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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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。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，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。氧化后的5-HTP产物与血管紧张素形成加合物。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。用平行LC/EC-LC/MS系统分析了共轭去肌红蛋白的结构，发现氧化后的5-HTP产物在m/z为1884.061的片段上形成加合物。我们发现，离线的EC合成池可以有效地产生5-HTP的氧化产物和与蛋白质的反应产物。加合物的特征是通过LTQ-Orbitrap上的向下测序和肽图。在第二项研究中，目前正在进行临床试验的治疗HD的药物苯丁酸钠的代谢物，以及SPB治疗后升高的内源性代谢物，在治疗过程中检测了患者血浆和尿样中的代谢物。该系统作为研究氧化还原反应和代谢途径的模型，这些反应和代谢途径是疾病状态的结果。一篇详细的论文描述了未知代谢物和其他成分的鉴定，这些成分在对照组和患者样本之间存在差异，发表在《分析生物化学》(EN Ebbel等人，肛门生物化学)上。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.).