常染色体显性多囊肾病患者与正常成人尿液的定量比较蛋白质组学分析

Comparative proteomic analysis of urine from autosomal dominant polycystic kidney disease patients and normal adults

目的搜索常染色体显性多囊肾病(ADPKD)患者与正常成人尿液蛋白质组中有丰度差异的蛋白质成分。方法制备ADPKD患者(PKD1突变)及正常成人尿液蛋白组样品,同位素编码的亲和力标签法标记蛋白质、筛选被标记肽段,二维液相色谱-串联质谱法分离鉴定被标记肽段,从而鉴定蛋白质。通过计算两同位素试剂标记的相同肽段指纹峰面积比,寻找有丰度差异的蛋白质。根据被鉴定肽段的可信度,从鉴定出的蛋白质中选出6种,免疫印迹法验证其在两尿液蛋白组样品中的丰度差异。结果两尿液蛋白质组共鉴定出尿蛋白质106种, ADPKD患者尿液中水平较正常上调者48种,下调者45种。其中多囊蛋白1、肝细胞生长因子、单核细胞趋化蛋白3及孕激素受体等5种蛋白丰度差异为免疫印迹所证实。结论包括多囊蛋白1在内的多种蛋白质成分在ADPKD患者尿液中丰度发生改变,差异蛋白包括生长因子、凋亡调节蛋白、细胞外基质成分、受体、参与胞浆运输的蛋白质、酶类、细胞信号蛋白、转录因子及转录调节因子等,这些蛋白质信息可能为寻找与ADPKD发病相关的蛋白提供部分实验依据。

Objective To identify proteins which have different abundance in the proteomes of patients with autosomal dominant polycystic kidney disease (ADPKD) and normal adults. Method Urine proteomes of ADPKD patients (PKD1 mutated) and normal adults were labeled on cysteine residues respectively with isotope-coded affinity tag (ICAT) heavy (13C) reagent and light (12C) reagent, and then mixed and digested by trypsin. The labeled cysteine-containing peplides were purified by affinity chromatography. They were separated and analyzed by two dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) for protein identification and quantification. According to the confidence of their peptides, abundance differentiation of 6 identified proteins were selected and were tested by Western blot.Results One hundred and six proteins were identified from two resources of urine proteomes. Among them, 48 were up-regulated with 45 down-regulated in ADPKD patients. Abundance differentiation of 5 identified proteins (polycystin-1, hepatocyte growth factor, monocyte chemotactic protein 3, progesterone receptor and retinoblastoma-like protein 2) among the 6 types were verified by Western blot. Conclusion Urine proteins differ in abundance between ADPKD patients and normal adults, including polycystin-1, growth factors, apoptosis regulators, matrix proteins, receptors, transporters, enzymes, cell signaling proteins, transcription factors and regulators, and so on. Data of this study may provide, at least partly, valuable experimental evidence of proteins involved in the pathogenesis of ADPKD.