Relative quantitation of protein nitration by liquid chromatography-mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation.

Relative quantitation of protein nitration by liquid chromatography-mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation.
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使用同位素编码的二甲基标记和化学沉淀,通过液相色谱-质谱法对蛋白质硝化进行相对定量。

DOI:
10.1016/j.chroma.2011.12.100
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发表时间:
2012
期刊:
Journal of chromatography. A
影响因子:
--
通讯作者:
Prokai,Laszlo
Prokai,Laszlo
中科院分区:
--
文献类型:
--
作者:
Guo,Jia;Prokai-Tatrai,Katalin;Prokai,Laszlo

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蛋白质硝化被认为是与各种疾病相关的氮氧化应激的重要生物标志物。虽然确定硝化作用的蛋白质靶点很重要,但其定量分析对于了解这种低丰度翻译后修饰的生物学影响也是必要的。我们以前曾报道过一种有效和简单的富集方法,硝基肽,以减少样品的复杂性,并允许明确的位点特异性鉴定的LC-MS分析。这种方法依赖于两个化学衍生化步骤:脂肪胺的特异性还原甲基化,然后,硝基酪氨酸转化为相应的氨基酪氨酸之前,通过我们以前介绍的固相试剂的选择性捕获。因此,该方法固有地提供了通过使用甲醛的同位素变体进行还原甲基化来相对定量硝基肽的机会。通过LC-MS分析不同的N-甲基化的硝基肽和nitrobiquitin作为一个模型的硝基蛋白富集从人血清白蛋白消化和人血浆,分别测试这种简单的方法。
Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LC–MS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LC–MS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively.
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