臺灣博碩士論文加值系統

蜂蜜為全球主要的民生食品之一，自古以來就被當作食品或藥物來使用，而蜂蜜的混摻與假冒對於蜂蜜質量安全是消費者普遍關心的問題，也影響蜂蜜產品的出口貿易。蜂蜜的蜜源及產地繁多，導致蜂蜜的理化組成歧異度大，因此提高鑑別的困難度。為了建立蜂蜜真偽鑑定及區分不同蜜源、產地及產國蜂蜜的平台，本研究針對各種蜂蜜進行蛋白質體學研究，探討真偽蜂蜜和不同蜜源、產地及產國蜂蜜之蛋白差異表現。超高效液相層析儀搭配四級棒暨飛行時間式質譜儀 (Ultra-performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry, UPLC-Q-TOF MS) 之質譜分析法，具有高靈敏度及高解析度等優點，可進行蛋白質之定性和定量，SWATH (Sequential window acquisition of all theoretical fragment ion spectra) 非數據依賴擷取 (Data independent acquisition, DIA) 模式為目前最新穎之非標定 (label-free) 質譜定量技術之一，具有良好的準確度、再現性以及完整保留所有母離子和子離子資訊。本實驗主要分為三部分，第一部份為台灣龍眼蜜資料庫的建立，從龍眼蜜中總共鑑定到74個蛋白，包含 27 個蜜蜂蛋白及47 個植物蛋白。從OPLS-DA model 結果可以成功區分台南、台中及彰化三個蜂蜜產地。第二部分，在不同蜜源及產國的結果中，共鑑定到 26 個蜜蜂蛋白及 48 個植物蛋白，於 PCA 及 OPLS-DA model 中皆可成功區分不同蜜源 (龍眼蜜及荔枝蜜) 及產國 (台灣及泰國) 之蜂蜜。第三部分，探討真蜜、調和蜜及不同比例的混和蜜，從 OPLS-DA model 中可以成功辨別真蜜、調和蜜及混摻 10 % 及 20 % 調和蜜的混合蜜。因此，本研究利用 SWATH data-independent acquisition 新階質譜技術進行蜂蜜的蛋白體學分析，建立可以區分蜂蜜真偽和蜜源、產地及產國鑑別的蛋白質資料庫，協助政府或業者提升國產蜂蜜品質，並降低國人對食品安全的疑慮。

Honey is widely consumed by humans. It has the major commercial attention due to its multiple application as a food constituent or therapeutic effects. Although there are several techniques for detecting several forms of syrup used in honey adulteration, the detection of adulteration with rice syrup is still a problem. Because honey has the widely variety of the nectar source and producing region, the physical and chemical composition of honey is difficult to be identified. Therefore, an effective screening technology for the detection of honey adulterated with syrup, producing region and botanical source is urgently needed. Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is a new label-free quantitative proteomic technique. SWATH was developed by the team of Aebersold in 2012. In this study, a simple, rapid and effective UHPLC/Q-TOF-MS method with SWATH label-free acquisition mode for the detection of proteins with different quantity. This research can be divided into three parts. First, a total of 74 proteins were identified in Taiwanese longan honey, among which 27 and 47 proteins were from Apidae and Viridiplantae taxonomy respectively. In results of OPLS-DA analysis using these honey proteomic signatures, three honey from different producing counties (Tainan, Changhua and Taichung) could be successfully distinguished. In the second part, 26 and 48 proteins from Apidae and Viridiplantae taxonomy respectively are identified in the honey from different botanical source and producing countries. In the result of PCA and OPLS-DA model, we successfully distinguish longan and litchi honey; Taiwanese and Thailand honey. Third, we analyzed different proportions of true and adulterated honey with proteomics. Overall, the honey proteomic signatures will help the government and industry for the identification of honey adulterated with syrup, producing country and nectar source.

