LC/EC-LC/MS STUDIES OF PROTEIN AND OXIDIZED NEUROTRANSMITTER INTERACTIONS

这个子项目是许多研究子项目中的一个 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。 亨廷顿病（HD）是一种以运动和精神功能障碍为特征的疾病。晚期的临床表型包括舞蹈动作和痴呆的发展增加。先前的研究表明HD中氧化损伤标志物水平升高。有证据表明，5-羟色胺途径中的中间体产生自由基并导致氧化损伤1，该途径中的其他中间体发挥神经保护作用。最初的研究表明，氧化5-羟色氨酸（5-HTP）的产品结合蛋白质在HD的可能性。我们研究了氧化的5-HTP与血管紧张素和马apomyoglobin使用新的高容量电化学（EC）合成细胞和平行LC/EC-LC/MS的相互作用。这项研究是针对确定在神经退行性疾病的生物标志物。5-HTP样品在EC合成细胞中与血管紧张素和马脱辅基肌红蛋白在各种电位下氧化。 发现最佳氧化电位为500 mV。 血管紧张素与5-HTP在EC合成细胞中离线反应。 收集洗脱液，并在平行LC/EC-LC/MS系统（ESA CoulArray 4通道EC系统和反相柱，配有Sciex QStar QoTOF MS）上进行分析。发现氧化的5-HTP产物（m/z 219.007和233.056）与血管紧张素形成加合物（图1）。氧化的5-HTP的建议反应位点位于酪氨酸的间位。 马脱辅基肌红蛋白在EC合成池中与5-HTP反应。 随后收集洗脱液并用胰蛋白酶消化。使用ExPASy测定和计算脱辅基肌红蛋白的各种胰蛋白酶消化片段的M/z值。 使用平行LC/EC-LC/MS系统（ESA CoulArray 4通道EC系统和具有Applied Biosystems Q-Trap 2000 MS的反相柱）来阐明缀合的脱辅基肌红蛋白的结构。发现氧化的5-HTP产物（m/z 203.058和217.037）在m/z 1884.061的片段上形成加合物。建议的反应位点是在间位上的酪氨酸和取代苯丙氨酸的邻位，间位，或对位。 我们已经发现，离线EC合成细胞可以有效地用于生产5-HTP的氧化产物和5-HTP与血管紧张素和马脱辅基肌红蛋白的反应产物。 同时，实验结果还表明，该平行LC/EC-LC/MS系统可用于监测5-HTP与多种蛋白质的氧化产物。该系统作为一个模型，用于研究作为疾病状态的结果生理发生的氧化还原反应。这方面的一个例子是5-HTP及其氧化产物在神经退行性疾病如HD的发展中的作用。 参考文献： 1.沃莱塞湖Langlais，P.，马特森，W.，Mark，K.，Gamache，P.，Archives of Neurology，42，1158 - 1161，1985. 2. Gamache，P.，史密斯，R.，麦卡锡河，Waraska，J.，阿克沃思岛Spectroscopy，18（6），14 - 21，2003. 3. Dryhurst，G.，汉弗莱斯，K.，药学科学杂志. 76（10），839 - 847，1987.

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. Huntington's disease (HD) is a disorder characterized by motor and psychiatric dysfunction. Clinical phenotypes at advanced stages include increased development of choreic movements and dementia. Previous studies showed increased levels of oxidative damage markers in HD. There is evidence that intermediates in the serotonin pathway produce free radicals and contribute to oxidative damage1 and that other intermediates in this pathway play a neuroprotective role. Initial studies suggested the possibility that oxidized 5-hydroxytryptophan (5-HTP) products bind to protein in HD. We studied interactions of oxidized 5-HTP with angiotensin and equine apomyoglobin using novel high capacity electrochemical (EC) synthesis cells and parallel LC/EC-LC/MS. This study is directed at determining biomarkers in neurodegenerative disease. Samples of 5-HTP were oxidized at a variety of potentials in an EC synthesis cell with angiotensin and equine apomyoglobin. Optimal oxidation potential was found to be 500 mV. Angiotensin, was reacted with 5-HTP offline in an EC synthesis cell. Eluent was collected and analyzed on the parallel LC/EC-LC/MS system (ESA CoulArray 4 channel EC system and reversed phase column with Sciex QStar QoTOF MS). Oxidized 5-HTP products (m/z 219.007 and 233.056) were found to form adducts with angiotensin (Figure 1). The suggested reaction site of the oxidized 5-HTP is at the meta-position on tyrosine. Equine apomyoglobin, was reacted with 5-HTP in an EC synthesis cell. The eluent was subsequently collected and digested with trypsin. M/z values for various tryptic digest fragments of apomyoglobin were determined calculated using ExPASy. The parallel LC/EC-LC/MS system was used to elucidate structures of conjugated apomyoglobin (ESA CoulArray 4 channel EC system and reversed phase column with Applied Biosystems Q-Trap 2000 MS). Oxidized 5-HTP products (m/z 203.058 and 217.037) were found to form adducts on a fragment with m/z 1884.061. The suggested reaction site was at the meta-position on tyrosine and substitution on phenylalanine at either the ortho, meta, or para-positions. We have found that an offline EC synthesis cell can be used effectively to produce oxidation products of 5-HTP and reaction products of 5-HTP with angiotensin and equine apomyoglobin. Also, the results also showed that the parallel LC/EC-LC/MS system can be used to monitor oxidation products of 5-HTP with various proteins. This system serves as a model for investigating redox reactions that occur physiologically as a consequence of disease state. An example of this would be the role of 5-HTP and its oxidation products in the development of neurodegenerative diseases such as HD. References: 1. Volicer, L., Langlais, P., Matson, W., Mark, K., Gamache, P., Archives of Neurology, 42, 1158-1161, 1985. 2. Gamache, P., Smith, R., McCarthy, R., Waraska, J., Acworth, I., Spectroscopy, 18(6), 14-21, 2003. 3. Dryhurst, G., Humphries, K., Journal of Pharmaceutical Sciences. 76(10), 839-847, 1987.