兔晶状体蛋白质组的双向电泳和质谱鉴定研究

Identification of rabbit lens proteins by two-dimensional gel electrophoreses and mass spectrometry

目的 探讨双向电泳和质谱鉴定在晶状体蛋白质组特性研究中的价值 ,为白内障的防治提供新的有效途径。方法 采用固相 pH梯度 (IPG)等电聚焦双向凝胶电泳方法对兔龄为 3个月的新西兰兔透明晶状体蛋白质进行分离 ,使用ImageMaster 2DElite 3.0 1图像分析软件分析电泳图像 ,使用基质辅助激光解吸附飞行时间质谱仪对主要的高丰度晶状体蛋白质进行鉴定。结果 双向电泳图谱显示 ,在 2 5 0 μg兔晶状体中蛋白质分布在等电点 (PI)为 pH值 5～ 9、相对分子质量为14 0 0 0～ 94 0 0 0的区域内 ;而高丰度晶状体蛋白质的相对分子质量为 14 0 0 0～ 4 0 0 0 0。图像分析软件定量检出 180个蛋白质斑点的近似PI、相对分子质量及蛋白质相对含量 ,质谱鉴定得到其中 16个高丰度晶状体蛋白质的种类。结论 双向电泳和质谱鉴定可有效分离和分析晶状体蛋白质组的特性 ,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径 ,为白内障的防治带来了新的前景。

Objective To study the role of lens proteomics using two-dimensional electrophoresis (2-DE) and mass spectrometry in order to exploring novel effective ways for cataract prevention and therapy. Methods The proteins of the three-month-year old rabbit lens were separated using immobilized pH gradients 2-DE. Image analysis was carried out using Image Master 2D Elite 3.01 software package. Most of the crystallines wasidentified by matrix assisted laser adsorption/ionization-time of-flight-mass spectrometry (MALDI-TOF-MS). Results The maps of 2-DE showed that lens proteins were in the section of pH 5-9 and the relative molecular weight was 14 000-94 000, while relative molecular weight of more abundant crystalline was localized at 14 000-40 000. About 180 protein spots were detected with the similar PI, molecular weight and quantity of each spot could be acquired by image analysis software. Sixteen crystallines were identified using MALDI-TOF-MS. Conclusion Proteomic analysis of lens can be accomplished and the proteins can be well separated and analyzed using 2-DE and mass spectrometry. This technique offers a new avenue for analyses of lens proteins and to assess their differential expression in cataract, and may thus provide a novel approach to cataract prevention and therapy.