SELDI蛋白质芯片筛选差异蛋白分子的二级鉴定

Further separating and identifying of differentially expressed proteins analyzed by SELDI

目的探讨以SELDI蛋白质芯片筛选后的细胞差异表达蛋白的分离和鉴定方法及其意义。方法以经体外培养及10μmol/L褪黑素药物干预的内皮祖细胞差异表达蛋白质为研究对象,分别采用Tricine-SDS-PAGE和双向凝胶电泳方法进行差异蛋白分离及结果比较,以FT-MS二级质谱鉴定。结果经Tricine-SDS-PAGE分离后,选取分子量为53 ku左右的趋势性差异表达蛋白进行FT-MS分析,质谱鉴定为微管蛋白-α3(吻合度评分为87)。经双向凝胶电泳分离后,于凝胶近酸性端、分子质量在72～95 ku之间区域,选取蛋白表达量较高的一点,质谱鉴定其为人热休克蛋白90-α(吻合度评分为91)。结论 Tricine-SDS-PAGE对于大致35～62 ku范围的蛋白分离较清晰、重复性好且样品用量少,2-DE对最低上样量有较严格要求,但它能提供分子质量、等电点双重参数来更准确定位所感兴趣蛋白点,且对大分子质量蛋白的分离效果更佳,经质谱鉴定结果理想。

Objective To further separate and identify the differentially expressed proteins analyzed by SELDI,and discuss the methodology significance.Methods Endothelial progenitor cells cultured in the medium containing melatonin in concentration of 10 μmol/L were used to extract proteins and analyze by SELDI for preliminary screening differentially expressed proteins.Tricine-SDS-PAGE and 2-DE was chosen to separate the target proteins,respectively,and FT-MS was used to identify the proteins.Results α-tubulin and human heat shock protein 90-α were detected by FT-MS after separated by Tricine-SDS-PAGE and 2-DE,respectively.Conclusions Tricine-SDS-PAGE is preferred for separating proteins in the mass range 35～62 ku,while 2-DE is better for separating bigger protein molecules.