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研究生:沈敬傑
研究生(外文):Ching-Chieh Shen
論文名稱:五種偵測含胺類代謝物的衍生化方法之比較
論文名稱(外文):Comparison of five derivatization methods for the detection of amine-containing metabolites
指導教授:戴桓青戴桓青引用關係
指導教授(外文):Tai, Hwan-Ching
口試委員:廖尉斯徐丞志
口試委員(外文):Liao, Wei-SsuHsu, Cheng-Chih
口試日期:2016-07-19
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
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:英文
論文頁數:84
中文關鍵詞:代謝體學液相層析串聯質譜胺基酸衍生化比較研究
外文關鍵詞:metabolomicsliquid-chromatography tandem mass spectroscopyamino acidderivatizationcomparative research
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代謝體學是後基因體世代的新興研究領域,複雜生物樣品的代謝體分析可以反映生物系統內即時的反應狀況,目前估計人體血漿有超過10000種可定量的代謝物,而目前研究代謝體的分析工具主要為液相層析質譜儀。但不是每種代謝物都適合用液相層析質譜分析,而將代謝物做適當的衍生化可以增強分析物的分離效果、游離效率、並利用二次質譜碎裂的圖譜做身份鑑定。在代謝體中許多分子具有胺類官能基,其中包含胺基酸及其衍生物和胜肽,經過文獻搜尋探討,我們鎖定五種胺基衍生化試劑:OPA、Dansyl、Dabsyl、Fmoc-Cl、Marfey試劑來進行分析比對。
在這份研究中,我們用液相層析質譜配合螢光偵測來比較不同胺基衍生化試劑的相對優劣性,藉由在相同儀器和最佳化的條件下,來找出這些試劑適合的實驗條件與實驗目的。我們比較了酸鹼性對螢光、紫外/可見光強度影響、產物疏水性、揮發性鹽類對層析分離效果及質譜游離效率的影響,還有不同衍生化試劑的二次質譜碎裂的能量及碎裂後產生的離子。在過去的文獻中,尚未建立如此系統性的研究。
在這份研究中,我們選擇三種常見的動向組成0.1%甲酸水溶液 (pH 2.6)、2 mM 醋酸銨水溶液 (pH 5)、2 mM 碳酸銨水溶液 (pH 8)當作共同的沖提條件。從實驗結果我們觀察到下列的相對強弱,紫外光可見光吸收 (Dansyl > Fmoc > Marfey > Dabsyl > OPA)、螢光強度 (Fmoc > OPA > Dansyl)、疏水性 (Dabsyl > Fmoc > Dansyl ≈ Marfey ≈ OPA)、游離效果 (Dansyl ≈ Dabsyl > OPA ≈ Fmoc > Marfey)。Fmoc和Dansyl在碰撞誘導碎裂室中都會產生固定荷質比的特徵離子,但OPA和Marfey則是產生失去固定的碎片的特徵離子,Dabsyl碎裂的位置太多因而產生複雜的質譜圖。
經過系統性的比較試劑間的優劣性我們發現Dansyl適合代謝體研究,包含螢光定量和多重反應偵測,Fmoc和Dansyl一樣,都是應用潛力比較大的衍生化試劑。OPA也是泛用的螢光試劑,OPA反應的條件可以再優化,未來值得更深入的探討和研究,而Marfey試劑主要可以應用在鏡像異構物的分離。Dabsyl的應用就比較狹隘,必須視分析目的而定,因為其分子碎裂能裂太高,從這份研究中得到的不同試劑間的比較可以當作一項參考工具,幫助未來想投入突觸代謝體學研究的人,在設計實驗上更順利。


