以穩定同位素稀釋法搭配奈升流速液相層析奈電噴灑游離串聯式質譜儀分析癌症病患DNA中多個DNA加成產物

Analysis of Multiple DNA Adducts in Cancer Patients by Stable Isotope Dilution Nanoflow UPLC-Nanospray Ionization Tandem Mass Spectrometry

王學鈞(Hsueh-Chun Wang)

指導教授 ： 陳皓君

國立中正大學/理學院/化學暨生物化學系/碩士(2014年)

摘要

本實驗室利用穩定同位素稀釋法並搭配奈升流速奈電噴灑游離串聯式質譜儀 (nanoLC-NSI/MS/MS)選用其中具有高特異性及高選擇性的反應偵測模式 (H-SRM)，用來評估癌症病患檢體中DNA加成產物的含量是否與癌症具有關聯性，進而作為癌症風險評的指標。所分析的DNA加成產物包含外環性DNA加成產物，其中與脂質過氧化相關的三個乙烯基加成產物1,N6-etheno-2'-deoxyadenosine (εdAdo)、3,N4-etheno-2'-deoxycytidine (εdCyd)、1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo)與丙烯醛、丁烯酸所形成的兩個1,N2-propano-2'-deoxyguanosine (AdG與CdG)、氧化性DNA加成產物8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG)以及與外生性環境暴露相關的乙基化DNA加成產物N7-ethylguanine (7-EtG)、 O2-, O4-, N3-ethylthymidine (edT)、與體內慢性發炎導致的內生性鹵化胞嘧啶5-chloro-2'-deoxycytidine (5-Cl-dC)與5-bromo-2'-deoxycytidine (5-Br-dC)。本研究分成兩部分。第一部分為分析五位第四期口腔癌病患唾液中的DNA，檢視其中外環性DNA加成產物及7-EtG共六種DNA加成產物的含量並與健康者做比較。第二部分為分析食道癌、胃癌、大腸癌病患的腫瘤組織與周圍的正常組織，用以比較上述的12種DNA加成產物在腫瘤及正常組織之間含量的差異。本研究的兩個部分，分別用來評估這些DNA加成產物是否可做為癌症風險評估的生化指標，以及研究在腫瘤形成過程中DNA加成產物所扮演的角色，且所需檢體量少，每次分析僅需25 μg癌症病患的DNA就可同時偵測多個DNA加成產物。

關鍵字

奈升流速液極致相層析奈電噴灑游離串聯式質譜法

並列摘要

We used stable isotope dilution method by nanoflow ultra performance liquid chromatography-nanospray ionization tandem mass spectrometry (nanoUPLC-NSI/MS/MS) under the highly-selective reaction monitoring (H-SRM) mode to analyze multiple DNA adducts in cancer patients DNA to examine the role of these adducts in cancer formation and their potential as the cancer biomarkers. We focus on four groups of DNA adducts, namely exocyclic adducts, oxidized adduct 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG), ethylated adducts, and halogenated adducts. Exocyclic adducts include three lipid peroxidation-related etheno adducts 1,N6-etheno-2'-deoxyadenosine (εdAdo), 3,N4-etheno-2'-deoxycytidine (εdCyd), 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) and two 1,N2-propano-2'-deoxyguanosine derived from acrolein (AdG) and crotonaldehyde (CdG). Ethylated adducts include, O2-, O4-, N3-ethylthymidine (edT), and N7-ethylguanine (7-EtGua). The halogenated adducts are 5-chloro-2'-deoxycytidine (5-Cl-dC) and 5-bromo-2'-deoxycytidine (5-Br-dC). These DNA adducts are associated with atherosclerosis, inflammation, oxidative-stress-induced DNA damage, environmental and chemical exposed. In the first part of the study, we quantified the levels of the five exocyclic DNA adducts and 7-EtGua in salivary DNA of five 4th stage oral cancer patients and five healthy donors. In the second part, we analyzed the twelve adducts in DNA from esophageal, gastric, and colorectal cancer patients’ tumor and normal tissue. Only 25 micrograms of DNA was use for each analysis, this highly sensitive, specific, and accurate nanoUPLC-NSI/MS/MS assay might be clinically feasible for simultaneous quantification of these adducts as potential biomarkers of cancer diagnosis and to investigate the role of these adducts in cancer etiology.

並列關鍵字

nanoUPLC-NSI/MS/MS

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