A. 基質輔助雷射脫附游離法的離子產生機制 B. 邏輯演繹序列串聯質譜法應用於地衣澱粉多醣之結構鑑定

A. Ion Generation Mechanism of Matrix-Assisted Laser Desorption/Ionization (MALDI) B. Structural Determination of Polysaccharide-Lichenan by Logically Derived Sequence Tandem Mass Spectrometry

林厚余(Hou-Yu Lin)

指導教授 ： 林志民

共同指導教授 ： 倪其焜(Chi-Kung Ni)

國立臺灣大學/理學院/化學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200201

摘要

A.
基質輔助雷射脫附游離法已被廣泛地使用於各類型樣品的分析，尤其是應用於高分子量分子的分析，例如高分子化合物或生物分子。由於此方法屬於「軟性」的游離方式，因此分子較容易在游離的過程中維持完整的狀態。自從基質輔助雷射脫附游離法發展以來，有很多模型被提出要解釋離子的生成方法。因此在本論文中將以量測基質的螢光生命期和不同溫度下基質分子的離子產率解釋離子生成的模型。

第一個實驗中我們要驗證一個過去在解釋基質輔助雷射脫附游離法常被提及的S1-S1 annihilation在離子化的過程中，是否為重要的反應機制。在這個實驗中，我們分別量測了12個常用的質基分子的時間解析螢光光譜，其中6個基質有產生S1-S1 annihilation，5個基質不會產生S1-S1 annihilation，1個基質因生命期太短無法確認，代表能量集結模型在基質輔助雷射脫附游離過程中，不是必須的反應途徑。

在過去的研究基質輔助雷射脫附游離法中的一項參數－離子產率，是一個驗證離子化機制的重要參數。根據我們實驗室先前的研究，提出了熱致質子轉移可能是主要的離子化機制。在第二個實驗中我們在不同的樣品起始溫度量測常用的基質分子2,5-DHB的離子產率。實驗的結果顯示了在不同樣品起始溫度改變雷射光通量，其離子產率的比例會符合熱致離子轉移模型的預測有相同的趨勢，因此2,5-DHB為基質在紫外光基質輔助雷射脫附游離法中熱致離子轉移才是離子生成的主要反應。

B.
一種新的質譜法－邏輯演繹序列串聯質譜法應用在多醣的基本結構鑑定。傳統分析多醣的方法包含了許多複雜的過程，例如衍生化、泛甲基化、氣相層析質譜法和核磁共振光譜法可以被邏輯演繹序列串聯質譜法所取代。在這種新的分析法中，多醣被水解成單醣、雙醣和各種不同尺寸的寡醣，然後使用高性能液相層析和邏輯演繹序列質譜法鑑定這些單醣、雙醣和寡醣。本實驗將邏輯演繹序列串聯質譜法應用到地衣澱粉多醣，決定了地衣澱粉的重複單元為An-Bn，A為β-Glc-(1→4)-β-Glc-(1→4)-β-Glc-(1→3)-Glc，B為β-Glc-(1→4)-β-Glc-(1→4)-β-Glc -(1→4)-β-Glc-(1→3)-Glc，n為重複單元數。邏輯演繹序列串聯質譜法能大幅的減少鑑定多醣時所需的時間、心力和樣品量。這種新方法將是鑑定多醣基本結構有力的工具。

關鍵字

基質輔助雷射脫附游離法 ； 熱致質子轉移 ； 螢光光譜 ； 離子產率 ； 邏輯演繹序列串聯質譜法 ； 地衣澱粉 ； 多醣

並列摘要

A.
Matrix-assisted laser desorption/ionization (MALDI) are widely used in mass analysis of various types of samples, for example polymers and biomolecules. This method is a “soft” ionization method, such that the molecules remain intact during ionization. Since the invention of MALDI, many theoretical models have been proposed to explain the primary ion generation mechanism of MALDI. Here, we performed two experiments to explain the ionization mechanism of MALDI. The first experiment was the fluorescence lifetime measurement of matrices, and the second experiment was the ion-to-neutral measurement of matrices at different temperatures.

In the first experiment, we examined a model proposed by previous studies which indicated that the S1-S1 annihilation might be the key process in the generation of primary ions. We investigated the S1-S1 annihilation of 12 matrices by measuring their time-resolved fluorescence spectra. We observed the S1-S1 annihilation in six matrices. We did not observe the S1-S1 annihilation for the other five matrices, while one of the matrices has very short excited-state lifetime for S1-S1 annihilation. The results indicate that the S1-S1 annihilation is not necessary for the ionization mechanism of MALDI.

The ion-to-neutral ratio is an important parameter to examine protonated ion generation mechanism in MALDI. In the second experiment, the initial temperature dependence of the ion-to-neutral ratio was studied. The results agree well with the temperature-dependent ion-to-neutral ratio predicted by the thermal model, indicating that thermally induced proton transfer is the dominant reaction in UV-MALDI of 2,5-DHB.
B.
A new mass spectrometry method, namely logically derived sequence (LODES) tandem mass spectrometry (MSn), was applied to determine the primary structures of polysaccharides.Conventional polysaccharide structural analysis involve complicated procedures, including chemical derivatization, permethylation, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectrometry. These processes can be replaced by the relatively simple method of LODES/MSn. In this new method, polysaccharides were first hydrolyzed into monosaccharides, disaccharides, and various sizes of oligosaccharides, and followed by the structural determination of these monosaccharides, disaccharides, and oligosaccharides using high performance liquid chromatography and LODES/MSn. The application of LODES/MSn on the primary structural determination of polysaccharide lichenan was demonstrated. The repeating unit of lichenan was determined as An-Bn by LODES/MSn, where A represents β-Glc-(1→→4)-β-Glc-(1→4)-β-Glc-(1→3)-Glc, B represents β-Glc-(1→4)-β-Glc -(1→4)-β-Glc-(1→4)-β-Glc-(1→3)-Glc, and n is the number of repeating units. Using LODES/MSn, the time, effort, and the amount of sample needed in structural determination of polysaccharides are greatly reduced compared to the conventional methods. We demonstrated that LODES/MSn can be a powerful tool in the primary structural determination of polysaccharides.

並列關鍵字

MALDI ； thermally induced proton transfer ； flurorescence spectroscopy ； ion-to-neutral ratio ； LODES/MSn ； lichenan ； polysaccharide

參考文獻

第一章 參考文獻

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