The study of complex metabolites in biological samples is a rapidly advancing field. Tremendous progress has been made using advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques to analyze human metabolomes. It is now believed that human serum alone contains over 10,000 quantifiable metabolites. However, there is no universal LC-MS/MS condition that suits all metabolites. Instead of constantly tuning LC-MS conditions for different metabolites, a better approach is to derivatize metabolites to give them more desirable properties such as better LC separation efficiency, enhanced ionization efficiency, and favorable MS/MS fragmentation patterns.
In this study we focus on identifying optimal derivatization methods for amine-containing metabolites in LC-MS/MS analysis coupled with fluorescence detection. These metabolites may include amino acids, their derivatives, and peptides, which may function as hormones, neurotransmitters, and other signaling molecules in the body. We surveyed a wide variety of amine-derivatization regents and narrowed the candidate list down to five: o-phthlaldehyde (OPA), Dansyl-Cl, Dabsyl-Cl, Fmoc-Cl and Marfey’s reagent. We compared them in terms of absorbance intensity, product hydrophobicity, fluorescence intensity and pH dependence, separation efficiency in reversed-phase LC, ionization efficiency and salt dependence, as well as MS/MS fragmentation energy and fingerprint. To our knowledge such detailed comparisons of amine derivatization methods have never been carried out before.
In this study we compared three general aqueous mobile phase compositions: 0.1% FA (pH 2.6), 2 mM AA (pH 5), 2 mM ABC (pH 8). Under respective optimal eluent conditions, we have observed these general trend in terms of absorbance intensity (Dansyl > Marfey > Fmoc > Dabsyl > OPA), fluorescence intensity (Fmoc > OPA > Dansyl), hydrophobicity (Dabsyl > Fmoc > Dansyl ≈ Marfey ≈ OPA), and ionization efficiency (Dansyl ≈ Dabsyl > OPA ≈ Fmoc > Marfey). Fmoc and Dansyl exhibit characteristic product ions with fixed m/z in collision-induced dissociation cell, while OPA and Marfey show characteristic fixed mass loss in fragment product ions. Dabsyl fragments at many positions to create a complex MS/MS spectrum.
After extensive comparisons, we found that Dansyl shows the greatest potential for a universal derivatization method for metabolomics studies, especially for quantitation by fluorescence and multiple-reaction monitoring. Fmoc is a similarly useful reagent and has the advantage of low collision energy. OPA is a versatile fluorogenic reagent and its chemistry can be fine-tuned using different thiol molecules, which is worth further investigating and optimizing. Marfey’s reagent is useful for the chromatographic separation of enantiomers due to its chiral nature. Dabsyl is very difficult to fragment in MS/MS experiemnts, which may be a strength or a weakness depending on analytical goals. The performance comparisons between different reagents derived from this study can serve as a guide for designing better metabolomics experiments under different contexts.


誌謝 i
中文摘要 ii
ABSTRACT iv
Table of Contents vi
LIST OF FIGURES ix
LIST OF TABLES xii
Abbreviations xiii
Chapter 1 INTRODUCTION 1
1.1 Introduction to metabolomics 1
1.2 LC-MS in metabolomics research 5
1.3 Pre-column derivatization method 9
1.4 Post-column derivatization method 21
1.5 Derivatization-reagent and amino acids selection 25
1.5.1 Derivatization-reagent selection criteria 25
1.5.2 Amino aicds selection criteria 25
1.6 Aim of this study 26
Chapter 2 RESULTS AND DISUSSION 27
2.1 UV/Vis absorbance and Fluorescence intensity assay 27
2.1.1 UV/Vis absorbance assay 27
2.1.2 Fluorescence intensity assay 28
2.1.3 Limit of detection of Fmoc method 30
2.2 Chromatographic performance 31
2.2.1 Retention time (hydrophobicity) 31
2.2.2 Separation performance assay 32
2.3 Ionization efficiency of different derivatization reagents 39
2.4 Tandem mass spectroscopy 41
2.4.1 Fragmentation energy and fragmentation pattern 41
2.5 Discussion 50
2.5.1 General description of five derivitization reagent at its optimal condition 50
2.5.2 Practical considerations 56
Chapter 3 CONCLUSION 59
Chapter 4 MATERIALS AND METHODS 61
4.1 Materials 61
4.1.1 Equipment 61
4.1.2 Reagents 61
4.1.3 Amino acid standards 62
4.1.4 Derivatizing reagent 62
4.2 Methods 63
4.2.1 Derivatization protocol for five different reagents 63
4.2.2 MS/MS experiment 65
4.2.3 LC-MS/MS experiment 64
4.2.4 LOD and LOQ for Fmoc method 66
REFERENCE 67
Appendix 80
Working protocol for derivatizing amino acid 80
1.1.1 Derivatization procedure of OPA method 80
1.1.2 Derivatization procedure of Fmoc method 81
1.1.3 Derivatization procedure of Dansyl method 82
1.1.4 Derivatization procedure of Marfey method 83
1.1.5 Derivatization procedure of Dabsyl method 84

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