Zhang Qibin, Qian Wei-Jun, Knyushko Tatyana V, Clauss Therese R W, Purvine Samuel O, Moore Ronald J, Sacksteder Colette A, Chin Mark H, Smith Desmond J, Camp David G, Bigelow Diana J, Smith Richard D
Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
J Proteome Res. 2007 Jun;6(6):2257-68. doi: 10.1021/pr0606934. Epub 2007 May 12.
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.
在各种神经退行性疾病和与年龄相关的病变中均发现蛋白质酪氨酸硝化水平升高。然而，直到最近，由于缺乏有效的富集方法，阻碍了对这种重要的低水平蛋白质修饰的分析。我们开发了一种方法，可特异性富集含硝基酪氨酸的肽段，从而能够通过液相色谱-串联质谱（LC-MS/MS）明确鉴定硝基酪氨酸肽段和特定的硝化位点。该过程包括将硝基酪氨酸衍生化为游离巯基，然后用硫丙基琼脂糖珠高效富集含巯基的肽段。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）对氨基酪氨酸进行衍生化，以及（4）去除SATA上的S-乙酰基以形成游离巯基。通过对比富集和未富集样品中鉴定的串联质谱图中硝基酪氨酸衍生化肽段的百分比，证明了该方法的高特异性。对未富集的体外硝化的人组蛋白H1.2、牛血清白蛋白（BSA）和小鼠脑匀浆样品进行的全局分析中，鉴定出的含硝基酪氨酸肽段分别占9%、9%和5.9%，而富集样品中分别为91%、62%和35%。对富集的小鼠脑匀浆进行的重复LC-MS/MS分析鉴定出150个独特的硝化肽段，覆盖102种蛋白质，估计错误发现率为3.3%。
