Meta-analysis of genome-wide association studies in East Asian-ancestry populations identifies four new loci for body mass index

Recent genetic association studies have identified 55 genetic loci associated with obesity or body mass index (BMI). The vast majority, 51 loci, however, were identified in European-ancestry populations. We conducted a meta-analysis of associations between BMI and ∼2.5 million genotyped or imputed single nucleotide polymorphisms among 86 757 individuals of Asian ancestry, followed by in silico and de novo replication among 7488–47 352 additional Asian-ancestry individuals. We identified four novel BMI-associated loci near the KCNQ1 (rs2237892, P = 9.29 × 10−13), ALDH2/MYL2 (rs671, P = 3.40 × 10−11; rs12229654, P = 4.56 × 10−9), ITIH4 (rs2535633, P = 1.77 × 10−10) and NT5C2 (rs11191580, P = 3.83 × 10−8) genes. The association of BMI with rs2237892, rs671 and rs12229654 was significantly stronger among men than among women. Of the 51 BMI-associated loci initially identified in European-ancestry populations, we confirmed eight loci at the genome-wide significance level (P < 5.0 × 10−8) and an additional 14 at P < 1.0 × 10−3 with the same direction of effect as reported previously. Findings from this analysis expand our knowledge of the genetic basis of obesity

Application of Electrode Extraction coupled to Mass Spectrometry

本研究主要是發展電極萃取法，配合液相層析質譜儀的分析技術，檢測尿液中肌酸酐化合物與環境水樣中巴拉松化合物。 在肌酸酐檢測部分，實驗結果顯示電極萃取技術於磷酸鹽電解質溶液中具有較佳的萃取效果，控制電解質溶液pH為3，預氧化電位+1.6 V，預氧化時間150秒，磁石攪拌速率972 rpm，以線上模式（on-line mode），於脫附腔中注入100 μL超純水靜態脫附1分鐘，並結合液相層析儀之動相動態脫附1分鐘，進入液相層析管柱分離後，以電灑游離法正離子模式進行偵測。應用此技術於水中肌酸酐化合物之偵測，其線性範圍為0.5-10 μg/mL，線性相關係數為0.9984，偵測極限可達0.4 μg/mL，精密度為12.5 %，相對回收率為98.4 %。並應用此技術於真實尿液樣品之偵測，將尿液樣品以pH 3磷酸鹽電解質溶液稀釋250倍，測得尿液中肌酸酐含量為1.66 mg/mL。 在巴拉松檢測部分，實驗結果顯示電極萃取技術是以0.1N HCl將水溶液酸化，分別控制水溶液pH為2，預濃縮電位-0.6 V，預濃縮時間60秒，磁石攪拌速率858 rpm，以線上模式（on-line mode）結合液相層析儀之動相動態脫附3分鐘，進入液相層析管柱分離後，以大氣壓化學游離法正離子模式進行偵測。應用此技術於水中巴拉松化合物之偵測，其線性範圍為50-1000 ng/mL，線性相關係數為0.9991，偵測極限可達3 ng/mL，精密度在2.6-19 % 之間，相對回收率在74.2-101 % 之間。In this study, we developed electrode extraction method coupled to liquid chromatography-mass spectrometry (LC-MS) to determine the amount of creatinine in human urine and parathion in environmental water sample. In creatinine study, extraction performed by electrode extraction at optimized conditions of preanodization potentional of +1.6V for 150s. The limit of detection for creatinine obtained in ultra pure water was 0.4 μg/mL. According to the analysis, the linear range was 0.5-10 μg/mL. To decrease the effect of matrix, in real sample analysis, the urine was diluted with pH 3 phosphate buffer solution to form ratio of 1:250 (v/v). The feasibility of applying the methods to determine creatinine in real samples was evaluated by analyzing