致謝 i
中文摘要 ii
Abstract iii
目次 iv
表目次 vii
附圖目次 viii
圖目次 ix
英文縮寫對照表 xvi
第一章 緒論 1
第一節 前言 1
第二節 蜂蜜簡介 2
第三節 前人研究 4
3.1 FT-IR (Fourier transform-infrared spectroscopy) 4
3.2 FT-Raman spectroscopy 5
3.3 NMR (Nuclear magnetic resonance) spectroscopy 5
3.4 GC-MS (Gas chromatography mass spectrometry) 5
3.5 SCIRA (Stable carbon isotope ratio analysis) 6
第四節 蛋白質體學 7
4.1 膠體分析方式 (gel-based) 10
4.2 非膠體分析方式 (gel-free) 10
4.3 標定質譜定量技術 (labeling) 11
4.4 非標定質譜定量技術 (label-free) 12
4.4.1 數據依賴擷取掃描模式 13
4.4.2 非數據依賴擷取掃描模式 14
4.5 蜂蜜蛋白質體研究 16
第五節 生物資訊學分析 (bioinformatics) 17
5.1 化學計量學分析 (chemometrics analysis) 17
5.1.1 主成分分析 17
5.1.2 結構正交投影辦別分析法 17
5.2 蛋白質功能性分析 (gene ontology analysis, GO analysis) 18
第六節 研究動機與目的 18
第七節 實驗流程 19
7.1 研究內容 19
7.2 研究策略 20
第二章 研究材料與方法 21
第一節 研究材料 21
第二節 實驗儀器與設備 23
第三節 分析軟體 24
第四節 蛋白質體分析 24
4.1 蜂蜜蛋白質萃取 (honey protein extraction) 24
4.2 蛋白質定量 (protein quantification) 26
4.3 膠體電泳 (gel electrophoresis) 26
4.4 銀染 (silver staining) 28
4.5 膠體內酵素水解 (in-gel digestion) 29
4.6 Zip Tip C18 去鹽 (zip-tip desalting) 31
第五節 液相層析串聯式質譜儀分析 (LC-MS/MS analysis) 31
5.1 超高效液相層析儀搭配四級棒暨飛行時間式質譜儀 (UPLC-Q-TOF MS) 31
5.1.1 層析梯度條件最佳化測試 32
5.1.2 內標準品濃度測試 34
5.1.3 真實樣品上機 34
5.2 即時直接分析法 (direct analysis in real time, DART) 37
5.2.1 DART 搭配線性離子阱質譜儀 (DART-LTQ XL) 38
5.2.2 DART 搭配飛行時間式質譜儀 (DART-TOF MS) 39
第六節 生物資訊學分析 (bioinformatics) 40
6.1 化學計量學分析 (chemometrics analysis) 40
6.1.1 主成分分析 40
6.1.2 結構正交投影辦別分析法 40
6.2 蛋白質功能性分析 (gene ontology analysis, GO analysis) 40
第三章 結果與討論 41
第一節 膠體電泳 41
1.1 11 % SDS-PAGE 41
1.2 28 % SDS-PAGE 43
第二節 液相層析串聯式質譜分析 52
2.1 超液相層析儀搭配四級棒暨飛行時間式質譜儀 (UPLC-Q-TOF MS) 52
2.1.1 蜂蜜蛋白質萃取方法測試 52
2.1.2 層析梯度最佳化測試 53
2.1.3 蛋白質萃取方法穩定度測試 54
2.1.4 內標準品濃度測試 56
2.1.5 真實樣品上機 57
2.1.6 化學計量學分析 61
2.1.7 蛋白質功能性分析 117
2.1.8 具有辨識性之蛋白質探討 119
2.2 即時直接分析法 (direct analysis in real time, DART) 122
2.2.1 DART 搭配線性離子阱質譜儀 (DART-LTQ XL) 122
2.2.2 DART 搭配飛行時間式質譜儀 (DART-TOF MS) 128
第四章 結論 134
參考文獻 154

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