白内障晶状体核蛋白质组学的初步研究

目的研究年龄相关性白内障晶状体核不同组分晶状体蛋白的组成。方法取12例年龄相关性白内障晶状体软核,提取晶状体核中的水溶性蛋白及尿溶性蛋白进行二维电泳及UPD-蛋白质染料染色试剂盒对凝胶染色后,用Image Master2D Platinum6.0软件对获得的蛋白图谱进行分析,再利用MALDI-TOF MS分析及数据库搜索对差异表达的蛋白质点进行鉴定。结果二维电泳结果显示,年龄相关性白内障晶状体核中水溶性蛋白和尿溶性蛋白大部分为相对分子质量14400~97400,pI5~9。高丰度蛋白质斑点相对分子质量分布在20000~31000,pI6~8区域内。年龄相关性白内障晶状体核水溶性蛋白二维电泳图识别出31个蛋白质斑点,尿溶性蛋白二维电泳图识别出26个蛋白质斑点,相差2倍以上表达的差异点有14个。对选取的蛋白质斑点进行MALDI-TOF MS分析,经数据库检索后鉴定出6种蛋白质,其中3种蛋白质表达下调（γS-晶状体蛋白、βA3-晶状体蛋白、βA4-晶状体蛋白）,1种蛋白质表达上调（βB1-晶状体蛋白）,2种蛋白质表达缺失（截取的人βB1-晶状体蛋白A链、截取的人γD-晶状体蛋白X链）。结论年龄相关性白内障晶状体核不同组分晶状体蛋白中主要由β-晶状体蛋白组成,与尿溶性晶状体蛋白相比,水溶性蛋白鉴定出γD-晶状体蛋白,而βA4-晶状体蛋白阙如。

Proteomic analysis of human lenses nucleus in cataractous eye

BackgroundThe research on function of protein is an important task as the achieve of human genome project.The whole lens was used in proteome study of crystallin in post,but it can not interpret the relationship of lens nucleus proteome and formation of age-related cataract.ObjectiveThe purpose of this study was to compare and analyze the composition of water soluble and urea soluble crystalline in the nucleus from human cataractous lenses.MethodsThe lens nucleus samples with the hardness grade Ⅱwere obtained from 12 lens with age-related cataract during the surgery.The water soluble and urea soluble fraction of the samples were analyzed by two-dimensional（2-D）gel electrophoresis.Image analysis was carried out using Image Master 2-D platinum 6.0 software package.The interested protein spots were excised from 2-D gels,and then digested and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF MS）.Written informed consent was obtained from each patient prior to the surgery.ResultsTwo-dimension result showed that the majority of crystallin proteins in the nucleus with age-related cataract had amolecular weight ranged from 14 400 to 97 400 and pI 5-9.Themolecular weight range of high level of proteome spots in samples distributed in the domain of 20 000-31 000 with the pI 6-8.According to the results obtained from Image Master 2-D software,total 31 protein spots were identified in water soluble proteins and 26 protein spots were identified in urea soluble proteins.Fourteen protein spots were found to be differentially expressed（2 times differences）in urea soluble proteins compared to water soluble proteins.Six differential crystalline species,including 3 down-regulated proteins（γS crystallin,βA3 crystallin,βA4 crystallin）,up-regulated protein（β B1 crystallin）,2 unmatched proteins（chain X,human gamma-D crystallin structure at 1.25 A resolution,chain A,crystal structure of truncated human beta-B1-crystallin）were positively identified by MALDI-TOF MS.ConclusionThe proteome compositions of crystalline species are mostly β-crystalline in the nucleus of age-related cataract;in contrast to urea soluble proteins,water soluble proteins contain gamma D crystalline while crystallin beta A4 is absent.