| Increased levels of 3-nitrotyrosine (3-NT), are associated with a broad variety of disease states including rheumatoid arthritis, atherosclerosis, inflammation, colon cancer, pancreatic cancer, Alzheimer's disease and many others. The increase in protein nitration in disease states occurs as a result of increased reactive nitrogen species (RNS). Nitration of proteins has been shown to be selective yet lacking sequence homology making nitration sites difficult to predict based on protein sequence. Current methods for the analysis of protein 3-NT are limited in their ability to yield both quantitative and structural information. Comparative quantitation methods such as isotope coded affinity tag (ICAT) are useful for determining the abundance of protein in one sample relative to another. Two methods for derivatization of 3-NT peptides and proteins for comparative quantitation were developed. The goal was to simultaneously determine sites of protein tyrosine nitration and analyze two samples for comparison of the extent of nitration at particular sites resulting from the physiological state of the samples (ie. aged vs. young, diseased vs. healthy, etc. The first method described in this dissertation involved the reduction of 3-NT to 3-aminotyrosine (3-AT). The amine was coupled with phenyl isothiocyanate (PITC) forming a thiourea (HPPT). The HPPT was then photocyclized to form anilinobenzoxazole. The second method also involved reduction of 3-NT to 3-AT. The 3-AT was derivatized to a fluorescent product, benzylamine phenylbenzoxazole, in the presence of potassium ferricyanide. During method development, a side product of reduction of 3-NT to 3-AT was discovered. An alternative method for the reduction was developed. The method was based on nickel platinum catalyzed sodium borohydride reduction of nitro compounds. Overall, the methods described here offer proof of concept for their utility for comparative quantitation and identification of sites of tyrosine nitration in biological samples. |
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