含胺類代謝物的化學衍生物與液相層析質譜分析策略

Abstract

逆向色層分析質譜法是分析含胺基代謝物的主要鑑定與定量方法。許多胺基代謝物因為高極性與低濃度而不易偵測，因此科學家開發了許多衍生化方法來加以改進。但是如何選擇最佳的衍生化策略，至今仍缺乏有效的準則。 論文第一部分針對五種常用的胺基衍生化試劑進行了系統性比較：Dansyl-Cl, ophthalaldehyde(OPA)、Fmoc-Cl、Dabsyl-Cl 與Marfey 試劑。在三種不同層析pH 條件下（2.6、5.0、8.0），比較了吸收度、螢光、層析分離效率與離子化效率。同時在串聯式質譜中以不同的碰撞能量比較裂解情形。將這五種衍生化方法，分析中藥黃連，可偵測到數百種胺基代謝物。五種方法偵測到的代謝物不盡相同，沒有單一試劑效果特別優異。唯有結合多種試劑，才能盡量提升偵測的覆蓋率。這些比較數據對於未來的代謝體實驗設計提供了可靠依據。 許多胺基代謝物具有掌性異構物，不易使用層析質譜法分離鑑。在論文第二部分，設計了以左旋DiCys配合OPA的新穎衍生化策略，用來分離掌性異構物。並與文獻中使用的五種掌性硫醇分子進行比較：N-acetyl-L-cysteine (NAC)、N-acetyl-D-penicillamine (NAP)、isobutyryl-L-cysteine (IBLC)、N-(tert-butoxycarbonyl)-L-cysteine methyl ester (NBC)與N-(tertbutylthiocarbamoyl)-L-cysteine ethyl ester (BTCC)。由分析結果來看，DiCys與IBLC的分離效果最佳，螢光訊號也最強。因此後續利用DiCys與IBLC對於洗米水中的胺基酸掌性異構物進行定量分析。 穩定同位素標定技術是代謝體學中質譜定量的重要工具。現有的胺基衍生化同位素標定試劑具有合成難度高或是售價昂貴等缺點。論文的第三部分設計了一種新穎的同位素標定iv技術，以DiCys與OPA的反應作為基礎，僅需非常簡單的合成方法，即可獲得十三重（13-plex）的標定試劑。在此選定了其中的四重試劑進行合成（+0 Da、+3 Da、+6 Da、+9 Da），並且尋找最佳標定條件，探討衍生化產物的穩定性。之後以酪蛋白水解產物驗證了此四重標定試劑的定量準確性，並應用於四種稻米的洗米水中的胺基酸掌性異構物之定量。

Reversed phase-liquid chromatography-mass spectrometry (RP-LC-MS) has become the standard analytical technique for the identification and quantification of amine-containing metabolites. However, many amine metabolites are challenging to detect due to strong polarityand low abundance. Many derivatization techniques have been introduced to assist their analyses by LC-MS, but there still lacks a clear guideline for selecting the appropriate derivatization method. In the first part of this thesis, five popular amine-derivatization reagents are selected for inter-comparison: Dansyl-Cl, o-phthalaldehyde (OPA), Fmoc-Cl, Dabsyl-Cl, and Marfey’s reagent. Their absorbance, fluorescence, chromatographic separation, and ionization properties are systematically compared under three pH conditions 2.6, 5.0, and 8.0. Their MS/MS fragmentation patterns are also examined under different collision energies. The relative stregnths ans shortcomings of each reagent is discused. These five reagents are applied to the analyses of Coptis chinensis, a Chinese medical herb, revealing hundreds of amine metabolites identified through MS/MS analyses. None of the five methods stands out as being clearly superior, and their compound coverage profiles are clearly different. The combination of multiple derivatization reagents is required for comprehensive coverage. These comparison data provide useful guidelines for choosing a suitable derivatization strategy for efficient metabolomics experiments. Many amine metabolites are chiral, but to resolve their enantiomeric counterparts is a challenging task for RP-LC-MS analyses. In the second part of the thesis, N,N-dimethyl-L-cysteine (DiCys)/OPA is introduced as as a novel derivatization method for chiral amines. Its performance is compared to five chiral thiols used in previous literature, including N-acetyl-L-cysteine (NAC), Nacetyl- D-penicillamine (NAP), isobutyryl-L-cysteine (IBLC), N-(tert-butoxycarbonyl)-L-cysteine methyl ester (NBC), and N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC). Among them, DiCys and IBLC give the best results for resolving a panel of D- and L-amino acids, as well as the strongest fluorescence. DiCys and IBLC are then applied to resolve amino acid enantiomers in rice water . Differential isotope labeling is an essential technique in quantitative metabolomics. However, the amine-reactive isotope labeling reagents currently available are either challenging to synthesize or costly to purchase. The third part of this thesis attempts to design an isotope labeling scheme based on OPA and DiCys. Based on straightforward synthetic procedures, 13-plex labeling reagents may be easily obtained (0~12 Da). As proof of principle, the 4-plex version has been synthesized (+0 Da, +3 Da, +6 Da, and +9 Da). Moreover, the optimal condition for OPA/DiCys derivatization and the properties of the resulting adduct have been investigated. The 4-plex labeling assay is successfully applied to the amino acid analysis of hydrolyzed casein protein and four different rice water samples.