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

蛋白质和氧化神经递质相互作用的 LC/EC-LC/MS 研究

基本信息

批准号：
7955958
负责人：
WAYNE R MATSON
金额：
$ 7.09万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-01 至 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7955958
关键词：
5-Hydroxytryptophan Angiotensins Binding Biological Markers Biology Butyrates Cells Chorea Clinical Treatment Clinical Trials Computer Retrieval of Information on Scientific Projects Database Dementia Development Disease Equus caballus Free Radicals Functional disorder Funding Grant Huntington Disease Institution Manuscripts Mass Spectrum Analysis Medicine Metabolic Pathway Modeling Motor Neurodegenerative Disorders Neurotransmitters Oxidation-Reduction Pathway interactions Patients Peptide Mapping Pharmaceutical Preparations Phase Plasma Play Positioning Attribute Proteins Publications Publishing Reaction Research Research Personnel Resources Role Sampling Serotonin Site Sodium Source Staging Structure System Trypsin Tyrosine United States National Institutes of Health Urine adduct apomyoglobin clinical phenotype novel oxidation oxidative damage

项目摘要

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. This study is directed at determining biomarkers in neurodegenerative disease. 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 damage 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. 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. 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. 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. 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. Adducts are being characterized by to-down sequencing on the LTQ-Orbitrap and by peptide mapping. In a second study, the metabolites of sodium phenyl butyrate, a drug currently in clinical trials for treatment of HD, and endogeneous metabolites elevated by SPB treatment, were determined in plasma and urine samples of patients over the course of treatment. This system serves as a model for investigating redox reactions and metabolic pathways that occur as a consequence of disease state. One manuscript describing the SPB studies has been published, and another, that describes the identification of unknown metabolites and other components that vary between controls and patient samples, has been submitted for publication.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。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。这项研究旨在确定神经退行性疾病的生物标志物。先前的研究表明，HD 中氧化损伤标记物的水平升高。有证据表明，血清素途径中的中间体会产生自由基并导致氧化损伤，并且该途径中的其他中间体发挥神经保护作用。初步研究表明氧化 5-羟色氨酸 (5-HTP) 产物可能与 HD 中的蛋白质结合。我们使用新型高容量电化学 (EC) 合成池和平行 LC/EC-LC/MS 研究了氧化 5-HTP 与血管紧张素和马脱肌红蛋白的相互作用。 5-HTP 样品在 EC 合成池中用血管紧张素和马脱肌红蛋白在多种电位下进行氧化。发现最佳氧化电位为 500 mV。血管紧张素在 EC 合成池中与 5-HTP 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 系统和带有 Sciex QStar QoTOF MS 的反相色谱柱）上进行分析。氧化 5-HTP 产物（m/z 219.007 和 233.056）被发现与血管紧张素形成加合物。氧化 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。使用平行 LC/EC-LC/MS 系统来阐明结合的脱辅基肌红蛋白的结构（ESA CoulArray 4 通道 EC 系统和配有 Applied Biosystems Q-Trap 2000 MS 的反相色谱柱）。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。我们发现离线EC合成池可以有效地用于生产5-HTP的氧化产物以及5-HTP与血管紧张素和马璜肌红蛋白的反应产物。加合物通过 LTQ-Orbitrap 上的自上而下测序和肽图谱进行表征。在第二项研究中，在治疗过程中测定了患者血浆和尿液样本中苯丁酸钠（目前正在临床试验中治疗 HD 的药物）的代谢物以及 SPB 治疗升高的内源性代谢物。该系统可作为研究因疾病状态而发生的氧化还原反应和代谢途径的模型。一份描述 SPB 研究的手稿已发表，另一份描述未知代谢物和对照样本与患者样本之间存在差异的其他成分的鉴定的手稿已提交出版。