CATARACT RELATED MODIFICATIONS OF LENS CRYSTALLINS

描述：拟定研究将使用新的分析方法 基于质谱法来鉴定人透镜蛋白， 表征其翻译后修饰。在第一级 两种新型的质谱仪，电喷雾离子化 （ESIMS）和基质辅助激光解吸（MALDI），将用于 精确测定完整蛋白质的分子量， 百分之零点零一。在第二级分析中，晶体蛋白将被消化 其分子量将通过直接测定 高效液相色谱-质谱联用 (HPLC/MS）。年轻、透明镜片的结果将用于制作数据 银行，结果从老化和白内障的镜头将是 比较了目前，来自年轻人的α-晶体蛋白的参考数据 和旧的清晰镜头几乎完成，大量的数据已经 收集β和γ-晶体蛋白。的具体目标 目前的建议是1）。为了完成β-和 γ-晶体蛋白在人透镜中表达，并定义晶状体后移。 这些蛋白质的翻译修饰与衰老有关。2）。 鉴定晶状体蛋白组分和翻译后修饰 的水不溶性盐酸胍可溶性级分 年轻人和老年人捐赠的透明镜片。3）。识别翻译后 α-、β-和γ-晶体蛋白的修饰与 白内障的早期症状4）。通过合作研究，确定 晶体蛋白组分和个体的翻译后修饰 通过二维凝胶电泳从人晶状体中分离斑点。

DESCRIPTION: The proposed investigation will use new analytical methods based on mass spectrometry to identify human lens proteins and characterize their post-translational modifications. In the first level of analysis, two new types of mass spectrometry, electrospray ionization (ESIMS) and matrix-assisted laser desorption (MALDI), will be used to determine the molecular weights of intact proteins with an accuracy to 0.01%. In the second level of analysis, crystallins will be digested into peptides whose molecular weights will be determined by directly coupled high performance liquid chromatography mass spectrometry (HPLC/MS). Results for young, clear lenses will be used to make a data bank, to which results from aged and cataractous lenses will be compared. At present, reference data for alpha-crystallins from young and old clear lenses are nearly complete and considerable data have been collected for beta and gamma-crystallins. The Specific Aims of the present proposal are 1). To complete the identification of beta- and gamma-crystallins that are expressed in the human lens and define post- translational modifications of these proteins associated with aging. 2). Identify the crystallin components and post-translational modifications of the water-insoluble, guanidine hydrochloride-soluble fraction of clear lenses from young and old donors. 3). Identify post-translational modifications of alpha-, beta-, and gamma-crystallins associated with early stages of cataract. 4). Through collaborative studies, identify the crystallin components and post-translational modifications of individual spots isolated from human lenses by 2-D gel electrophoresis.