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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 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。本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，并有助于氧化损伤，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。发现氧化的5-HTP产物与血管紧张素形成加合物。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。使用平行LC/EC-LC/MS系统来阐明缀合的脱辅基肌红蛋白的结构。发现氧化的5-HTP产物在m/z 1884.061的片段上形成加合物。我们已经发现，离线EC合成池可以有效地用于生产5-HTP的氧化产物和与蛋白质的反应产物。通过LTQ-Orbitrap上的自下而上测序和肽图谱对加合物进行表征。在第二项研究中，在治疗过程中，在患者的血浆和尿液样本中测定了苯丁酸钠（目前用于治疗HD的临床试验药物）的代谢物和SPB治疗升高的内源性代谢物。该系统作为一个模型，用于研究氧化还原反应和代谢途径发生的疾病状态的结果。描述未知代谢物和在对照和患者样品之间变化的其他组分的鉴定的详细论文发表在Analytical Biochemistry（EN Ebbel et al.，Anal Biochem.2010，399，152-161）。

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.).