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研究生:黃詠筠
研究生(外文):Yung-Yun Huang
論文名稱:氧化錳鐵磁性奈米粒子結合質譜術應用於磷酸化蛋白質分析
論文名稱(外文):The Manganese Ferrite Magnetic Nanoparticles in Combination with Mass Spectrometry for the Analysis of Protein Phosphorylation
指導教授:蕭鶴軒
指導教授(外文):He-Hsuan Hsiao
口試委員:李茂榮何銘益
口試委員(外文):Maw-Rong LeeMing-Yi Ho
口試日期:2016-07-12
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:83
中文關鍵詞:質譜技術磷酸化蛋白質錳鐵奈米粒子
外文關鍵詞:Mass SpectrometryPhosphoproteinManganese Ferrite Magnetic Nanoparticles
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摘要
生物樣品的分析往往伴隨著極為複雜基質因而影響偵測,此時一個強而有力的分析工具對研究而言是很重要的。質譜技術具備高選擇性與高靈敏度的特性儼然成為現代生化分析的最佳利器之一。蛋白質磷酸化 (Protein phosphorylation) 為常見且重要的轉譯後修飾 (Post-Translational Modifications,PTMs) 之一。 藉由蛋白質的磷酸化與去磷酸化調控細胞中許多的生理變化,因此研究蛋白質磷酸化一直都是個熱門的研究主題。由於生物體中磷酸化蛋白含量較少,且磷酸根帶負電荷特性會降低質譜正離子模式下分析之感度,因此需開發出一簡單、快速且具高回收率與高選擇性的方法來純化磷酸化蛋白質。
本研究利用水熱法合成出MnFe2O4奈米粒子,建立於過度金屬與磷酸根之間螯合的原理,藉以純化磷酸化胜肽片段。MnFe2O4奈米粒子應用在純化磷酸化胜肽中具有良好的靈敏度與純化專一性,即使樣品濃度在500 attomol的條件下,也能分離出微量的磷酸化胜肽,即便樣品中存在大量未經磷酸化修飾的胜肽片段,於BSA和β-casein莫爾數比10:1的條件下也能夠專一的純化出磷酸化胜肽片段,此外奈米粒子對樣品的承載量為3.6 μg/mg,並且回收率高於85%。應用於真實樣品牛乳、血清與細胞裂解蛋白質分析中,分別成功地純化出18條、3條以及1331條磷酸化胜肽片段。此外MnFe2O4奈米粒子也易於保存,在空氣或水溶液中皆不易因氧化作用而質變,可以保有良好的磁性,因其磁性,純化的操作時,只須外加磁場即可將磁性奈米粒子與液體分離,相較於傳統固相式微萃取法,使用上更為便利。


Abstract
Analysis of biological samples frequently has accompanied with the complex matrices that impacts the identification and quantification of biological analytes. Therefore, a powerful mass spectrometry-based tool for research is necessary, due to its high sensitivity and high selectivity. Protein phosphorylation is one of the most important post-transitional modifications (PTMs), which plays a key role in the regulation of many cellular processes, such as phosphorylation enzymes, kinase and phosphatase are critical in the modulation of cellular transduction pathway. Over the past decade, the investigation of protein phosphorylation have been one of most popular research topics. However, the major challenges for phosphoproteomic analysis include the low abundance of phosphoproteins in biological samples and the ion suppression of phosphopeptide signals due to the large amount of non-phosphoryated peptides. For this reason, a sensitive and selective pretreatment method is essential for the facile and rapid analysis of protein phosphorylation.
