LC/EC-LC/MS Studies of Protein and Oxidized Neurotransmitter Interactions

摘要

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. 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. To model these reactions, 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. The application presented here is directed at determining biomarkers in neurodegenerative disease. Briefly, 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.