高質量解析基質輔助雷射脫附飛行時間質譜儀之發展及其於醣分子分析之應用

Abstract

本論文主要致力於構建一完整之分析流程以改善基質輔助雷射脫附游離質譜法 (MALDI MS) 於生物醣分子之分析能力。第一章從樣品製備著手，介紹醣分子之基本架構及質譜分析方法，詳細討論了於 MALDI 中，常用於分析醣分子之樣品製備方法，並對其製備流程進行最佳化，以獲得最高之偵測靈敏度。 第二章探討一醣分子裂解之新技術，本裂解技術於樣品製備完成後進行光處理反應，待光處理結束後再進入質譜儀進行分析，因此可適用於所有配置 MALDI 游離源之質譜儀。與商業儀器提供之常規裂解技術相比，本方法之簡易性更高、泛用性更廣。而本實驗也將此裂解技術應用至多種醣分子樣品，結果表明本技術可有效地獲得醣分子之母離子及其裂解片段離子，其中包含豐富的醣環內裂解之資訊，對醣分子結構鑑定極有助益。此外，本實驗也選定多個醣分子異構物，透過本裂解技術加以分辨，更加展現了本技術對醣分子異構物辨別之優異性能。 第三章針對飛行時間 (TOF) 質譜儀之儀器架構進行詳細討論，透過理論模型，重新檢視傳統線性 TOF 質譜儀之儀器架構，進而改善其設計缺陷。根據此理論模型，本實驗亦建構出一優化之線性 MALDI TOF 質譜儀來加以驗證，透過系統性地最佳化各個實驗參數，可有效地獲得高品質且具高質量解析度之質譜圖。本研究也將此線性 MALDI TOF 質譜儀應用於多種不同質量之生物分子樣品，結果顯示此質譜儀具有高質量解析性能及高動態範圍能力。本實驗不僅驗證了本理論模型，並且證實經優化之線性 TOF 質譜儀可成為具潛力之生物分析工具。

This thesis is dedicated to evaluating the potential and the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for biomolecular analysis, particularly for carbohydrates. Chapter 1 introduces the general functions and roles of carbohydrates as well as the nomenclatures for their representation. The fundamentals of mass spectrometry are also mentioned in detail. Furthermore, the commonly used sample preparation methods for carbohydrate analyses in MALDI are also discussed and optimized for acquiring the best ion sensitivity. The present procedures with excellent ionization efficiency are then applied on several important native carbohydrate analytes, their results are carefully compared and discussed. Chapter 2 introduces a new concept of fragment-based analyses for carbohydrates in MALDI MS. In contrast to regular fragmentation method provided by commercialized instrument, the present fragmentation strategy is suitable for all MALDI-based instrument. The fragmentation technique introduced herein presents as a sample pretreatment method which decomposing the analytes prior to the analysis of mass spectrometry. By this strategy, the intact ions and their fragments of carbohydrate can be effectively obtained and can generate unambiguous fingerprints for characterization. This allows reliable and fast identification of common carbohydrate motifs. In addition, six native carbohydrate isomers were investigated to systematically examine which structural elements of carbohydrates can be differentiated. Furthermore, the general experimental aspects that need to be considered during this analysis are discussed in detail. Chapter 3 discusses the instrumental aspects for enhancing mass resolving power (Rm) in linear TOF MS. Different instrumental factors were carefully optimized according to a comprehensive calculation model to extend the ability and performance for biomolecule analyses. The established linear MALDI TOF MS was then applied to several important biomolecules with diverse sizes. Complex standard sample such as bacteria test sample (BTS) was also tested to reveal the high dynamic range of the instrument. This shows the robustness of the present approach and that the lab-built linear MALDI TOF MS can be a versatile and highly valuable analytical tool for carbohydrate and other bio-analyses.