,respectively.Bioinformatics analysis showed that 86 differentially expressed proteins were mainly related to biological processes such as complement activation and regulation,acute inflammatory response,and extracellular matrix tissue remodeling.Among them,21 differentially expressed proteins were enriched in the complement and coagulation cascades pathways,15 in the extracellular matrix-receptor interaction pathway,and 8 in the PI3K-Akt signaling pathway.ELISA results showed that the expression trends of three randomly selected differentially expressed proteins of the two groups were consistent with the results of label-free quantitative proteomic analysis.Conclusions There are significant changes in proteomic profiles of aqueous humor between the high myopia cataract patients and simple cataract patients.High myopia is closely associated with inflammation and immune interactions,and remodeling of extracellular matrix.