urines sample from human. The relative recovery of creatinine was 98.4 %. The creatinine determined in urine samples was 1.66 mg/mL. Electrode extraction combined with liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI/MS/MS) was used to determine the trace levels of parathion in river water and ground water. In extraction preconcentration potentional was at -0.6V and preconcentration for 60s. Then the analytes were desorbed with dynamic desorption in modified SPME/HPLC desorption chamber for 3 min and determined with LC-APCI/MS/MS. From results show, the linear range is from 50 to 1000 ng/mL. The limit of detection for parathion in water was 3 ng/mL. The relative recovery of parathion was between 74.2 and 101 %.摘要......................................................i Abstract.................................................ii 目錄....................................................iii 表次.....................................................vi 圖次....................................................vii 第一章、緒論..............................................1 1.1、分析物.............................................1 1.1.1、肌酸酐.........................................1 1.1.1.1、腎臟.......................................1 1.1.1.2、肌酸酐.....................................3 1.1.1.3、偵測肌酸酐的方法回顧.......................6 1.1.2、巴拉松.........................................8 1.1.2.1、有機磷農藥簡介.............................8 1.1.2.2、有機磷農藥的危害...........................8 1.1.2.3、巴拉松簡介.................................8 1.2、儀器原理..........................................12 1.2.1、電灑游離法....................................13 1.2.2、大氣壓化學游離法..............................15 1.2.3、四極矩質量分析器..............................17 1.2.4、串聯質譜儀....................................17 1.3、前處理技術簡介....................................19 1.4、電化學前處理之回顧................................23 1.5、電化學結合質譜儀技術回顧..........................26 第二章、實驗部分.........................................28 2.1、藥品與實驗器材....................................28 2.1.1、藥品..........................................28 2.1.2、實驗器材與設備................................28 2.1.3、電極萃取吸附裝置..............................28 2.2、儀器設備..........................................30 2.3、實驗條件..........................................30 2.3.1、層析條件......................................30 2.3.2、質譜條件......................................30 2.4、藥品配製..........................................30 2.4.1、配製creatinine標準溶..........................30 2.4.2、配製parathion標準溶液.........................31 2.5、實驗溶液之配製....................................31 2.6、化合物於液相層析質譜儀分析條件....................32 2.6.1、肌酸酐於液相層析質譜儀條件之測定..............32 2.6.2、巴拉松於液相層析質譜儀條件之測定..............32 2.7、液相層析質譜儀參數之探討..........................32 2.7.1、肌酸酐之電灑游離法正離子模式..................32 