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標題: | 新穎質譜影像樣品前處理方法—增加解析度及輔助蛋白質偵測 Novel Sample Preparations for Mass Spectrometry Imaging – To Increase Spatial Resolution and Facilitate Protein Detection |
作者: | Li-Cyun Chen 陳禮群 |
指導教授: | 徐丞志(Cheng-Chih Hsu) 徐丞志(Cheng-Chih Hsu | ccrhsu@ntu.edu.tw | ), |
關鍵字: | 質譜影像,樣品前處理,空間解析度,胰蛋白酶,原位蛋白質水解,常壓游離法,脫附型電噴灑游離法,奈米脫附型電噴灑游離法, Mass spectrometry imaging,Spatial resolution,Tissue expansion,Sample preparation,DESI,nanoDESI,In situ trypsin digestion, |
出版年 : | 2022 |
學位: | 碩士 |
摘要: | 樣品前處理在許多實驗中都扮演著重要的角色,尤其是質譜影像實驗。相較於其他質譜分析,質譜影像的待測物具有低含量、高背景的特質,必須採取樣品前處理以降低基質效應,保留分析物訊號。以基質輔助雷射游離質譜影像(MALDI-MSI)實驗為例,一般組織切片在進行質譜影像實驗時,觀測到的主要訊號會以脂質為主,並不是因為蛋白質不存在於組織中,而是因為過多的脂質訊號抑制了蛋白質的訊號,而如果在樣品前處理中,先行利用選擇性清洗方式移除脂質與鹽類,蛋白質訊號才得以顯現,另外在樣品前處理時基質的選擇也會大幅影響分析結果,以此為例可以彰顯樣品前處理的重要性。在過去的研究中,樣品前處理的功能已經從簡單的去除基質、增強訊號,演變成能夠解決實驗上遇到的複雜問題。本研究大方向即為發展並運用新穎的樣品前處理方法,解決實驗上所遇到的問題。 本論文第一部分立志於增進質譜影像的空間解析度。不同於傳統上以更新儀器、優化系統、或利用影像後處理來增進空間解析度,本研究發展並運用樣品前處理,對於組織樣品進行放大,進而得到增加解析度之結果。第二段則引進一廣泛使用於基質輔助雷射游離質譜影像(MALDI-MSI)中分析蛋白質樣品之技術,在樣品表面上利用胰蛋白酶將蛋白質水解成胜肽片段,以利常壓游離方法例如脫附型電噴灑游離法(DESI)產生較強之訊號,有助於質譜影像之分析。 Mass spectrometry imaging (MSI) visualizes spatial distribution of molecules in a biological tissue. Sample preparation is one of the most important procedure, which significantly affects the performance in MSI. Typically, biological samples are complicated, containing a variety of biomolecules such as carbohydrates, lipids, proteins, and nucleic acids. However, these biological samples contain complicated components, which often causes significant matrix effects for MSI data acquisition. Thus, common sample preparation methods are utilized to remove undesirable matrices and enrich target molecules. For example, washing step is necessary to remove abundant lipids to enhance protein signals in matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI). For the past few decades, sample preparation methods have evolved to not only reduce matrix suppression but also solve complex problems such as increase spatial resolution. In this research, we aimed to develop novel sample preparation methods to deal with several different problems in MSI experiments. In the first part, we aimed to improve spatial resolution of MSI samples through sample preparation. The improvement of spatial resolution was commonly carried out by improving instrumental design or developing software processing. However, in our attempt, we addressed the problem unconventionally, we developed a tissue expansion method that expands sample size during sample preparation, and it is compatible with MSI experiments, a result of a two to threefold linear expansion of mouse organs was achieved on a mouse kidney sample. For the second part, we introduced in situ trypsin digestion, which was mostly used in MALDI experiments, into DESI-MSI sample preparation to overcome the difficulties in protein DESI-MSI. Detection of large molecules such as protein using DESI has proven challenging, most studies reported the modification of ionization source into microextraction-based ionization, including nanoDESI and liquid extraction surface analysis (LESA) to overcome the problem. In our approach we apply in situ digestion to digest proteins into peptides to improve ionization, hundreds of peptide MSI images were generated by one single sample. Coupled with in situ peptide MSMS results using nanoDESI and protein identification software, several unique protein species could be identified, which were rarely seen in top-down microextraction-based protein MSI. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84709 |
DOI: | 10.6342/NTU202204067 |
全文授權: | 同意授權(限校園內公開) |
電子全文公開日期: | 2022-10-20 |
顯示於系所單位: | 化學系 |
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