In this study, MnFe2O4 magnetic nanoparticles (MNPs) were synthesized by using one-pot hydrothermal method for the rapid and selective enrichment of phosphorylated peptides. MnFe2O4 MNPs possessed high selectivity and high selectivity for the isolation of phosphopeptides based on chelation interaction. The phosphopeptides derived from ultralow concentration β-casein (500 attomol) could be captured with MnFe¬2O4 MNPs and even in a mixture of BSA peptides and β-casein peptides mixture at molar ratio 10:1, the phosphopeptides were still isolated as well. The bind capacity and recovery for this method are 3.6 μg/mg (amount of proteins/amount of MNPs) and over 85%, respectively. To demonstrate the performance of MnFe2O4 MNPs, tryptic digests of milk, human serum and total cell lysate were employed as the complex mixture. We successful enriched 18 phosphopeptides from milk, and identified 3 endogenous phosphopeptides from human serum, 1331 phosphopeptides from total cell lysate as well. The workflow for the enrichment is similar to solid phase extraction (SPE) but the procedure is more convenient than traditional SPE. In brief, strong points of MnFe2O¬4 MNPs are the facile fabrication, durable and inexpensiveness, and possess high sensitivity and high specificity for the isolation of low concentration of phosphopeptides from the complex biological samples.


目錄
摘要………………………………………………………………….….
Abstract………………………………………………………….………
圖目錄………………………………………………………...……...…
表目錄……………………………………………………………..……
第一章、 緒論……………………………………………….……..….1
1.1蛋白質體學介紹……………………………...……………………1
1.2蛋白質轉譯後修飾介紹…………………………………………..4
1.3磷酸化轉譯後修飾分析…………………………………………..5
1.4磁性奈米粒子簡介……………………………………………..…8
1.4.1有限小尺寸效應………………………………...………..…8
1.4.2表面效應……………………………………..……………...9
1.4.3磁性奈米粒子合成……………………………………....….9
1.5質譜儀簡介及其原理………………………………………..…..10
1.5.1基質輔助雷射脫附游離法介紹…………………………...10
1.5.2飛行時間質量分析器………………………………..……..15
1.5.3奈米電灑游離法介紹…………………………………..….17
1.5.4四極矩軌道阱串聯質譜儀……………………………...….18
1.6研究動機……………………………………………………..…..22
第二章、 實驗材料與實驗流程……………………………..………23
2.1蛋白質樣品、前處理及磷酸化蛋白純化試劑……………....….23
2.2實驗器材………………………………………………………....24
2.3基質輔助雷射脫附飛行時間是串聯質譜儀設備與試劑……….25
2.4液相層析串聯質譜儀設備與試劑……………...…………..…....25
2.5樣品前處理…………………………………………………….....25
2.5.1 藥品配置…………………………………………..…..…....25
2.5.2 牛血清蛋白與β牛乳酪蛋白之水解………………….…....26
2.5.3 牛乳蛋白水解……………………………………………....26
2.5.4 人類腎上上皮細胞 (HEK293) 蛋白質萃取與水解.......…27
2.6 錳鐵奈米粒子合成……………………………………………….30
2.7 β牛乳酪蛋白磷酸化胜肽片段之純化…………………………...30
2.8 質譜分析與數據分析…………………………………………….33
2.8.1 MALDI-TOF分析……………………………………….…...33
2.8.2 Nano-UPLC 結合Q ExactiveTM Plus 分析………………….33