2.7.2、肌酸酐之大氣壓化學游離法正離子模式............33 2.7.3、巴拉松之大氣壓化學游離法正離子模式............35 2.8、肌酸酐最佳離子化方法之探討........................36 2.9、電極萃取條件之探討................................37 2.9.1、肌酸酐之電極萃取技術最佳化條件探討............37 2.9.2、巴拉松之電極萃取技術最佳化條件探討............38 2.10、利用電灑游離法正離子模式，評估肌酸酐之線性範圍、線 性方程式、線性相關係數和偵測極限.................39 2.11、分析水中肌酸酐化合物之精密度.....................39 2.12、分析水中肌酸酐化合物之回收率.....................40 2.13、分析水樣中巴拉松化合物之線性範圍、線性方程式、線性 相關係數和極偵測極限.............................40 2.14、分析水樣中巴拉松化合物之精密度...................40 2.15、分析水中巴拉松化合物之回收率.....................40 2.16、真實水樣中巴拉松之偵測...........................40 第三章、結果與討論.......................................41 3.1、肌酸酐化合物之液相層析質譜儀參數探討..............41 3.1.1、肌酸酐化合物標準品溶液之液相層析質譜分析......41 3.1.1.1、電灑游離法之質譜分析......................41 3.1.1.2、大氣壓化學游離法之質譜分析................50 3.1.2、最佳離子化方法之探討..........................64 3.1.3、電極萃取法條件之探討..........................64 3.1.4、評估尿液中基質干擾之影響......................70 3.1.5、利用電灑游離法正離子模式，評估肌酸酐之線性範圍、 線性方程式、線性相關係數和偵測極限............75 3.1.6、分析尿液中肌酸酐化合物之精密度................75 3.1.7、分析尿液中肌酸酐化合物之回收率................75 3.1.8、偵測真實尿液中機酸酐之含量....................79 3.2、巴拉松化合物之液相層析質譜儀參數探討..............79 3.2.1、巴拉松化合物標準品溶液之液相層析質譜分析......79 3.2.1.1、大氣壓化學游離法參數之探討................79 3.2.2、電極萃取法條件之探討..........................90 3.2.3、分析水樣中巴拉松化合物之線性範圍、線性方程式、線性相關係數和極偵測極限..................................101 3.2.4. 分析水樣中巴拉松化合物之精密度...............101 3.2.5. 分析水中巴拉松化合物之回收率.................101 3.2.6. 真實水樣中巴拉松之偵測.......................101 第四章、結論............................................106 第五章、參考資料........................................10

Nanomaterial-Based Mass Spectrometry for Analysis of Ultraviolet Absorbers in Urine and the Charge-State Distribution of Protein

In this thesis, nanomaterial-based electrospray mass spectrometric approach was utilized for small molecular and macromolecular analysis. The thesis is divided in five chapters. The functionalized magnetic nanoparticles, Fe3O4/SiO2 was synthesized and then utilized for analysis of eleven ultraviolet absorbers in urine. The feasibility of the presented method was also evaluated. The linear range of proposed method were 10-1000 ng mL-1 for HBP, BP-3, BP-2, BP-6 and 4-MBC; 0.5-1000 ng mL-1, 0.5-500 ng L-1, 0.75-1000 ng mL-1 and 0.75-500 ng mL-1 for DHB, BP-12, DHMB and OD-PABA, respectively. LODs were ranged from 0.1 ng mL-1 to 8.8 ng mL-1 with R.S.D. below 10.8%. This approach was also applied for real samples analysis. The trace amount of DHMB in urine was detected and the concentration is 277.1 ng mL-1. The ambient mass spectrometry for one-droplet analysis was developed in this thesis. A 3.5 μL solution was dipped on a laboratory-made screen-printed plate coated with silver or graphite gel. The voltage of +5 kV