Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: Does deamidation contribute to crystallin insolubility?
Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: Does deamidation contribute to crystallin insolubility?
复制标题
DOI：
10.1021/pr050473a
复制
发表时间：
2006-10-06
影响因子：
4.4
通讯作者：
David, L. L.
中科院分区：
文献类型：
--
作者：
Wilmarth, P. A.;Tanner, S.;David, L. L.
我们采用了最近开发的盲修改搜索技术，以产生最全面的地图的翻译后修饰（PTM）在人类透镜构建的日期。使用多维液相色谱-质谱法分析了三个老化晶状体（其中两个具有中度白内障）和一个年轻对照透镜。总共鉴定了透镜蛋白中的491个修饰位点。在晶体蛋白中有155个PTM位点：77个先前报道的位点和78个新检测到的PTM位点。这些位点中的几个具有先前在透镜中未通过质谱法检测到的修饰，包括羧甲基赖氨酸（+58 Da）、羧乙基赖氨酸（+72 Da）和具有未知化学结构的+55 Da精氨酸修饰。在所有三种老化晶状体中均观察到这些新变化，但在新透镜中未发现。鉴定了几个新的半胱氨酸甲基化位点，表明这种修饰在透镜中比以前认为的更广泛。结果被用来估计在特定网站的光谱计数的修改程度。我们通过比较老化晶状体的水溶性和水不溶性部分之间的光谱计数来测试长期存在的假设，即PTM有助于与年龄相关的晶状体蛋白溶解度的损失，并且发现在水不溶性部分中脱酰胺的程度显著增加。根据光谱计数，老化晶状体中最丰富的PTM是脱酰胺和甲基化半胱氨酸，其他PTM的水平较低。
We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+ 58 Da), carboxyethyl lysine (+ 72 Da), and an arginine modification of + 55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.