2.8.3 資料庫搜尋及比對之參數…………………………………..35
第三章、 結果與討論………………………………………...……...36
3.1 磁性奈米粒子鑑定………………………………………………36
3.1.1 MnFe2O4 磁性奈米粒子大小鑑定………………………36
3.1.2 MnFe2O4磁性奈米粒子離子鍵鑑定………………………39
3.1.3 MnFe2O4磁性奈米粒子晶格鑑定…………………………41
3.1.4 MnFe2O4磁性奈米粒子磁力鑑定…………………………44
3.2 純化磷酸化蛋白條件探討………………………………………46
3.2.1 螯合時間條件探討………………………………………..46
3.2.2 沖提條件探討……………………………………………..49
3.2.3 MnFe2O4磁性奈米粒子最大吸附量測試…………………52
3.2.4內標準品添加量探討……………………………………...54
3.2.5 萃取回收率測試…………………………………………..57
3.3 MALDI-TOF分析條件與質譜參數探討………………………...59
3.4 MALDI-TOF質譜圖分析………………………………………...62
3.4.1 β-casein胰蛋白酶水解胜肽片段分析……………………...62
3.4.2混合β-casein與牛血清白蛋白BSA水解胜肽片段分析...65
3.4.3 真實樣品:牛奶蛋白中的水解磷酸化胜肽分析…………..69
3.4.4真實樣品:人類血清中內源性磷酸化胜肽分析……………73
3.5胰蛋白酶水解人類腎臟上皮細胞全蛋白方法選擇與純化…….76
第四章、 結論………………………………………………...……...80
第五章、 參考文獻……………………………………………...…...81


圖目錄
圖一、蛋白質體質譜分析流程圖..............................................................3
圖二、實驗總體流程圖..............................................................................7
圖三、三種MALDI基質的化學結構與晶體形狀………..………....…14
圖四、飛行時間質量分析器內部結構……………………….……...…16
圖五、四極矩質量分析器……………………………......………….….20
圖六、軌道阱質量分析器………………………………….……….…..20
圖七、Q ExactiveTM Plus內部結構示意圖………………………...……21
圖八、磷酸化胜肽純化流程…………………………………............…32
圖九、不同水熱條件下合成MnFe2O4磁性奈米粒子比較…………....38
圖十、MnFe2O4磁性奈米粒子之FT-IR圖譜………………………....40
圖十一、MnFe2O4磁性奈米粒子XRD圖譜………………………...…42
圖十二、MnFe2O4粒子ICSD資料庫……………………………….…..42
圖十三、MnFe2O4磁性奈米粒子磁滯曲線圖………………………….45
圖十四、不同螯合時間下上清液中胜肽之質譜圖…….………….…..48
圖十五、m/z 2061磷酸化胜肽片段不同沖提條件的探討…………….51
圖十六、m/z 3122磷酸化胜肽片段不同沖提條件的探討…………….51
圖十七、MnFe2O4磁性奈米粒子最大吸附量趨勢圖………………….53
圖十八、Sweet spot effect……………………………………………….56
圖十九、內標準品添加量比較圖………………………………...…...56
圖二十、磷酸化胜肽回收率測試圖………………………………......58
圖二十一、不同MALDI參數與磷酸化胜肽訊號強度比較………...61
圖二十二、β-casein 50 fmol以MnFe2O4奈米粒子純化前後MALDI質
譜圖……………………...……………………………...…63
圖二十三、β-casein 0.5 fmol以MnFe2O4奈米粒子純化前後MALDI
質譜圖…………………..……………………………....…64
圖二十四、BSA與β-casein 莫爾數比1:1混合樣品以MnFe2O4奈米
粒子純化前後MALDI質譜圖….………….....………….66
圖二十五、BSA與β-casein 莫爾數比10:1混合樣品以MnFe2O4奈
米粒子純化前後MALDI質譜圖………..…………….…67
圖二十六、BSA與β-casein 莫爾數比100:1混合樣品以MnFe2O4奈
米粒子純化前後MALDI質譜圖…………….................…68
圖二十七、真實樣品-牛乳蛋白水解胜肽以MnFe2O4奈米粒子純化前
後MALDI質譜圖……………………………………...…70
圖二十八、真實樣品-人類血清中內源性胜肽以MnFe2O4奈米粒子純
化前後MALDI質譜圖…………………………………...74
圖二十九、全細胞蛋白所鑑定出的胜肽片段之比較圖…………..…77
圖三十、全細胞蛋白所鑑定出的蛋白質之比較圖…………………..77
表目錄
表一、Nano-LC沖提梯度表………………………………………….....34
表二、XRD對應晶格平面及其對應角度………………………..…….43
表三、使用MALDI游離法鑑定以MnFe2O4奈米粒子純化出牛乳中的
酪蛋白磷酸化胜肽…………………………………………..…71
表四、使用ESI與MALDI游離法比較以MnFe2O4奈米粒子純化出牛
乳中的酪蛋白磷酸化胜肽…………………………..………....72
表五、經磁珠純化後人類血清中磷酸化胜肽片段之序列………..…75


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