Development of Mass Spectrometry-based Analytical Methods for the Analysis of Small Organics, Large Biomolecules, and Bacterial Cells
|關鍵字:||質譜;定量分析;致病菌;奈米粒子;表面輔助雷射脫附游離法;mass spectrometry;quantitative analysis;pathogenic bacteria;nanoparticle;surface-assist laser desorption/ionization|
|摘要:||質譜法是可用於決定分析物的質量、結構和組成的有效分析技術。過去數十年以來，質譜法已廣泛應用於小的有機分子或是大分子分析，也已有許多新穎的游離法被開發來可適用於不同性質的樣品分析。通常樣品游離的過程可在大氣壓下或是高真空的環境下進行，其中電噴灑游離法是一個最廣為應用的大氣壓下游離法。在電噴灑游離法的樣品進樣端通常需要施加高電壓以輔助誘導液相樣品游離成氣態離子。此外，如要進行定量分析，通常需要搭配層析儀的應用。此論文的第一部分是以開發一個簡單易操作的游離方法為目標，並期望在沒有施加電壓和組合層析方法的輔助下，可進行定性與定量的質譜分析。論文中利用了電動的移液管操控吸管尖做為直接質譜進樣裝置，並當作噴灑頭將電動排出的液滴直接在質譜前端進行噴灑產生氣相離子，此方法已用實驗證明可用於分析有機小分子和蛋白質，另外也嘗試了此方法在定量分析上的可能性。此論文的第二部分則是利用在真空下進行以奈米粒子為基材的親和表面輔助雷射脫附游離法用於分析三種不同的細菌 (即金黃色葡萄球菌、大腸桿菌及綠膿桿菌)。通常使用質譜法分析細菌的偵測極限約為104 隻細菌左右，因此此部分的目標為利用本方法降低質譜分析的偵測極限。由於包覆氧化鐵的磁性氧化鋁奈米粒子已被證實具備抓取葛蘭氏陰性和葛蘭氏陽性菌的能力，因此使用此奈米粒子做為濃縮細菌的親和探針，緊接著進行氧化反應產生氧化分解產物以做為辨別不同細菌的小生物標幟分子，再搭配適合分析小分子量的表面雷射輔助脫附游離法進行檢測。結果發現這三種選用的細菌各會產生代表各細菌的氧化分解小分子，因此可根據這些小分子的偵測判定各細菌在樣品中是否存在。此外此方法之偵測極限也降低到只需要約500隻細菌便可偵測。總括而言，此論文開發的兩種新型質譜法可應用於小分子、胜肽、蛋白質以及細菌的分析。|
Mass spectrometry (MS) is a powerful technology for determination of molecular weights, structures, and composition of analytes. The applications of using MS to the analysis of small organics and large molecules have been extensively studied in the past decades. A variety of ionization methods have been developed to suit the analysis of different properties of analytes. Generally, ionization occurs either in atmospheric pressure or high vacuum conditions. Electrospray ionization has been used as one of popular ion sources operated at atmospheric pressure for mass spectrometric analysis. Generally, electric contact on the sample introduction part applied with a high voltage is required for facilitating ionization processes of the sample from liquid phase to gas phase ions. Additionally, chromatography is required to combine with MS for reliable quantitative analysis. In the first part of this thesis, the goal was aimed to develop a simplified ionization method that can be used to conduct qualitative and quantitative analysis without applying electric contact on the sample introduction site and combining with any chromatography systems. An electronic pipette-operated tip was demonstrated to be as the sampling tube and as the emitter to generate gas phase ions in the proximity of a mass spectrometer for MS analysis. This approach can be used to analyze small organics and large proteins. Furthermore, the possibility of using this approach for quantitative analysis was examined. In the second part of this thesis, a new analytical method that can be used to characterize different bacteria by nanoparticle (NP)-based affinity surface-assisted laser desorption/ionization (SALDI) MS, which is operated under vacuum, was proposed. Generally, the detection limit for bacterial identification by MS is ~104 cells. Thus, the goal was aimed to improve the detection limit of bacteria by the NP-based SALDI-MS. It has been explored that alumina coated iron oxide magnetic NPs (Fe3O4@Al2O3 MNPs) were capable to concentrate pathogenic bacteria including Gram-positive and Gram-negative bacteria. Thus, Fe3O4@Al2O3 MNPs were used as affinity probes to enrich bacteria cells followed by oxidation reaction. The generated oxidation products with low molecular weights can be readily analyzed by SALDI-MS. The results show that the oxidation products from three selected model bacteria, i.e. Staphylococcus aureus, Escherichia coli O157:H7, and Pseudomonas aeruginosa, are unique, which can be used to represent these three model bacteria. Furthermore, the detection limit was as low as ~500 cells. In summary, two MS-based methods developed in this study can be used for the analysis of small organics, peptides, proteins, and bacterial cells.