人胰液蛋白质组的双向电泳

Two-dimensional gel electrophoresis of proteome in human pancreatic juice.

目的建立优化的用于胰液蛋白质组研究的双向电泳方法。方法经内镜逆行胰胆管造影(ERCP)术收集纯胰液标本5例,分别进行多次双向电泳,比较不同标本保存时间、制备方法、泡胀液组成、固相pH胶条、等电聚焦条件及染色方案等因素对双向电泳图谱质量的影响。结果胰液标本在-80℃保存4个月做双向电泳对图谱没有明显影响。根据图谱质量比较,三氯乙酸/丙酮沉淀继以含硫脲的泡胀液复溶是优化的标本制备方法;应用窄范围固相pH胶条可以在增加标本上样量的同时改善分辨率;银染和兼容质谱的银染方案所获得的图谱同样具有良好的重复性和分辨率;不同病理状态的胰液标本的双向电泳图谱表现出一定程度的相似性。结论标本制备是胰液双向电泳的关键,优化的方法可以获得重复性好、分辨率高的双向电泳图谱,为不同病理状态下胰液蛋白质组学差异的研究奠定基础。

Objective To establish an optimized strategy of two-dimensional gel eleetrophoresis （2-DE） for proteome analysis of human pancreatic juice. Methods Pure pancreatic juice samples were aspirated from 5 patients undergoing endoscopic retrograde eholangiopaneretography （ERCP） procedures. 2-DE was carried out repeatedly and the effectiveness of different running conditions was compared according to the final results on 2-DE maps. The influences of preserving time. sample preparation techniques, compositions of rehydration solutions, immobilized pH gradient （IPG） strips. isoelectric focusing, and gel staining protocols on the quality of 2 DE maps were compared. Results The 2 DE patterns showed no significant difference after samples were preserved at 80C for 4 months. Deposition of triehloroaeetie acid/acetone followed by resolubilization with rehydration buffer containing thiourea was the best sample preparation protoeol; narrow-range IPG strips had higher separation efficiency with increase of loading protein in 2 DE; Silver staining protocol and its reformative form compatible with mass spectrometry had same reproducibility and high resolution in 2 DE maps. The 2-DE patterns of pancreatic juice from different patients shared some kind of similarity. Conclusions The key step of 2-DE is sample preparation technique. Well-resolved 2-DE maps with a high reproducibility can be obtained by the optimized strategy in this study, which provide a basis for differential proteome analysis of human pancreatic juice.