PROTEIN 3-NY ID METHOD OF AFFINITY LABEL, SOLID PHASE CAPTURE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 3-硝基酪氨酸是一种翻译后修饰，由活性氮(主要是过氧亚硝酸根)与酪氨酸反应产生，在生理条件下存在，在许多疾病中含量增加。因此，蛋白质中的3-硝基酪氨酸修饰是氧化应激的一个潜在的生物标志物。许多具有这种修饰的蛋白质(如前列环素合成酶和锰超氧化物歧化酶)表现出其酶活性的变化。在化学修饰的蛋白质中，经常有相同的酪氨酸被修饰。这些结果表明，在修饰过程中可能存在一定的特异性。在目前可用的方法中，只有质谱仪能够特定地定位这些位置。然而，只有一小部分特定蛋白质的种群可能会被修改，这使得检测变得困难。即使对于丰富的、几乎完全修饰的蛋白质，基于硝基酪氨酸或靶蛋白质特定亲和力的纯化也是必要的。 亲和捕获的一种方法包括用连二亚硫酸钠将硝基酪氨酸还原为氨基酪氨酸，然后在降低的pH下与胺特定的生物素标记(磺基-NHS-S-S-生物素)反应。氨基酪氨酸的pKa为4.7(而其他蛋白质胺的pKa约为7~11)。当标记反应在降低的pH(例如，pH 5)下进行时，非酪氨酸胺的活性要小得多。一旦标记的蛋白质被消化，标记的多肽就可以用亲和素/链霉亲和素柱捕获，并通过还原标记中的二硫键进行洗脱。在我们手中，这种溶液相标记方法被证明具有有限的灵敏度。固相捕集已被证明比溶液标记后亲和捕获具有更高的回收率，并且有可能省去几个纯化步骤，潜在地提高方法的灵敏度。尝试了几种基质和连接物策略，但商业上可获得的硫醇捕获树脂的改进版本被证明产生了最好的结果。首先使用合成的含有硝基酪氨酸的胰酶BSA多肽对所测试的树脂的化学成分进行了评估。以四硝基甲烷处理的牛血清白蛋白(BSA)为模型蛋白，用MALDI-TOF MS对标记多肽进行了分析。结果表明，用化学方法捕获含硝基酪氨酸的多肽是可行的，比溶液相标记和捕获的灵敏度提高了10倍，但该方法的灵敏度可能仍不足以检测生物体系中的内源性硝基酪氨酸水平。这种方法代表着朝着表征复杂蛋白质混合物中特定的硝基酪氨酸修饰的目标迈出了重要的一步，但还需要进一步的优化。这个项目构成了海贝克博士论文的核心，他现在已经接受了PNNL的博士后职位。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 3-Nitrotyrosine is a post-translational modification produced by the reaction of reactive nitrogen species (predominantly peroxynitrite) with tyrosine, present under physiological conditions, and in increased amounts in many diseases. Thus, 3-nitrotyrosine modification in proteins is a potential biomarker for oxidative stress. Many proteins exhibiting this modification (e.g. prostacyclin synthase and manganese superoxide dismutase) display a change in their enzymatic activity. In chemically modified proteins often times the same tyrosines are modified. These results suggest that there may be some specificity in modification. Of the currently available methods, only mass spectrometry is able to locate these sites specifically. However, only a relatively small portion of the population for a specific protein may be modified, which makes detection difficult. Even for abundant, almost fully modified proteins a nitrotyrosine or target protein specific affinity-based purification is necessary. One method of affinity capture involves the reduction of nitrotyrosine with sodium dithionite to aminotyrosine followed by reaction with an amine specific biotin label (Sulfo-NHS-S-S-Biotin) at reduced pH. The pKa of aminotyrosine is 4.7 (compared to the pKa of other protein amines being ca. 7 to 11). When the labeling reaction is performed at a reduced pH (e.g., pH 5), non-tyrosine amines are much less reactive. Once the labeled protein is digested, the labeled peptides can then be captured with an avidin/streptavidin column and eluted by reduction of the disulfide bond in the label. In our hands, this solution phase labeling method proved to have limited sensitivity. Solid-phase capture has been shown to have a higher recovery than solution-phase labeling followed by affinity capture and has the potential to eliminate several purification steps, potentially increasing the sensitivity of the method. Several matrices and linker strategies were attempted, but a modified version of a commercially available thiol capture resin proved to yield the best results. The chemistry of the resins tested was first evaluated by using a synthetic tryptic BSA peptide with an incorporated nitrotyrosine. The tagged peptide was analyzed using MALDI-TOF MS. To test the biological applicability of the resin, tetranitromethane treated bovine serum albumin (BSA) was used as a model protein. The results indicated that the chemistry for the capture of nitrotyrosine containing peptides is feasible, and 10-fold sensitivity increase over solution phase labeling and capture was achieved, but the sensitivity of the method may still not be adequate to detect endogenous levels of nitrotyrosine in a biological system. This method represents a significant step toward the goal of characterizing specific nitrotyrosine modifications in complex protein mixtures, but further optimization is needed. This project formed the core of the Phd thesis of Mr. Heibeck, who has now accepted a postdoctoral position at PNNL.