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

这个子项目是众多研究子项目之一

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. Huntingtons disease (HD) is a disorder characterized by motor and psychiatric dysfunction. Clinical phenotypes at advanced stages include increased development of choreic movements and dementia. 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 damage1 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. This study is directed at determining biomarkers in neurodegenerative disease. 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 (Figure 1). 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. M/z values for various tryptic digest fragments of apomyoglobin were determined calculated using ExPASy. 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. The suggested reaction site was at the meta-position on tyrosine and substitution on phenylalanine at either the ortho, meta, or para-positions. 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. Also, the results also showed that the parallel LC/EC-LC/MS system can be used to monitor oxidation products of 5-HTP with various proteins. This system serves as a model for investigating redox reactions that occur physiologically as a consequence of disease state. An example of this would be the role of 5-HTP and its oxidation products in the development of neurodegenerative diseases such as HD. References: 1. Volicer, L., Langlais, P., Matson, W., Mark, K., Gamache, P., Archives of Neurology, 42, 1158-1161, 1985. 2. Gamache, P., Smith, R., McCarthy, R., Waraska, J., Acworth, I., Spectroscopy, 18(6), 14-21, 2003. 3. Dryhurst, G., Humphries, K., Journal of Pharmaceutical Sciences. 76(10), 839-847, 1987.