1.以穩定同位素稀釋奈升流速奈電噴灑游離串聯式質譜法分析人體中外環性DNA加成產物以及血紅蛋白之加成產物. 2.利用硝基酪胺酸免疫親和性管柱搭配奈升流速奈電噴灑游離串聯式質譜法分析唾液蛋白的硝化與亞硝化轉譯後修飾

1.Quantification Of Exocyclic DNA Adducts And Hemoglobin Adduct In Human By NanoLC-NSI/MS/ MS.2.Post-translational Nitration And Nitrosylation Of Human Salivary Proteins Identified By 3-NT Immunoaffinity Column coupled with nanoLC-NSI/MS/MS

林文棚(Wen-Peng Lin)

指導教授 ： 陳皓君

國立中正大學/理學院/化學暨生物化學系/博士(2012年)

摘要

人類的生活環境中充斥著許多對人體有危害性的具反應性化學物質，當這些具反應性的化學物質進入生物體中，會與去氧核醣核酸或是蛋白質等生物分子進行反應，形成不同修飾結果並造成生物分子的損傷，例如DNA與蛋白質的加成產物以及蛋白質的轉譯後修飾。其中DNA的損傷可能會導致DNA的結構不穩定、染色體異常、複製時的鹼基配對錯誤等突變情況，甚至會引起癌症等疾病的發生；至於蛋白質的轉譯後修飾則會導致蛋白質功能喪失影響細胞功能，在疾病過程中扮演著重要的角色。除了外生性的來源外，氧化壓力所誘導的脂質過氧化過程中亦會生成類似的危害物質，是重要的內生性來源。為了瞭解DNA或蛋白質的損傷與疾病之相關性，必須在不同的疾病過程中針對這些生物分子的損傷進行定性與定量分析，才能找出與疾病相關之生物指標。然而不管是DNA、蛋白質加成產物或是蛋白質轉譯後修飾在人體中的含量皆非常的微量，加上生物樣品極為珍貴不容易取得，因此極需要高靈敏度與高準確度的分析儀器來進行低微量分析物之準確定量。奈升流速液相層析奈電噴灑游離串聯式質譜法 (簡稱nanoLC-NSI/MS/MS)的問世解決了微量分析之靈敏度與準確度的問題，隨著液相層析儀分析管柱內徑的微小化，降低了樣品需求；而質譜儀的發展中，奈電噴灑游離源改進了電噴灑游離時分析物離化效率低的問題，加上串聯式質譜儀的使用，搭配高特異性之選擇反應偵測的分析模式 (簡稱SRM)，使得nanoLC-NSI/MS/MS的分析系統提供了高靈敏度與高準確性的優點。在本論文所描述的研究中，可劃分成三個部分。第一個部分是針對人體DNA中由丙烯醛、丁烯醛與具反應性環氧化物所反應形成的DNA加成產物；第二部分則是在人類血紅蛋白中分析由乙醛所產生的蛋白質乙基化加成產物；至於第三部分為針對唾液蛋白中硝化與亞硝化等轉譯後修飾的研究。在前兩部分研究中，除了利用nanoLC-NSI/MS/MS的分析系統搭配SRM模式之外，分析流程中也藉由添加穩定同位素內標準品，提升了定量分析的靈敏度與分析過程中分析物的回收率。第三部分中，主要是利用3-硝基酪胺酸免疫親和性管柱，在複雜的唾液樣品中純化出具有硝化與亞硝化的唾液蛋白，經過蛋白質水解過程後利用nanoLC-NSI/MS/MS進行蛋白質的辨識。本論文中著重在分析方法的開發，儘管目前所偵測的生物樣品皆來自於健康的捐贈者，但我們期待此發展之分析技術可以應用在疾病中尋找生物指標，並運用在疾病不同過程中的診斷以及預防。

關鍵字

奈升流速液相層析奈電噴灑游離串聯式質譜法

並列摘要

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.

並列關鍵字

nanoLC-NSI/MS/MS

參考文獻

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