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。 亨廷顿病(HD)是一种以运动和精神障碍为特征的疾病。晚期的临床表型包括舞蹈动作和痴呆症的增加。先前的研究表明HD患者体内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，而这一途径中的其他中间体起到神经保护作用。初步研究表明，在HD中，氧化的5-羟色氨酸(5-HTP)产物可能与蛋白质结合。我们利用新型高容量电化学合成池和平行LC/EC-LC/MS研究了氧化的5-HTP与血管紧张素和马的脱脂蛋白的相互作用，旨在确定神经退行性疾病的生物标志物。5-羟色胺的样品在EC合成室中以血管紧张素和马去肌红蛋白在不同的电位下氧化。最佳氧化电位为500 mV。血管紧张素在EC合成池中与5-HTP脱机反应。洗脱液在平行LC/EC-LC/MS系统(ESA CoulArray4通道EC系统和反相柱，Sciex QStar QoTOF MS)上收集和分析。氧化的5-羟色胺产物(m/z 219.007和233.056)与血管紧张素形成加合物(图1)。氧化后的5-HTP的反应中心位于酪氨酸的间位。 马去肌红蛋白在EC合成池中与5-HTP反应。随后收集洗脱液并用胰酶消化。用ExPASy计算不同胰酶消化片段的m/z值。利用平行LC/EC-LC/MS系统(ESA Coularray4通道EC系统和反相柱与应用生物系统Q-Trap 2000 MS)对共轭脱脂蛋白的结构进行了研究。氧化的5-羟色胺产物(m/z为203.058和217.037)在m/z为1884.061的片段上形成加合物。建议的反应部位是酪氨酸的间位和苯丙氨酸的邻位、间位或对位取代。我们发现，一个离线的EC合成池可以有效地产生5-HTP的氧化产物以及5-HTP与血管紧张素和马去肌红蛋白的反应产物。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而在生理上发生的氧化还原反应。这方面的一个例子是5-羟色胺及其氧化产物在神经退行性疾病(如HD)的发展中的作用。 参考资料： 1.沃利格，L.，朗莱，P.，马特森，W.，马克，K.，伽马奇，P.，神经病学档案，42,1158-1161,1985。 2.Gamache，P.，Smith，R.，McCarthy，R.，Waraska，J.，Acworth，I.，光谱学，18(6)，14-21,2003 3.Dryhurst，G.，Humphries，K.，《药学杂志》。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. Huntingtons 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.