Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA.
J Chromatogr A. 2012 Apr 6;1232:266-75. doi: 10.1016/j.chroma.2011.12.100. Epub 2012 Jan 9.
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.
蛋白质硝化已被认为是与各种疾病相关的氮氧化应激的一个重要生物标志物。虽然鉴定硝化的蛋白质靶标很重要，但为了了解这种低丰度翻译后修饰的生物学影响，还需要对其进行定量分析。我们之前报道了一种有效的、简单的硝化肽富集方法，可减少样品复杂性，并通过 LC-MS 分析允许明确的位点特异性鉴定。该方法依赖于两个化学衍生化步骤：特别是脂肪族胺的还原甲基化，然后将硝基酪氨酸转化为相应的氨基酪氨酸，然后再用我们之前引入的固相试剂选择性捕获。因此，该方法通过使用甲醛的同位素变体进行还原甲基化，为硝化肽的相对定量提供了内在机会。该简单方法通过 LC-MS 分析不同 N-甲基化硝化肽和硝化泛素进行了测试，硝化泛素分别作为从人血清白蛋白消化物和人血浆中富集的模型硝化蛋白。
