1. Analysis of DNA adducts, 3-nitrotyrosine and 3-bromotyrosine by mass spectrometry.2. Study of post-translational modifications of lysozyme induced by oxanine

Autor: Wei-Loong Chiu, 邱偉倫
Rok vydání: 2007
Druh dokumentu: 學位論文 ; thesis
Popis: 95
1(a). Analysis of 1,N 6-ethenoadenine and 1,N2-ethenoguanine in human body by mass spectrometry Abstract Etheno DNA adducts are promutagenic DNA lesions derived from exogenous as well as endogenous sources. The levels of etheno adducts in tissue DNA are elevated in cancer prone tissues, and the urinary excretion of etheno adducts is associated with oxidative stress. 1,N 6-ethenoadenine and 1,N2-ethenoguanine are promutagenic lesion detected in tissue DNA. it has been shown that these adducts can be repaired by human DNA glycosylases, and they are expected to be excreted in urine. In this study, we have developed for the accurate quantifi-cation of eAde and 1,N 2-eGua in human urine samples by the highly sensitive and specific stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometric assay (GC/NICI/MS) and liquid chromatography-electrospray ionization/ tandem mass spectrometry. (LC-ESI/MS/MS). After adjusting for creatinine levels and body weight, statistically significant associations were observed between urinary levels of these two adducts and cigarette smoking. Three highly specific and sensitive assays based on mass spectrometry should be valuable in measuring urinary etheno adducts as a potential noninvasive biomarker for oxidative DNA damage.During analysis of eAde and/or eCyt in DNA by GC/MS or LC/MS, adenosine deaminase is used to convert adenine or deoxyadenosine to the deaminated analog. It is due to the fact that abundant unmodified adenine or deoxy-adenosine could interfere with analysis of the trace amount of etheno adducts and that adenine and deoxyadenosine have the same mass as those of eCyt and its dexoyribonucleoside, respectively. We herein report identification of a glycosylase activity for eAde, but not eCyt, as a contaminant in a commercialized adenosine deaminase preparation. Enzyme hydrolysis of chloroacetaldehyde treated calf thymus DNA and human placental DNA followed by adenosine deaminase treatment yielded ede determined by isotope dilution GC/NICI/MS and LC/ESI/MS/MS. 1(b). Simultaneous quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography/ electrospray ionization-tandem mass spectrometry Abstract Upon stimulation by invasion of pathogens, reactive species are generated by activated phagocytes, which can damage normal tissue and contribute to inflammatory injury. Myeloperoxidase and eosinophil peroxidase are abundant leukocyte proteins which catalyze oxidation of nitrite and Br－ by H2O2 to produce nitrating and brominating agents. Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In this study, an assay based on isotope dilution liquid chromatography/electrospray ionization-tandem mass spectrometry was developed for simultaneous analysis of 3NT and 3BT in human urinary proteins as noninvasive biomarkers for protein nitration and bromination in vivo. The study is the first measureing 3BT level in human urinary protein by LC/MS/MS. The detection limits (S/N =3) of this assay were 9.3 pg for 3NT and 5.0 pg for 3BT. This assay also allows quantification of 20 pg of 3NT and 3BT in 1.0 mL of the urine sample, which is equivalent to corresponding the concentration quantification limit of 88 pM (3NT) and 77 pM (3BT). Using this assay, the levels of 3NT and 3BT ranged from 1.6 ± 0.1 to 39 ± 3 3NT / 105 Tyr and 0.3 ± 0.01 to 16 ± 1.7 3BT / 103 Tyr in the 24 h urinary protein samples of healthy individuals. After levels of 3NT and 3BT were adjusted for creatinine contents, a statistically significant correlation was found between 3BT level of urinary protein and cotinine, the metabolite of nicotine in human urine. To the best our knowledge, this study is the first measurement of 3BT levels in human urinary protein by LC/MS/MS. 2. Study of post-translational modifications of lysozyme induced by oxanine Abstract Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion derived from the guanine base with nitric oxide and nitrous acid. It was shown by gel electrophoresis that oxanine mediated the formation of DNA-protein cross-links (DPCs) with DNA-binding proteins and in the cell extract. Although 2’-deoxyoxanosine was shown to react with amines including the N-terminal amino group of glycine, the structures of DNA-protein cross-links induced by oxanine have not been characterized. In this study, we chose lysozyme as a protein model to study the modification of DNA-protein cross-links and identified the oxanine modified residues by using HPLC coupled with tandem mass spectrometry. Our results revealed that the side chain groups of Lys-13, Lys-97, Lys-116, Ser-85 and Ser-86 of lysozyme were reactive toward 2’-deoxyoxanosine, and the major form of modification is modified by oxanine molecule but not 2’-deoxyoxanosine. In addition, only three residues (Lys-116, Ser-85 and Ser-86) are reactive with dOxo-DNA. According the side chain group exposed area and secondary structure of hen egg white lysozyme, we propose that the modification between dOxo-DNA and lysozyme correlates with the extents of solvent exposure of amino acid in protein. Because the repair mechanism of DPCs is not extensively investigated, the characterization of oxanine-derived DPC structures should facilitate studies of their detection in vivo and their biological consequences.
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