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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。我们利用新型的高容量电化学合成池和平行LC/EC-LC/MS研究了氧化后的5-HTP与血管紧张素和马去肌红蛋白的相互作用。最佳氧化电位为500 mV。血管紧张素在EC合成池中与5-HTP脱机反应。洗脱液在平行LC/EC-LC/MS系统(ESA CoulArray4通道EC系统和反相柱，Sciex QStar QoTOF MS)上收集和分析。氧化的5-羟色胺产物(m/z为219.007和233.056)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。利用平行LC/EC-LC/MS系统(ESA Coularray4通道EC系统和反相柱与应用生物系统Q-Trap 2000 MS)对共轭脱脂蛋白的结构进行了研究。氧化的5-羟色胺产物(m/z为203.058和217.037)在m/z为1884.061的片段上形成加合物。我们发现，一个离线的EC合成池可以有效地产生5-HTP的氧化产物以及5-HTP与血管紧张素和马去肌红蛋白的反应产物。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。去年发表了一份描述SPB研究的手稿。在当前时期，一篇详细的论文描述了未知代谢物和其他成分的鉴定，这些成分在对照和患者样本之间存在差异，发表在分析生物化学(EN Ebbel等人，使用平行液相色谱/电化学阵列/质谱仪和离线串联质谱仪鉴定亨廷顿病患者中苯丁酸酯生成的代谢物)上。肛门生物化学。2010,399,152-161。)。

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 was published last year. In the current period, 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., Identification of phenylbutyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography/electrochemical array/mass spectrometry and off-line tandem mass spectrometry. Anal Biochem. 2010, 399, 152-161.).