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目的建立和比较尿毒症患者及正常人的血清蛋白质双向电泳图谱,寻找和鉴定尿毒症患者差异表达的血清蛋白质谱。方法以固相pH梯度(IPG)等电聚焦(IEF)为第一向和垂直十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)为第二向,分别对尿毒症患者和正常者血清蛋白质样品进行二维双向电泳,经银染显色和ImageMaster2D5.0软件分析凝胶图像,对有差异性表达的尿毒症者的蛋白质用基质辅助激光解吸电离飞行时间串联质谱进行鉴定。结果成功获得了重复性较好的双向电泳银染图谱。尿毒症患者血清蛋白质表达谱发生了改变,将其中26个明显差异性表达的蛋白质点进行肽质指纹图谱和串联质谱分析,在InternationalProteinIndex(IPI)human数据库中检索,鉴定出其中20个蛋白。结论固相pH梯度双向凝胶电泳分离尿毒症患者血清总蛋白获得重复性较好的结果。尿毒症患者血清蛋白质表达谱发生了明显改变,基质辅助激光解吸电离飞行时间串联质谱分析鉴定的结果为尿毒症蛋白质毒素的研究提供了依据。
Objective To establish and compare two-dimensional gel electrophoresis profiles of serum proteins in patients with uremia and normal subjects, and to find and identify differentially expressed serum protein profiles in uremic patients. Methods The first and the second SDS-PAGE were obtained by isoelectric focusing (IEF) of solid phase pH gradient (IPG) Two-dimensional two-dimensional electrophoresis of normal serum protein samples was performed. The silver staining and ImageMaster2D5.0 software were used to analyze the gel images. Proteins from differentially expressed uremic patients were identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. The results obtained successfully repeated two-dimensional electrophoresis silver stain map. Serum protein profiles of patients with uremia were changed. Twenty-six of the protein spots differentially expressed were analyzed by peptide fingerprinting and tandem mass spectrometry, and 20 of them were searched in International ProteinIndex (IPI) human database. Conclusion Solid-phase pH gradient two-dimensional gel electrophoresis separation of serum total protein in patients with uremia to obtain better reproducibility results. Serum protein expression profiles of uremic patients changed significantly. The results of matrix-assisted laser desorption / ionization time of flight tandem mass spectrometry analysis provided the basis for the study of uremic toxins.