尿毒症患者血清蛋白质组份的双向凝胶电泳-飞行时间串联质谱分析研究

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International Protein Index（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。

Analysis of proteomic components of sera from patients with uremia by two dimensional electrophoresis and matrix assisted laser desorption/ionization time of flying mass spectrometry

Objective To establish and optimize two-dimensional gel electrophoresis technical platform, and compare the different sera proteomic components between uremic patients and normals. Methods Immobiline pH gradients isoelectric focusing was used as 1D, and vertical SDS-PAGE as 2D. Some applications, such as sample preparation and volume of loading sample, choice of IPC gel, were improved. Sliver staining, ImageMaster 2D S.O analysis software, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS) and IPI human database searching were used to separate and indentify the proteome from uremic patients sera.Results Satisfactory 2DE patterns of the uremic serum proteins were obtained. Twenty-six protein spots were remarkably changed in uremic patients, and 20 protein spots were identified by MALDI-TOF-TOF-MS. Conclusions Good reproducibility could be obtained by applying immobilized pH gradient 2DE to seperate and identify the proteome in serum, which contributes to further study on uremia toxins pertaining to protein.