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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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。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。我们研究了氧化的5-HTP与血管紧张素和马apomyoglobin的相互作用，使用新的高容量电化学（EC）合成细胞和平行LC/EC-LC/MS。5-HTP的样品在各种电位的EC合成细胞与血管紧张素和马apomyoglobin氧化。 发现最佳氧化电位为500 mV。 血管紧张素与5-HTP在EC合成细胞中离线反应。 收集洗脱液，并在平行LC/EC-LC/MS系统（ESA CoulArray 4通道EC系统和反相柱，配有Sciex QStar QoTOF MS）上进行分析。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。氧化的5-HTP的建议反应位点在酪氨酸的间位。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（ESA CoulArray 4通道EC系统和具有Applied Biosystems Q-Trap 2000 MS的反相柱）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（m/z 203.058和217.037）在m/z 1884.061的片段上形成加合物。 我们已经发现，离线EC合成细胞可以有效地用于生产5-HTP的氧化产物和5-HTP与血管紧张素和马脱辅基肌红蛋白的反应产物。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。一份描述SPB研究的手稿已经发表，另一份描述未知代谢物和其他组分的鉴定（在对照和患者样本之间存在差异）的手稿已提交出版。

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.