was applied on the surface of the droplet and electrospray was performed when applying the voltage. The ultraviolet absorbers studied are including 3-(4-methylbenzylidene)-camphor (4-MBC), 2-ethyl-4-(dimethylamino)benzoate (OD-PABA) and 2-hydroxy-4-(octyloxy)-benzophenone (BP-12). To evaluate the effect on the ionization efficiency, the solution containing analytes and nanomaterials was dipped on the screen-printed substrate. From the results, the MWCNTs can enhance the intensity of the signal detected and short the analytical time. This technique demonstrates that the nanoparticles assisted electrospray ionization offers a high specific and high throughput screening for trace analysis. The analysis of protein conformation by ESI-MS was devoted in this study. This work outlines the changes to the charge-state distribution (CSD) and to improvement of the resolution of isotopic peak when the nanomaterial solution is infused to the ion source and co-electrosprayed with the protein-contained solution. The various conformation gold nanoparticles (AuNPs) and titanium dioxide nanoparticles (TiO2) were used in this study. Generally the ESI-MS of protein solution often show a monomodal distribution of charge-state peaks with a maximum charge-state and a most-intense charge-state. When AuNPs mixed with protein such as cytochrome c and ubiquitin, the mass resolution of isotopic peak in the mass spectrum were improved. The ESI-MS of protein solution was shown the average distribution of isotopic peaks with the most-abundant charge-state. For the assessment of myoglobin with AuNPs and TiO2, the charge-stated peaks of protein were shifted. The present study was demonstrated the effect of nanomaterials on the charge-states distribution of protein and the characteristics of isotopic peaks of protein in ESI-MS.本論文研究主要是利用各式奈米材料結合電噴灑質譜法於小分子與生化分子分析之研究。利用二氧化矽修飾磁性奈米粒子應用於尿液中微量紫外光吸收劑之偵測，研究中首先針對合成之材料進行各項鑑定，包含IR、TEM、XRD、ESCA等光譜鑑定，之後進行樣品前處理之最佳化條件探討，並於實驗最佳化條件下，評估方法的可行性，根據實驗結果顯示，本實驗方法之線性範圍分別為於HBP、BP-7、BP-3、BP-2、BP-6與4-MBC等分析物介於10-1000 ng mL-1；對於DHB與BP-12分析物分別為0.5-1000 ng mL-1與0.5-500 ng L-1；對於DHMB與OD-PABA分析物分別為0.75-1000 ng mL-1與0.75-500 ng mL-1，線性相關係數為0.991以上，偵測極限介於0.1 ng mL-1 (OD-PABA)與 8.8 ng mL-1 (BP-2)之間，定量極限介於0.2 ng mL-1 (OD-PABA)與29.3 ng mL-1 (BP-2)之間，精密度介於1.8 %與10.8 %之間。應用於16個尿液樣品分析，於S5樣品中，測得尿液樣品中含有微量DHMB之成份，濃度為283.4 ng mL-1。 本實驗另發展一滴溶液分析大氣壓質譜法，於透明膠片上塗覆一層疏水性導電材料，根據實驗結果顯示，當在3.5 μL液滴表面施予不同電位時，液面與塗覆材料間的接觸角會隨著電位大小而改變，出現類似電潤濕的現象，且由離子訊號強度與CCD攝影機觀察其噴灑狀態，其訊號穩定度佳且持續時間較長，將其應用於奈米材料混合溶液分析，於尿液樣品中添加Fe3O4/SiO2與Fe3O4奈米材料對於分析物的訊號並無明顯提升，但在兩種不同磁性材料於分析物離子強度比較結果，使用Fe3O4/SiO2對於分析物的訊號可提升1.3(4-MBC)-2.1(BP-12)倍；添加MWCNTs則是分析物離子訊號相較於直接尿液樣品分析可提升2(4-MBC)-7(OD-PABA)倍，根據此實驗條件進行方法評估，方法線性範圍介於0.25-5 μg mL-1，且相對標準偏差低於16%。 