非小細胞肺癌中第一型人類白血球抗原相關胜肽之蛋白基因體分析

Taipei Medical University Institutional Repository > 藥學院 > 臨床藥物基因體學暨蛋白質體學碩士學位學程 > 博碩士論文 > Item 987654321/61432

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題名:	非小細胞肺癌中第一型人類白血球抗原相關胜肽之蛋白基因體分析 Proteogenomics analysis of class I human leukocyte antigen associated peptides in non-small cell lung cancer.
作者:	陳佩汝 CHEN, PEI-RU
貢獻者:	臨床藥物基因體學暨蛋白質體學碩士學位學程韓嘉莉
關鍵詞:	蛋白質基因組學;免疫沉澱法;質譜儀;新生抗原;免疫胜肽組學;非小細胞肺癌 proteogenomics;immunoprecipitation;mass spectrometry;neoantigen;immunopeptidomics;non-small cell lung cancer
日期:	2021-08-26
上傳時間:	2022-03-07 22:40:10 (UTC+8)
摘要:	近年來免疫療法已成為癌症治療的新趨勢，意即利用患者自身的免疫系統辨識呈現於人類白血球抗原(human leukocyte antigen, HLA)上帶有突變的腫瘤抗原後，即會啟動自身的免疫反應來消滅”非我”的癌細胞。這些來自於體細胞突變的新生抗原(neoantigen)，具有高度的腫瘤專一性，很適合作為發展個人化免疫療法的標靶。過去的研究主要透過基因體分析來預測會呈現在人類白血球抗原上之新生抗原，可是準確率並不高，近年發展迅速的質譜分析(mass spectrometric analysis)能提供直接的證據證明真實呈現在人類白血球抗原上的胜肽。在本論文中，我們的目標是建立一個蛋白基因體平台，用於鑑定非小細胞肺癌細胞中第一型人類白血球抗原的相關胜胜。此分析策略結合全外顯子定序(whole exome sequencing, WES)與液相層析質譜儀(liquid chromatography-mass spectrometry, LC-MS)，經由全外顯子定序確認突變位點並轉換為可用於蛋白質鑑定之突變蛋白質序列資料庫；液相層析質譜儀分析則可鑑定純化後的胜肽。為了提高第一型人類白血球抗原胜肽的純化效率，我們對免疫沉澱法(immunoprecipitation)的實驗步驟進行調整與優化，包含親和純化的材料、材料與抗體的比例、洗滌液組合、洗滌次數調整及洗脫酸的選擇。我們使用優化後方法可從PC9細胞株中鑑定到的人類白血球抗原胜肽由48條增加至426條，且大部分的胜肽長度皆介於8至11個胺基酸間，這些胜肽的第二、第三與最終位的氨基酸分佈也符合第一型人類白血球抗原胜肽的特徵。我們將優化後的免疫沉澱法應用於帶有EGFR突變之非小細胞肺癌細胞–H75及H35，可分別鑑定到304條及324條胜肽，且兩個細胞都各自鑑定到一條突變肽。經由預測，它們對人類白血球抗原(HLA-A*01:01)的結合能力為強結合與非結合。此外，將兩個細胞的純化結果與蛋白體分析一起比較發現人類白血球抗原胜肽的強度與蛋白質的表現量有類似的表現趨勢–在H75強度較強的胜肽其來源蛋白質在H75的表現量也較H35高，顯示由免疫沉澱法純化出的胜肽豐度可能與來源蛋白的表現量相關。我們期望此蛋白基因體平台可以應用在各種癌細胞或腫瘤組織成為有效鑑定新生抗原的工具之一，以促進個人化免疫療法的發展。 In the past decade, immunotherapy emerges as a new paradigm in cancer treatment which stimulates immunological elimination of cancer cells by the host’s immune system via recognition of cancer-specific antigens bound to human leukocyte antigen (HLA) molecules on the cell surface. The neoantigens derived from somatic mutations in the cancer cell offer high tumor specificity for developing personalized immunotherapy. However, the cancer genomics analysis cannot predict accurately the presented neoantigens. The mass spectrometric analysis of HLA-binding peptide provides direct evidence for antigen identification. In this thesis, we aim to establish a proteogenomics platform for identification of HLA-binding peptides in the EGFR-mutant non-small cell lung cancer cell (NSCLC). The proteogenomics strategy integrates whole exome sequencing (WES) and liquid chromatography-mass spectrometry (LC-MS)-based immunopeptidomics analyses to identify the HLA class I associated peptides. The WES analysis on the cancer cells identified HLA alleles and generated the mutant protein sequences as customized protein sequence database. We optimized the immunoprecipitation protocol in terms of the material of affinity purification, ratio of beads to antibody, washing buffers, number of washing steps, and elution acids for improving the purification of HLA class I-binding peptides. The purified peptides were analyzed by LC-MS/MS and then the MS/MS data was searched against the sequence database that merges the reference proteins and the mutant protein sequences generated from WES for peptide identification. After optimization, we can improve the identification of HLA-binding from 48 to 426 peptides in PC9. Most of identified peptides have length of 8 – 11 amino acids that match the feature of HLA class I binding peptides and displayed HLA-specific binding motif at position 2, 3 and at terminal position. The optimized protocol was further applied in two EGFR-mutant NSCLC cells, H75 and H35, to identify 304 and 324 peptides, respectively. In the proteome analysis, glycolysis I and gluconeogenesis I are the enriched pathway that related to energy storage and showed overexpression in H75. Two mutant peptides were detected in the two cell lines that were predicted as strong binder and non-binder with HLA-A0101. Additionally, we analyzed the proteomic profiles in these two cell lines and found that the protein expression profiles in two cancer cells had similar expression trend as the intensity of purified HLA-binding peptides. We expect that our established proteogenomics platform would be a better tool for identification of neoantigens in different cancer cells or tumors and facilitate the development of personalized immunotherapy.
描述:	碩士指導教授：韓嘉莉委員：陳璿宇委員：王三源
資料類型:	thesis
顯示於類別:	[臨床藥物基因體學暨蛋白質體學碩士學位學程] 博碩士論文

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