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研究生： 賴盈宏
Lai, Yin-Hung
論文名稱： Experimental and Theoretical Studies on Ionization Mechanisms of MALDI Mass Spectrometry
基質輔助雷射脫附游離質譜離化機制之 實驗與理論探討
指導教授： 李遠哲
Lee, Yuan-Tesh
林聖賢
Lin, Sheng-Hsien
王亦生
Wang, Yi-Sheng
倪其焜
Ni, Chi-Kung
口試委員: 李遠哲
林聖賢
王亦生
倪其焜
陳仲瑄
林俊利
學位類別： 博士
Doctor
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2012
畢業學年度： 100
語文別： 中文
論文頁數： 106
中文關鍵詞： 基質輔助雷射脫附游離質譜 、離化機制
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    • This dissertation presents experimental and theoretical studies of mechanisms of matrix-assisted laser desorption/ionization (MALDI), encompassing the primary reaction, secondary reaction, and desorption process.
    In the first part, the chemical reactions that affect the primary ionization of matrix in MALDI are addressed. It focuses on relaxations of photon energy on a timescale comparable with that of ionization. These relaxations consume the available energy and change the ionization conditions. They include fluorescence and fragmentation, as well as internal conversion followed by vibration relaxation, which build up the thermal energy in the ground state. With high absorption cross-section and long excited state lifetime, photoionization is important to the production of ions of the matrix; otherwise the photon energy is predicted to be converted to the thermal energy in the ground-state, promoting thermal-induced reaction that includes thermal ionization and fragmentation. The chemical properties of matrices and the excitation conditions alter the branching ratio of the aforementioned reactions. The chemical reactions of four commonly used matrices were discussed with reference to the obtained solid-phase absorption spectra of mixed matrix crystal, fluorescence properties, infrared emission, and abundance of fragments that were detected by a mass spectrometer. Evidence of change in the primary reactions at three laser wavelengths (266, 337, and 355 nm) was systematically analyzed. This concept may explain the diversity of experimental results observed in the MALDI experiments, providing insight into the ensemble of chemical reactions that influence ion production.
    In the second part, we focus on the study of 2,4,6-trihydroxyacetophenone (THAP), in which most of the absorbed energy in this matrix contributes to the non-radiation decay channel and is converted into thermal energy. Evaluation of the thermal contribution to MALDI was studied on THAP-based MALDI. This work demonstrates quantitatively the desorption of ions and neutral molecules in MALDI. Theoretical modeling incorporates transition state theory to predict the desorption of both ions and neutral molecules, assuming that chemical and thermodynamic equilibrium are established in the solid state prior to desorption. The utilized model differs from conventional models that assume chemical equilibrium is established in the gas phase. A quantitative thermodynamic interpretation in the solid state was proposed to predict the desorption of neutral matrix molecules, matrix ions, and analyte ions (angiotensin I) which are embedded in matrix molecules. The variation in ion yield with effective temperature under various conditions of laser fluence and initial temperature is predicted by the thermal model. The analysis also reveals the essential role of ion concentration in the modeling for the best fitting. The divergence of the ion beam with varying laser fluence was examined using an imaging detection method and the signal saturation that was normally observed at high fluence was appropriately reduced by ion focusing. Simplified but deceptive theoretical interpretations were obtained when the analysis was carried out without adequate calibration of the instrument bias. Finally, the mass spectrum of the mixture of THAP and C60 that were irradiated by 450 nm photons that were absorbed by C60 but not by THAP provides further experimental evidence to acknowledge the thermal contribution to proton disproportionation reaction between matrix molecules.

    1. Introduction 1 1.1 Development of MALDI Mass Spectrometry 2 1.2 General Features of MALDI Mass Spectrometry 6 1.2.1 Commonly Used Matrices and Sample Preparation 6 1.2.2 Laser Conditions in Conventional MALDI 7 1.3 Proposed Model for MALDI 9 1.3.1 Primary Reaction 11 1.3.2 Secondary Reaction 18 1.4 Overview 21 2. Primary reaction 23 2.1 Introduction 23 2.2 Experimental Section 26 2.2.1 Spectroscopic Measurements 26 2.2.2 Infrared Emission Detection 27 2.2.3 Lifetime Analysis of First Excited State 27 2.2.4 Mass Spectrometric Analysis 28 2.2.5 Quantum Chemical Calculations 30 2.2.6 Materials 30 2.3 Results and Discussion 30 2.3.1 Rapid Energy Dissipation Pathways in MALDI 30 2.3.2 Mixed Crystal Absorption Spectra 33 2.3.3 Radiative Relaxation – Fluorescence 36 2.3.4 Excited State Fragmentation 38 2.3.5 Non-Radiative Relaxation 39 2.3.6 Photoionization 48 2.3.7 Partitions of Branching Ratio among Matrices 49 2.4 Brief Summary 51 3. Thermal contribution to MALDI 53 3.1 Introduction 53 3.2 Experimental Section 54 3.3 Results and Discussion 59 3.3.1 Divergence of Ion Beam 59 3.3.2 Establishment of Quantitative Model 60 3.3.3 Examination of Reaction Model with Various Temperatures 66 3.3.4 Examination of Thermal Contribution in Proton Disproportionation Reaction 68 3.3.5 Application of Quantitative Model to Two-component System 71 3.4 Brief Summary 75 4. Conclusion and Perspective 76 Appendix A: Supporting Information for Primary Reaction 81 Appendix B Mathematic Formulae of Thermodynamic Model for MALDI Mass Spectrometry 89 Publication List 95 Reference 97

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