A method for selective enrichment and analysis of nitrotyrosine-containing peptides in complex proteome samples
A method for selective enrichment and analysis of nitrotyrosine-containing peptides in complex proteome samples
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DOI：
10.1021/pr0606934
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发表时间：
2007-01-01
影响因子：
4.4
通讯作者：
Smith, Richard D.
中科院分区：
文献类型：
--
作者：
Zhang, Qibin;Qian, Wei-Jun;Smith, Richard D.
在各种神经退行性疾病和与年龄相关的病理学中已经发现蛋白质酪氨酸硝化水平升高。然而，直到最近，缺乏一个有效的富集方法，阻止了这种重要的低水平蛋白质修饰的分析。我们已经开发了一种方法，专门富集含硝基酪氨酸的肽，使硝基酪氨酸肽和特定的硝化位点可以明确确定与LC-MS/MS。该程序包括衍生的硝基酪氨酸成游离巯基，然后由高效率的富集巯基的肽与硫丙基琼脂糖珠。衍生化过程包括：（1）用乙酸酐乙酰化以封闭所有伯胺，（2）将硝基酪氨酸还原为氨基酪氨酸，（3）用N-琥珀酰亚胺基S-乙酰硫代乙酸酯（SATA）衍生氨基酪氨酸，以及（4）SATA上的S-乙酰基脱保护以形成游离巯基。通过富集和未富集样品之间鉴别的串联质谱中硝基酪氨酸衍生肽的对比百分比证明了该方法的高度专属性。未富集的体外硝化人组蛋白H1.2、牛血清白蛋白（BSA）和小鼠脑匀浆样品的全球分析分别有9%、9%和5.9%的识别出的含硝基酪氨酸的肽，而富集的样品分别有91%、62%和35%。富集的小鼠脑匀浆的重复LC-MS/MS分析鉴定了150种独特的硝化肽，覆盖102种蛋白质，估计错误发现率为3.3%。
Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and age-related pathologies. Until recently, however, the lack of an efficient enrichment method has prevented the analysis of this important low-level protein modification. We have developed a method that specifically enriches nitrotyrosine-containing peptides so that both nitrotyrosine peptides and specific nitration sites can be unambiguously identified with LC-MS/MS. The procedure consists of the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process includes: ( 1) acetylation with acetic anhydride to block all primary amines, ( 2) reduction of nitrotyrosine to aminotyrosine, ( 3) derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and ( 4) deprotection of S-acetyl on SATA to form free sulfhydryl groups. The high specificity of this method is demonstrated by the contrasting percentage of nitrotyrosine-derivatized peptides in the identified tandem mass spectra between enriched and unenriched samples. Global analysis of unenriched in vitro nitrated human histone H1.2, bovine serum albumin ( BSA), and mouse brain homogenate samples had 9%, 9%, and 5.9% of identified nitrotyrosine-containing peptides, while the enriched samples had 91%, 62%, and 35%, respectively. Duplicate LC-MS/MS analyses of the enriched mouse brain homogenate identified 150 unique nitrated peptides covering 102 proteins with an estimated 3.3% false discovery rate.