由於蛋白質的電荷分佈與構型有關，因此利用電噴灑質譜法探討在不同奈米材料環境下，對於蛋白質電荷分佈型態之影響，實驗中分別探討不同尺寸金奈米粒子與二氧化鈦奈米粒子對於細胞色素C、肌紅蛋白與泛素電荷分佈之影響。於金奈米粒子研究部分，金奈米粒子對於細胞色素C與泛素於電噴灑質譜法分析下，其電荷分佈範圍並無顯著的改變，顯示這些蛋白質於金奈米材料環境下，其構型並不會因此受到改變，但是觀測細胞色素C與泛素之ESI-MS質譜圖則顯示其差異性。於細胞色素C之分析結果中，其每一帶電荷離子訊號峰由原先單一同位素訊號(monoisotopic peak)，隨著金奈米粒子的比例增加，每一電荷離子訊號會呈現一高斯分佈型態，且同位素峰皆明顯表現；在泛素的分析結果則顯示於電噴灑游離法下會與金奈米粒子形成水合離子訊號，如[Ubq+Au+6H2O+6H]6+與[Ubq+Au+6H2O]6+。 在肌紅蛋白中分析結果則顯示隨著金奈米粒子比例的增加，其帶電荷數會隨著減少且落於高質量區，但在二氧化鈦溶液環境下，肌紅蛋白其帶電荷價數介於+11價與+20價之間，相較於在金奈米環境下，最強訊號之電荷價數由+21價趨於+17價分佈不同，推測二氧化鈦於ESI-MS分析過程中對肌紅蛋白帶電荷數的提升有所助益，且在此兩種奈米材料環境下，由於都未觀察到heme group (m/z 616.4)訊號峰的出現，因此推測肌紅蛋白結構並無任何改變。目錄 中文摘要 ………………………………………………………………………….i Abstract ……………………………………………………………………………...iii 目錄 ………………………………………………………………………………v 表目錄 ……………………………………………………………………………...xi 圖目錄 …………………………………………………………………….……….xii 第一章 、序論 ……………………………………………………………………1 1.1. 奈米材料簡介 …………………………………………………………1 1.2. 奈米粒子製備方法 ……………………………………………………1 1.2.1. 磁性奈米粒子(magnetic nanoparticles, MNPs) ………………3 1.2.2. 金奈米粒子(gold nanoparticles, AuNPs) ……………………4 1.2.3. 二氧化鈦奈米粒子(titanium dioxide, TiO2 NPs) ………………5 1.2.4. 銀奈米粒子(silver nanoparticles, AgNPs) ……………………5 1.3. 質譜術簡介 ………………………………………………………………6 1.3.1. 離子化方法介紹 ………………………………………………6 1.3.1.1. 電灑游離法簡介 …………………………………………6 1.3.1.2. 離子形成機制 …………………………………………6 1.3.2. 質譜儀簡介 ……………………………………………………9 1.3.2.1. 四極矩式質量分析器(triple-quadrupole mass analyzer)…..9 1.3.2.1.1. 四極矩式質量分析器簡介 …………………………9 1.3.2.1.2. 串聯質譜儀(tandem mass spectrometer, MS/MS) ……9 1.3.2.2. 離子阱質量分析器 …………………………………..13 1.3.2.2.1. 三維離子阱質量分析器 ………………………..13 1.3.2.2.2. 二維離子阱(線性離子阱)質量分析器 ……………..14 1.4. 研究動機 ……………………………………………………………..19 1.5. 參考文獻 ……………………………………………………………..20 第二章、 …………………………………………………………………..22 2.1. 前言 ……………………………………………………………..22 2.2. 實驗部分 ………………………………………………………..30 2.2.1. 藥品與試劑 ……………………………………………..30 2.2.1.1. 紫外光吸收劑標準品 ……………………………..30 2.2.1.2. Fe3O4@ SiO2合成藥品 ……………………………..30 2.2.1.3. 溶劑 ……………………………………………..30 2.2.2. 藥品配製 ……………………………………………..30 2.2.2.1. 配製100 μg/mL 11種紫外光吸收劑單一標準溶液 ………………………………………………………..30 2.2.2.2. 配製10 μg/mL混合標準品工作溶液 ……………..32 2.2.2.3. 配製檢量線尿液工作溶液 ………………………..32 2.2.3. 儀器設備 ……………………………………………..32 2.2.4. Fe3O4/SiO2磁性奈米粒子合成與製備 …………………..33 2.2.4.1. 磁性奈米粒子合成 ……………………………..33 2.2.4.2. SiO2修飾磁性奈米粒子合成 …………………..33 2.2.5. Fe3O4/SiO2奈米粒子鑑定分析與樣品製備 ……………..36 2.2.5.1. 穿透式電子顯微鏡(transmission electron microscopy, TEM)鑑定分析 …………………………………..36 2.2.5.2. 高解析X光粉末繞射儀(high resolution X-ray powder diffraction, HR-XRD) ……………………………..36 2.2.5.3. 化學分析能譜儀(electron spectroscopy for chemical analysis, ESCA)鑑定分析 ………………………..36 2.2.6. 樣品前處理流程 ………………………………………..36 2.2.7. 尿液樣品 ……………………………………………..37 2.3. 結果與討論 ………………………………………………………..39 2.3.1. 修飾性磁性奈米粒子表面特性分析 …………………..39 2.3.2. 前處理方法之最佳化 …………………………………..39 2.3.2.1. 探討Fe3O4/SiO2奈米粒子添加量對尿液中紫外光吸收劑效率之影響 ………………………………………..39 2.3.2.2. 探討pH值對於尿液中紫外光吸收劑之萃取效率影響 ………………………………………………………..44 2.3.2.3. 探討萃取時間對於尿液中紫外光吸收劑之萃取效率影響 …………………………………………………..44 2.3.2.4. 