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.

该副本是利用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这不一定是调查员的机构。亨廷顿氏病（HD）是一种以运动和精神功能障碍为特征的疾病。高级阶段的临床表型包括增加的曲折运动和痴呆症的发展。这项研究旨在确定神经退行性疾病中的生物标志物。先前的研究表明，HD中氧化损伤标记的水平增加。有证据表明，在5-羟色胺途径中的中间体产生自由基并导致氧化损伤，并且该途径中的其他中间体起神经保护作用。最初的研究表明，氧化5-羟色氨酸（5-HTP）产物的可能性与HD中的蛋白质结合。我们使用新型的高容量电化学（EC）合成细胞和平行的LC/EC-LC/MS研究了氧化的5-HTP与血管紧张素和马匹阿素蛋白的相互作用。 5-HTP的样品在具有血管紧张素和马替玛蛋白的EC合成细胞中的多种电势处被氧化。发现最佳氧化电位为500 mV。血管紧张素在EC合成细胞中与5-HTP离线反应。在平行的LC/EC-LC/MS系统（ESA Coularray 4通道EC系统和Sciex QStar Qotof MS）上收集和分析了洗脱液。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。氧化5-HTP的建议的反应位点在酪氨酸的元位置处。在EC合成细胞中与5-HTP反应马中果蛋白。随后收集洗脱液并用胰蛋白酶消化。平行的LC/EC-LC/MS系统用于阐明共轭Apomyoglobin的结构（ESA Coularray 4通道EC系统和具有应用生物系统Q-TRAP 2000 ms的反向相柱）。发现氧化的5-HTP产物（M/Z 203.058和217.037）在M/z 1884.061的碎片上形成加合物。我们发现，一个离线EC合成细胞可有效地用于生产5-HTP的氧化产物，以及5-HTP与血管紧张素和马匹磷脂的反应产物。加合物的表征是通过在LTQ-Orbitrap上和肽映射上进行待命的测序来表征。在第二项研究中，在治疗过程中，在血浆和患者的尿液样本中确定了目前正在用于HD治疗的临床试验中的药物，目前正在接受HD治疗的临床试验中的药物的代谢产物。该系统是研究由于疾病状态而发生的氧化还原反应和代谢途径的模型。一项描述了SPB研究的手稿已经发表，另一种描述了对控件和患者样本之间不同代谢产物和其他成分的识别，已提交出版。