| Summary: | 博士 === 國立中正大學 === 化學暨生物化學研究所 === 100 === Human living environment fills with many reactive chemicals, and may react with biological molecules such as DNA or protein. It causes biological molecules damage and forms DNA, protein adducts and post-translational modification of proteins. DNA lesions may lead to the instability of the DNA structure, base pairing mismatching, and even cause the occurrence of cancer. The protein posttranslational modification may result in the protein dysfunction and affects cell function as well as plays an important role in the disease process. In addition to the exogenous source, the endogenous source such as oxidative stress-induced lipid peroxidation process also generate similar reactive chemicals. In order to understand the relationship of DNA or protein damage and disease. It should be done for qualitative and quantitative analysis of these biomolecules damage and identify disease-related biomarker. However, whether DNA, protein adducts or protein posttranslational modification, the content exist in the human body at low levels. In addition, precious biological sample is also not easy to obtain. Nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was delevoped to overcome the problem. With decreasing diameter of the analytical column reduce the sample requirements and the development of the mass spectrometer, nanospray ionization source improve the low efficiency of analyte ionization on conventional electrospray ionization. In addition, coupling with the selective reaction monitoring (SRM) promote high sensitivity and high accuracy on nanoLC-NSI/MS/MS analysis system. In the paper, the study can be divided into three parts. The first part is simultaneous detection and quantification of exocyclic DNA adducts in human placenta, leukocytes and saliva DNA; The second part is measuring the stable N-terminal N-ethylvaline (Et-Val) hemoglobin adduct after hydride reduction of the unstable Schiff base adduct. The third part is posttranslational modification study of nitration and nitrosation in salivary proteins. In addition to nanoLC-NSI/MS/MS analysis system with the SRM mode, the isotope internal standards were added in samples enhancing the accurate analysis and analytes recovery at previous two parts of study. For third part, a 3-nitrotyrosine immunoaffinity column was used to isolate nitrated and nitrosylated salivary proteins prior to desalting and trypsin digestion. The peptides were analyzed on a nanoflow liquid chromatography nanospray linear ion trap mass spectrometry and SEQUEST database searching to identify the sequence and modification sites. These paper focuses on the development of analytical methods. Although the use of biological samples were from healthy donors, but we expect that these analytical techniques can be applied to find noninvasive biomarkers for DNA and protein damage resulting from oxidative stress and for evaluating their roles in disease formation and prevention.
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