表面輔助雷射脫附游離質譜法暨奈米材料之生物分析

Bioanalysis of Surface-Assisted Laser Desorption/Ionization Mass Spectrometry Using Nanomaterials

陳玟岑(Wen-Tsen Chen)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00829

摘要

本論文主要是利用合成的奈米材料結合表面輔助雷射脫附游離質譜法(SALDI-MS)來偵測尿液中卡特普、茶葉中茶氨酸與四種兒茶素分子及寡聚去氧核苷酸分子濃度，且應用於蛋白質間相互作用之分析。論文分成五個章節，第一章內容包括質譜儀基本原理、基質輔助雷射脫附游離法、表面輔助雷射脫附游離法的簡介及待測分析物(卡特普，兒茶素，蛋白質與蛋白質-蛋白質之複合體，寡聚去氧核苷酸)的介紹。第二章利用修飾內標準品(4-MBA)之金奈米粒子(14 nm)作為SALDI-MS之內標準品，來分析尿液中卡特普之含量。先利用未經修飾之金奈米粒子抓取分析物後，再混合吸附4-MBA之金奈米粒子來進行分析，其偵測極限為1 uM (訊雜比為3)，線性範圍2.5–25 μM，顯示此法對卡特普偵測具有良好之線性範圍與再現性(相對標準誤差: <10%)，更可準確定量尿液樣品中卡特普濃度。第三章利用二氧化鈦奈米粒子與卡特普分別作為無機基質與內標準品，偵測茶葉樣品中茶氨酸和四種兒茶素分子濃度(如兒茶素、(_)-表沒食子兒茶素、(_)-表兒茶素沒食子兒茶素沒食子酸酯和(_)-表沒食子兒茶素沒食子兒茶素沒食子酸酯)。此法不僅提供良好的定量線性和靈敏度(偵測極限:介於femtomole-to-picomole之間)，亦展現再現性佳及變異性小(<13%)等特點，由實驗結果說明台灣和其他四個國家的茶葉樣品之鑑定有不同的質譜圖譜表現，不同季節和城市採收(台灣)的茶葉亦會有不同的成份組成。第四章利用汞-碲(HgTe)奈米材料為無機基質，在介面活性劑(Brij 76)和鋅離子存在下，分析蛋白質與蛋白質-蛋白質複合體(如a1-抗胰蛋白酶-胰蛋白酶和免疫球蛋白G -蛋白質G複合體)。此方法可以在溫和條件與低濃度(picomole)下，進行蛋白質及其複合體的分析，也可分別觀察到此兩種複合體的多價態訊號。此方法具簡單和再現性（RSD<25％）之特性，對於蛋白質體學上的其他蛋白質及其複合體之分析具有極大潛力。第五章利用汞-碲(HgTe)奈米材料為無機基質，偵測單股與雙股寡聚去氧核苷酸分子。此方法具有高靈敏度（偵測極限於femtomole-to-picomole level）和再現性（變異性：<23％）佳等特點，可用於偵測單股(最大達50-mer)與雙股(最大達30 base pairs)寡聚去氧核苷酸分子，並應用於分析一單核苷酸多態性鐮狀巨幼紅細胞中，b-珠蛋白(b-globin)上纈氨酸殘基之表現，相信未來應用於基因診斷具極大潛力。

關鍵字

金奈米粒子 ； 汞-碲奈米材料 ； 表面輔助雷射脫附游離質譜法 ； 卡特普 ； 兒茶素 ； 蛋白質-蛋白質複合體 ； 寡聚去氧核苷酸

並列摘要

This thesis focuses on synthesizing nanomaterials through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) for the analysis of captopril and catechins in urines and tea samples, respectively, and for the investigation of protein-protein interaction in addition to the detection of single- and double-stranded oligodeoxynucleotides (ss- and ds-ODNs, respectively). The thesis is divided into five parts. Chapter one introduces the framework and background of basic principles of mass spectrometry, the soft ionization method, MALDI, SALDI, generally applied in MS, and analysis of captopril, tea catechins, proteins and protein-protein complexes, and ODNs. In chapter two, an internal standard comprised captopril (CAP) mixed with 14-nm-diameter gold nanoparticles (Au NPs) was introduced. The analytes were first captured using the unmodified 14-nm Au NPs; followed by addition of the internal standard (CAP-Au NPs) and the analysis of the sample using SALDI-MS. This approach provided linearity for CAP over the concentration range 2.5–25 μM, with a limit of detection (signal-to-noise ratio = 3) of 1 μM. This approach provided good quantitative linearity and reproducibility (relative standard deviations: <10%) of CAP for the determination of the levels of CAP in human urine samples. In chapter three, SALDI-MS using titanium dioxide nanoparticles (TiO2 NPs) as the matrix and captopril (CAP) as internal standard were used for the determination of the concentrations of theanine and four catechins—catechin, (_)-epigallocatechin (EGC), (_)-epicatechin gallate (ECG), and (_)-epigallocatechin gallate (EGCG). This SALDI-MS approach provides good quantitative linearity and sensitivity (LOD at the femtomole-to-picomole level) for these five analytes. It also provides good reproducibility, spot-to-spot and batch-to-batch variations of less than 10 and 13%, respectively, for the analysis of tea samples, with identified peaks for theanine and four catechins. Tea samples from Taiwan and four other countries have various SALDI-MS profiles, showing their potential for differentiation of tea samples from different sources. The result also shows that tea samples harvested in different seasons and counties in Taiwan provide significantly different MS profiles. The fourth chapter utilizes the mercury-tellurium (HgTe) nanomaterials chosen as SALDI-MS matrices in the detection of proteins and weak protein–protein complexes, such as a1-antitrypsin–trypsin and IgG–protein G complexes, in the presence of stabilizing Brij 76 surfactant and Zn(II) ions. This soft and sensitive technique allows the detection of weak protein complexes at the picomole level under mild conditions. In addition, we observed multiple charged states for these two complexes, respectively. This simple and reproducible (RSD <25%) approach holds great potential for the detection of other proteins and their complexes for various omics such as proteomics. The fifth chapter describes the use of SALDI-MS with HgTe nanostructures acting as the matrix for the detection of ss- and ds-ODNs. The present approach provides good sensitivity (LOD at the femtomole-to-picomole level) and reproducibility (variation: <23%) for the detection of ss-ODNs (up to 50-mer) and ds-ODNs (up to 30 base pairs). Furthermore, the practicality of this approach was applied for the analysis of a single nucleotide polymorphism (SNP) that determines the fate of the valine residue in the b-globin of sickle cell megaloblasts. This simple and reproducible approach employing HgTe nanostructures as matrices appears to hold great potential for use in genomic diagnosis.

並列關鍵字

gold nanoparticles (Au NPs) ； mercury-tellurium (HgTe) nanomaterials ； surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) ； captopril (CAP) ； catechin ； protein-protein complex ； oligodeoxynucleotides (ODNs)

參考文獻

Chapter 1

(1)Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301.

(5)Cho,W. C. Genom. Proteom. Bioinformat. 2007, 5, 77–85.

(6)Fenselau, C.; Demirev, P. A. Mass Spectrom. Rev. 2001, 20, 157–171.

(7)Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349–451.