小鼠晶体蛋白质组的双向电泳和质谱鉴定研究

目的应用双向电泳和质谱技术研究5周龄小鼠晶体蛋白质组。方法提取小鼠晶体总蛋白，进行固相pH梯度（IPG）等电聚焦双向电泳，胶体考马斯亮蓝R-250染色，使用PDQuest7．30图像分析软件分析电泳图像。选择主要蛋白点胶上酶解，应用基质辅助激光解析电离飞行时间／飞行时间（MALDI—TOF／TOF）仪器进行串联质谱（MS／MS）鉴定。结果上样量为882μg和190μg时，分别检测370±41蛋白点（n=3）和57±5个蛋白点（n=3）。高上样量能够较好地分离晶体低丰度蛋白，如念珠状纤维结构蛋白BFSP；低上样量可很好地分离高丰度蛋白-晶体蛋白（包括αA、αB；βA1～βA4；βB1～βB3；γA～γF和γS等）。质谱鉴定得到1种细胞骨架蛋白和16种高丰度晶体蛋白。结论双向电泳和质谱技术有效考察了晶体总蛋白质，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。

Identification of Mouse Lens Proteins by Two-Dimensional Gel Electrophoresis and Mass Spectrometry

Objective To separate and identify the lens proteomics in 5 weeks old mice using two-dimensional electrophoresis （2-DE） and mass spectrometry. Methods The proteins of mouse lens were separated using immobilized pH gradient （IPG） two-dimensional electrophoresis （2-DE） and colloidal Coomassie brilliant blue （CBB） staining. Image analysis was carried out using PDQuest 7.30 software package. Most of the proteins in gel were identified by matrix assisted laser adsorption/ ionization-time of flight-tandem mass spectrometry （MALDI-TOF-MS/MS）. Results When a 882 μg sample or a 190 μg sample was added,370 ± 41 spots and 57 ±5 spots were detected, respectively. When a higher sample was applied, low-abundance proteins such as beaded filament structure protein （BFSP） could be separated. With a lower sample, the high-abundance proteins were separated, such as αA、αB;βA1～βA4;βB1～βB3;γA～γF and γS crystallins. Protein spots were identified by MALDI- TOF/TOF, including one cytoskeletal protein and sixteen high-abundance crystallins. Conclusions The lens proteins can be well separated and analyzed using 2-DE and mass spectrometry. Proteomic analysis technique offers a new avenue for analysis of lens proteins and to assess their differential expression in cataract.