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系統識別號 U0026-2908201921342400
論文名稱(中文) 利用穩定同位素標定胜肽之液相層析串聯多重反應監測質譜分析證實血液中肺腺癌蛋白質生物標記
論文名稱(英文) Verification of serological lung adenocarcinoma protein markers using liquid chromatography multiple reaction monitoring mass spectrometry
校院名稱 成功大學
系所名稱(中) 環境醫學研究所
系所名稱(英) Institute of Environmental and Occupational Health
學年度 107
學期 2
出版年 108
研究生(中文) 王靖勛
研究生(英文) Ching-Hsun Wang
學號 S76061070
學位類別 碩士
語文別 英文
論文頁數 44頁
口試委員 指導教授-廖寶琦
口試委員-陳皓君
口試委員-張權發
口試委員-馬瀰嘉
口試委員-林隆晟
中文關鍵字 肺腺癌  生物標記蛋白  多重反應監測  穩定同位素標定胜肽 
英文關鍵字 lung adenocarcinoma  protein biomarkers  multiple reaction monitoring  stable isotope-labeled peptides 
學科別分類
中文摘要 肺癌是第二常見的癌症也是癌症死亡的主要原因。在所有肺癌之中,肺腺癌占了大多數。雖然已經有一些肺癌生物標記已經被研究,但是沒有任何適合早期診斷的標記被證實。這導致迫切需要有肺癌更專一的早期偵測的生物標記。依據先前研究共有三十五個穩定同位素標記胜肽被合成並且作為內標準品用在液相層析多反應監測質譜中。整體而言本研究目的為(1)使用穩定同位素標記胜肽的液相層析多反應監測質譜,在血漿中比較肺腺癌病患與健康控制組的潛在生物標記。(2)利用曼-惠特尼U檢定與接收者操作特徵曲線去驗證與早期癌症發展相關的生物標記。(3)使用存活分析去發掘潛在生物標記的其他潛在功能。液相層析多反應監測質譜化驗被用來在血漿中測量一百零二個肺腺癌病患與八十四個健康控制組三十五個候選蛋白的水平。有七個蛋白被認為是有能力將第一期病患與健康控制組區別開來。這些蛋白被整合成一個蛋白標記小組,可以增加區別第一期病患與健康控制組的靈敏度並且以交叉驗證曲線下面積為0.76呈現高度分類表現。此外,真核起始因子的低表現量與光蛋白聚糖的高表現量在整體的生存上有顯著較差的預後(p值分別為0.012 與 0.0074),能作為預後的生物標記。七個蛋白標記的小組在大型的世代研究驗證能作為偵測早期肺腺癌或對未來研究治療方法的發展有幫助。
英文摘要 Lung cancer is the second most common cancer and the leading cause of cancer death in America. Among all diagnosed lung cancer, lung adenocarcinoma is the most common one. Although several lung cancer markers have been studied, no markers suitable for early detection have been validated, which leads to an urgent need to discover more lung cancer-specific biomarkers for early detection. A total of 35 stable isotope-labeled peptides are synthesized based on the previous study and are used as internal standards in liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) analysis. Overall, the specific aims of this study are (1) to compare the potential biomarkers between lung adenocarcinoma patients and healthy control in plasma using LC-MRM-MS with stable isotope-labeled peptides. (2) To verify the biomarkers associated with early development of lung cancer using Mann-Whitney U test and receiver operating characteristic (ROC) curve. (3) To discover other potentials of these biomarker candidates using survival analysis. An LC-MRM-MS assay was used for measuring the level of 35 candidate peptides in plasma from 102 lung adenocarcinoma patients and 84 healthy controls. Seven proteins were found to be able to distinguish stage I patients from controls. These proteins were combined into a protein marker panel which improved the sensitivity to discriminate stage I patients from controls and resulted in a high classification performance with cross-validated area under the curve=0.76. Besides, the low expression of eukaryotic initiation factor 4A-I and high expression of lumican showed significantly poor prognosis in overall survival (p=0.012 and 0.0074, respectively), which may be used as prognostic biomarkers for lung cancer. The seven-protein panel may be used for early detection of lung adenocarcinoma or therapeutics development after validation in a larger cohort.
論文目次 口試合格證明 I
摘要 II
Abstract III
致謝 V
Content VI
List of tables VIII
List of figures VIII
Abbreviation X
1. Introduction 1
1.1 Lung cancer 1
1.2 Early detection of lung cancer 1
1.3 Targeted mass spectrometry in biomarker verification 2
2. Objective 4
3. Materials and methods 5
3.1 Study design 5
3.2 Experiment section 7
3.2.1 Patients and specimens 7
3.2.2 Sample preparation 7
3.2.3 Proteolytic enzyme digestion and addition of internal standard peptides 8
3.2.4 Liquid chromatography coupled with scheduled multiple reaction monitoring mass spectrometry (LC-MRM-MS) 11
3.2.5 LC-MRM-MS Data Processing 12
4. Results and Discussions 14
4.1 Clinical Characteristics 14
4.2 Target peptide selection and assay development or biomarker candidate assessment 16
4.3 Selection of biomarker candidates for diagnosing early stage of lung adenocarcinoma 20
5. Disscusion 27
6. Conclusion 30
References 31
Appendix: 35 peptides transition list 36
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