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

• 批准号: 8365553
• 负责人: WAYNE R MATSON
• 金额: $ 6.46万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365553
• 关键词: 5-Hydroxytryptophan; Analytical Biochemistry; Angiotensins; Anus; Binding; Biological Markers; Biology; Butyrates; Cells; Chorea; Clinical Treatment; Clinical Trials; Dementia; Development; Disease; Equus caballus; Free Radicals; Functional disorder; Funding; Grant; Huntington Disease; Mass Spectrum Analysis; Medicine; Metabolic Pathway; Modeling; Motor; National Center for Research Resources; Neurodegenerative Disorders; Neurotransmitters; Oxidation-Reduction; Paper; Pathway interactions; Patients; Peptide Mapping; Pharmaceutical Preparations; Plasma; Play; Positioning Attribute; Principal Investigator; Proteins; Publishing; Reaction; Research; Research Infrastructure; Resources; Role; Sampling; Serotonin; Site; Sodium; Source; Staging; Structure; System; Trypsin; Tyrosine; United States National Institutes of Health; Urine; adduct; apomyoglobin; clinical phenotype; cost; liquid chromatography mass spectrometry; oxidation; oxidative damage

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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. Oxidized 5-HTP products 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 Oxidized 5-HTP products 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 with proteins. 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. A detailed paper that describes the identification of unknown metabolites and other components that vary between controls and patient samples, was published in Analytical Biochemistry (EN Ebbel et al., Anal Biochem. 2010, 399, 152-161.).

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 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。发现氧化的5-HTP产物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系统用于阐明氧化的5-HTP产物的共轭磷脂蛋白的结构，它们在M/z 1884.061的片段上形成加合物。我们发现，离线EC合成细胞可有效地用于生产5-HTP和与蛋白质反应产物的氧化产物。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。一篇详细的论文描述了对控制和患者样品之间未知代谢物和其他成分的鉴定，在分析生物化学中发表（En Ebbel等，Anal Biochem。2010，399，152-161。）。