人尿液蛋白质的超滤离心制备及其双向凝胶电泳分析

Ultrafiltration and two-dimensional gel electrophoresis analysis of human urinary protein

摘要:

目的：优化尿液蛋白质制备方法以获得蛋白点数多且分辨率高的尿液双向凝胶电泳（two dimensional gel electrophore－sis，２-DE）图谱，为尿液蛋白质组学研究提供技术支撑。方法：首先，使用6种不同方法制备尿液蛋白质后比较蛋白质得率，并分析这些方法的２-DE谱蛋白点数及３次重复匹配率，进行统计分析计算P值；最后利用2种固相pH梯度（immobilized pH gra－dient，IPG）胶条和不同的聚丙烯酰胺凝胶（polyacrylamide gel electrophoresis，PAGE）胶浓度进行2-DE分析，比较所得蛋白图谱的蛋白点数及分辨率。结果：与其他方法相比，超滤离心法制备尿液蛋白能获得更多的蛋白总量[（791±17） μg，P＝0.00０]，得到的电泳图谱蛋白点数也更多[（724±29）个，P＝0.00０]；利用pH 3~10 IPG胶条和12.5% PAGE分离有利于尿液蛋白质的整体分析，特别是酸性和碱性蛋白质点信息，而pH 4~7 IPG胶条能使等电点为4~7蛋白质的2-DE谱分辨率更高（P<0.05）。结论：建立了与2-DE兼容的尿液蛋白质制备方法，获得了更为全面的尿液蛋白点信息，为临床生理和疾病的尿液蛋白质组学研究奠定了基础。

Abstract:

Objective：To optimize urinary protein preparation method，to obtain a two-dimensional gel electrophoresis（2-DE） map with more urinary protein spots and higher resolution，and to provide technical supports for urinary proteomic researches. Methods：Firstly，urinary protein was extracted by 6 different preparation methods and then the total protein content between them were compared；secondly，protein spots as well as three-time repeated matching rate of 2-DE maps of these six methods were ana－lyzed，and P value for statistical analysis was set；finally，for the best method，2 different polyacrylamide gel electrophoresis（PAGE） concentrations and 2 different immobilized pH gradient（IPG） strips were utilized to compare protein spots and resolution of 2-DE maps. Results：Method named ultrafiltration got the most proteins（（791±17） μg，P=0.000） as well as maximum protein spots（724±29，P=0.000）. Utilization of 12.5% PAGE gel combined with IPG pH 3-10 strip was beneficial for the whole analysis of urinary proteins，es－pecially for the acid and basic proteins，however，when combined with pH 4-7 strip，a 2-DE map of higher resolution within the iso－electric point range of 4-7（P<0.05） can be obtained. Conclusion：This research establishes a 2-DE compatible urinary protein prepa－ration method and obtains more comprehensive urinary protein spots information，which lays a solid foundation for urinary proteomics research of clinical physiology and diseases.