慢性间歇性低氧大鼠肾组织差异蛋白的定量分析:基于TMT-PRM技术

Quantitative analysis of differential proteins in renal tissues of rats with chronic intermittent hypoxic exposure based on TMT and PRM technology

目的基于TMT-PRM技术研究慢性间歇性低氧大鼠肾脏组织的蛋白质组学,筛选差异表达蛋白以鉴定潜在的生物标志物。方法将20只SD大鼠随机分为正常组、实验组(n=10)。实验组放入低氧舱内建立慢性间歇性低氧(CIH)大鼠模型。12周后采集肾脏样本,HE染色观察大鼠肾组织形态变化;提取蛋白并酶解和TMT标记,采用LC-MS/MS鉴定蛋白,软件Proteome Discoverer2.4处理数据,进行生物信息学分析,以倍数变化1.2以上或低于0.83为标准,t检验P<0.05作为差异表达蛋白,并应用PRM技术验证候选蛋白的表达情况。结果CIH引起肾脏损伤,肾小球形态不规则,肾小管上皮细胞肿胀等。实验组与正常组比较,筛选出差异表达蛋白有31种,其中表达上调的22种,表达下调的9种。GO功能富集分析显示差异表达蛋白主要参与细胞黏附、缺氧应激、钙离子等生物学过程。KEGG通路富集分析结果显示差异表达蛋白涉及PPAR、AMPK、代谢途径等信号通路。PRM相对定量分析的目标蛋白中进行筛选,P07379(PCK1)、P19132(Fth1)、Q5XI79(Ndufaf7)3个蛋白与TMT定量蛋白质组结果趋势一致。结论应用TMT-PRM技术在慢性间歇性低氧大鼠肾组织筛选出差异表达蛋白有31种,其中P07379(PCK1)、P19132(Fth1)、Q5XI79(Ndufaf7)可能是与慢性间歇性低氧大鼠肾损伤密切相关的关键蛋白。

Objective To screen the differentially expressed proteins in the kidneys of rats exposed to chronic intermittent hypoxia(CIH)based on TMT-PRM technology to identify the potential biomarkers of renal injuries induced by CIH.Methods Twenty SD rats were randomized into two groups(n=10)and exposed to CIH or normoxia.After 12 weeks of exposure,the kidneys of the rats were collected for observing pathological changes.The renal proteins were extracted,enzymatically digested and labelled with TMT,and LC-MS/MS was used to identify the proteins.The data were processed using Proteome Discoverer2.4 for bioinformatic analysis of the differentially expressed proteins.PRM technology was used to verify the expression of the candidate proteins.Results CIH induced obvious renal injury in the rats,manifested by irregular glomerulus and swelling of the epithelial cells of the renal tubules.A total of 31 differentially expressed proteins were identified in CIH rats,including 22 up-regulated and 9 down-regulated proteins.Enrichment analysis of GO function and KEGG pathway analysis showed that the differentially expressed proteins participated mainly in the biological processes of cell adhesion,hypoxic stress,and calcium ions and were involved in the PPAR,AMPK,and metabolic pathways.In PRM quantitative analysis,3 proteins,namely P07379(PCK1),P19132(Fth1)and Q5XI79(Ndufaf7),showed results consistent with those of TMT quantitative proteomic analysis.Conclusion Thirty-one differentially expressed proteins were identified in the renal tissues of rats with CIH exposure,and among them P07379(PCK1),P19132(Fth1),and Q5XI79(Ndufaf7)may be the key proteins closely related to the renal injury in induced by CIH.