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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 3-硝基酪氨酸是由活性氮物质（主要是过氧亚硝酸盐）与酪氨酸反应产生的翻译后修饰，在生理条件下存在，并且在许多疾病中数量增加。 因此，蛋白质中的3-硝基酪氨酸修饰是氧化应激的潜在生物标志物。 许多表现出这种修饰的蛋白质（例如前列环素合酶和锰超氧化物歧化酶）显示出其酶活性的变化。 在化学修饰的蛋白质中，通常相同的酪氨酸被修饰。 这些结果表明，修饰可能具有一定的特异性。 在目前可用的方法中，只有质谱法能够具体定位这些位点。 然而，对于特定蛋白质，只有相对小部分的群体可以被修饰，这使得检测困难。 即使对于丰富的、几乎完全修饰的蛋白质，硝基酪氨酸或靶蛋白特异性的基于亲和力的纯化也是必要的。 一种亲和捕获的方法包括用连二亚硫酸钠将硝基酪氨酸还原成氨基酪氨酸，然后在降低的pH下与胺特异性生物素标记（磺基-NHS-S-S-生物素）反应。7至11）。 当标记反应在降低的pH下进行时（例如，pH 5），非酪氨酸胺的反应性低得多。一旦标记的蛋白质被消化，标记的肽可以用亲和素/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。 固相捕获已被证明具有比液相标记随后亲和捕获更高的回收率，并且具有消除几个纯化步骤的潜力，从而潜在地增加该方法的灵敏度。尝试了几种基质和接头策略，但市售硫醇捕获树脂的改性版本证明产生最佳结果。 首先通过使用具有掺入的硝基酪氨酸的合成胰蛋白酶BSA肽来评价测试的树脂的化学性质。 使用MALDI-TOF MS分析标记的肽。为了测试树脂的生物适用性，使用四硝基甲烷处理的牛血清白蛋白（BSA）作为模型蛋白。结果表明，用于捕获含硝基酪氨酸的肽的化学是可行的，并且实现了比溶液相标记和捕获高10倍的灵敏度，但是该方法的灵敏度可能仍然不足以检测生物系统中硝基酪氨酸的内源性水平。这种方法代表了一个重要的一步，在复杂的蛋白质混合物中的特定硝基酪氨酸修饰的特征的目标，但需要进一步优化。这个项目构成了Heibeck先生博士论文的核心，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.