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研究生: 蘇怡瑄
Su, Yi-Hsuan
論文名稱: 利用SWATH及iTRAQ技術進行小鼠腎臟中塑膠微粒毒性之差異蛋白質體學分析
Differential Proteomic Analysis of Microplastic Toxicity in Mouse Kidney by SWATH and iTRAQ Technology
指導教授: 陳頌方
Chen, Sung-Fang
口試委員: 陳頌方
Chen, Sung-Fang
蕭伊倫
Hsiao, I-Lun
陳百昇
Chen, Pai-Sheng
梁恭豪
Liang, Kung-Hao
口試日期: 2023/07/17
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2023
畢業學年度: 111
語文別: 中文
論文頁數: 107
中文關鍵詞: 塑膠微粒SWATHiTRAQ蛋白質體學
英文關鍵詞: microplastic, SWATH, iTRAQ, proteomics
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202301090
論文種類: 學術論文
相關次數: 點閱:68下載:0
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  • 根據聯合國的數據,每年有超過800萬噸的塑料垃圾被丟棄於海洋。這些塑膠垃圾會逐漸分解成塑膠微粒,最終可能積聚在人類食物鏈中。已有相關研究指出,塑膠微粒會積累在肝臟、腎臟和腸道等器官中。本研究使用了SWATH和iTRAQ兩種差異蛋白質技術,分析塑膠微粒投餵組和對照組的蛋白質體學。為了減少樣品的複雜度,腎臟蛋白質樣品經胰蛋白酶水解過後的胜肽,先經鹼性逆相層析法分離,接著進行奈米液相層析串聯質譜分析(nanoLC-MS/MS)。SWATH和iTRAQ在相對定量方面具有相似的趨勢,並分別成功定量了1984和2162個蛋白質,塑膠微粒投餵組和對照組之間有顯著差異的蛋白質有489個。透過GO及Metascape分析,發現這些差異表達的蛋白質主要與細胞過程、代謝過程和生物調節途徑有關。未來這些蛋白質有望成為塑膠微粒引起的腎損傷的潛在生物標記物,這項研究為塑膠微粒誘導腎損傷的機制提供新的見解。

    According to the United Nations, more than 8 million tons of plastic waste are discarded in ocean every year. It will be physically or chemically decomposed into microplastics, which may eventually accumulate in the human food chain. It has been reported that it will accumulate in organs such as liver, kidney and intestine. Two differential proteomic technologies, SWATH and iTRAQ, were used to investigate protein profiles in microplastic-treated groups and control group in this study. To reduce sample complexity, the proteins from kidney samples were tryptic digested and fractionated by alkaline reverse phase chromatography, followed by nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The SWATH and the iTRAQ gave similar analytical performance. Overall, 1984 and 2162 proteins were successfully quantified by the SWATH and the iTRAQ, respectively. Microplastic-treated groups showed significant differences in 489 proteins treated and control groups. Based on the Gene Ontology and Metascape analysis, these differential expressed proteins were found to be mainly related to cellular process, metabolic process, and biological regulation pathways. These proteins are expected to serve as potential biomarkers of microplastic-induced kidney injury.

    第一章 序論 1 第一節 前言 1 第二節 環境中的塑膠微粒 2 第三節 塑膠微粒對生物體的影響 4 第四節 塑膠微粒誘導之動物實驗 6 第五節 質譜儀技術 8 第六節 電灑游離法(Electrospray Ionization, ESI) 9 第七節 四極桿飛行時間質譜儀(Quadrupole-Time of Flight mass spectrometry, Q-TOF) 11 第八節 掃描模式 13 一、數據依賴採集模式(Information/Data-dependent Acquisition, IDA/DDA) 13 二、數據非依賴採集模式(Data-independent Acquisition, DIA) 14 第九節 液相層析分離技術 16 一、鹼性逆相層析法(Alkaline reverse phase chromatography) 17 第十節 蛋白質身分鑑定 18 一、胜肽質量指紋(Peptide Mass Fingerprinting, PMF) 18 二、胜肽碎片指紋(Peptide Fragment Fingerprinting, PFF) 19 第十一節 差異蛋白質體學 20 一、二維凝膠電泳(Two-Dimensional gel Eletrophoresis, 2-DE) 21 二、同重元素相對與絕對定量(isobaric Tags for Relative and Absolute Quantitation, iTRAQ) 22 三、SWATH (Sequential Window Acquisition of all Theoretical Mass Spectra) 24 第十二節 研究動機 25 第二章 實驗材料與方法 27 第一節 小鼠試驗 27 第二節 樣品製備 28 第三節 樣品純化濃縮 29 第四節 蛋白質濃度測定(BCA protein assay) 30 第五節 蛋白質水解(In-solution digestion) 31 一、蛋白質水解(In-solution digestion) 31 二、蛋白質水解和化學標定iTRAQ試劑 32 第六節 第一維分析策略 33 第七節 自製C18離心管柱去鹽(C18 spin column) 35 第八節 奈升級超高液相層析電噴灑串聯質譜儀(NanoLC-ESI tandem mass spectrometry) 37 一、奈升級超高效液相層析儀(NanoAcquity UPLC system, Waters) 37 二、四極桿飛行時間混成質譜儀(TripleTOF 6600+ system, Sciex) 38 第九節 數據分析 43 一、分析軟體 43 二、資料庫搜尋參數 43 第三章 結果與討論 46 第一節 小鼠試驗重覆劑量毒性評估 46 一、體重變化 46 二、血清生化 47 三、組織病理切片 48 第二節 蛋白質樣品濃度測定 49 第三節 第一維液相分離-鹼性液相層析法 51 第四節 蛋白質鑑定及SWATH資料庫建立 52 第五節 相對定量分析 54 一、基於SWATH方法的相對定量分析 54 二、基於iTRAQ方法的相對定量分析 56 第六節 SWATH與iTRAQ結果比較 58 第七節 生物途徑分析 61 一、Gene Ontology分析 61 二、Metascape分析 63 三、差異蛋白質功能分析 66 第四章 結論與未來展望 76 參考文獻 77 附錄 86

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