Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteornic approach
Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteornic approach
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DOI：
10.1016/j.mad.2006.08.006
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发表时间：
2006-11-01
影响因子：
5.3
通讯作者：
Richardson, Arlan
中科院分区：
文献类型：
--
作者：
Chaudhuri, Asish R.;de Waal, Eric M.;Richardson, Arlan
蛋白质羰基通常被用作细胞和组织中蛋白质氧化的标记。目前，2,4-二硝基苯肼（DNPH）被广泛用于（分光光度法或免疫学）定量蛋白质中羰基水平和鉴定羰基化的特定蛋白质。我们采用了一种基于荧光的方法，使用荧光素-5-硫代氨基脲（FTC）来量化蛋白质组中整体蛋白质羰基以及单个蛋白质上的羰基水平。体外生成的蛋白羰基通过用FTC标记氧化蛋白，然后用凝胶电泳将FTC标记的蛋白与游离探针分离来定量。发现FTC与蛋白质羰基的反应对羰基具有特异性。我们测量了年轻和年老小鼠肝脏中的蛋白质羰基水平，发现随着年龄的增长，全球蛋白质羰基水平显著增加（两倍）。使用二维凝胶电泳，我们使用该方法直接测量特定蛋白质中蛋白质羰基水平的机会。我们鉴定出12种蛋白质，其羰基含量（每μ g蛋白质的羰基摩尔数）随着年龄的增长增加了两倍以上。12种蛋白大部分含有过渡金属结合位点，其中Cu/Zn超氧化物歧化酶在老年小鼠中含有最高的羰基摩尔比。因此，基于荧光的测定使研究人员能够识别在不同病理和生理条件下被氧化的潜在靶蛋白。2006爱思唯尔爱尔兰有限公司版权所有。
Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the chances in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/mu g of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions. (c) 2006 Elsevier Ireland Ltd. All rights reserved.