探討脫附溶劑種類對於尿液中紫外光吸收劑脫附效率影響 ……………………………………………..48 2.3.2.5. 探討脫附溶劑體積對尿液中紫外光吸收劑之脫附效率影響 ……………………………………………..48 2.3.2.6. 探討脫附時間對於尿液中紫外光吸收劑之脫附效率影響 …………………………………………………..51 2.3.2.7. 尿液樣品基質效應 (matrix effect)之探討 ………..51 2.3.3. 分析尿液樣品中紫外光吸收劑及其衍生物之線性範圍、線性方程式、線性相關係數與偵測極限之方法確效 …..56 2.3.4. 分析尿液樣品中紫外光吸收劑及其衍生物之精密度 ………………………………………………………..56 2.3.5. 樣品分析 ……………………………………………..58 2.4. 結論 ……………………………………………………………..61 2.5. 參考文獻 ………………………………………………………..62 第三章、 …………………………………………………………………..66 3.1. 前言 ……………………………………………………………..66 3.1.1 大氣壓力質譜法(ambient mass spectrometry)發展 …..66 3.1.2 研究目的 ……………………………………….…….68 3.2. 實驗部分 ……………………………………………………….68 3.2.1. 藥品與試劑 …………………………………………….68 3.2.2. 儀器設備 …………………………………………….68 3.2.3. 多層奈米碳管(multiwall carbon nanotubes, MWCNTs)純化 ……………………………………………………….69 3.2.4. 實驗用基板製作 ……………………………………….69 3.2.5. 樣品製備 …………………………………………….71 3.2.6. 實驗流程 …………………………………………….71 3.3. 結果與討論 ……………………………………………………….73 3.3.1. 一滴溶液分析介面探討 …………………………….73 3.3.2. 探討樣品置放方式對分析物訊號的影響 …………….77 3.3.2.1. 層層堆疊模式(layer-by-layer mode) …………….77 3.3.2.2. 均勻分散模式(homogeneous mode) …………….77 3.3.3. 比較不同奈米材料對紫外光吸收劑分析訊號強度之影響 ………………………………………………………..77 3.4. 結論 ……………………………………………………………..87 3.5. 參考文獻 ………………………………………………………..88 第四章、 …………………………………………………………………..90 4.1. 前言 ……………………………………………………………..90 4.1.1. 蛋白質結構簡介 ………………………………………..90 4.1.1.1. 血質蛋白(heme protein) ……………………………..91 4.1.1.1.1. 細胞色素C(cytochrome c, Cyt. c) ……………..93 4.1.1.1.2. 肌紅蛋白(myoglobin, Myo) …………………..93 4.1.1.2. 泛素(ubiquitin, Ubq) ……………………………..96 4.1.2. 電噴灑質譜法於蛋白質結構分析 …………………..96 4.1.3. 奈米材料與蛋白質間相互作用之研究 ……………..98 4.1.4. 研究目的…………………………………………………100 4.2. 實驗部份 ………………………………………………………101 4.2.1. 藥品與試劑 ……………………………………………101 4.2.2. 儀器裝置 ……………………………………………101 4.2.2.1. 質譜儀 ……………………………………………101 4.2.2.2. 電子顯微鏡 ………………………………………102 4.2.3. 奈米材料製備 ………………………………………102 4.2.3.1. 金奈米粒子溶液製備 ……………………………102 4.2.3.1.1. 10 nm金奈米粒子合成(I) …………………102 4.2.3.1.2. 50 nm金奈米粒子合成(II) …………………102 4.2.3.1.3. 硫辛酸修飾金奈米粒子合成 ……………102 4.2.3.2. 銀奈米粒子溶液製備 ……………………………103 4.2.3.3. 二氧化鈦奈米粒子溶液製備 …………………103 4.2.4. 奈米粒子鑑定與分析 …………………………………104 4.2.4.1. 穿透式電子顯微鏡(transmission electron microscopy, TEM)鑑定分析 …………………………………104 4.2.5. 藥品配製 ……………………………………………104 4.2.5.1. 50 mM牛血清白蛋白(bovine serum albumin, BSA)配製 …………………………………………………104 4.2.5.2. 30 mM細胞色素C(cytochrome c)溶液配製 ....104 4.2.5.3. 50 μM血紅素(myoglobin)配製 …………………104 4.2.5.4. 100 μM乳清蛋白(α-lactalbumin, BLA) ………104 4.2.5.5. 200 μg mL-1泛素(ubiquitin)配製 ……………104 4.2.6. 研究方法 ……………………………………………105 4.3. 結果與討論 ………………………………………………………106 4.3.1. 合成奈米材料光譜與電子顯微鏡之鑑定 ……………106 4.3.2. 探討金奈米粒子於蛋白質之電荷分佈效應影響 …106 4.3.2.1. 10 nm金奈米粒子(酒紅色) ………………………106 4.3.3. 二氧化鈦奈米粒子對蛋白質之電荷分佈效應之影響 ………………………………………………………122 4.4. 結論 ……………………………………………………………126 4.5. 參考文獻 ………………………………………………………128 第五章、總結 ……………………………………………………………131   表目錄 表2-1 電噴灑游離法偵測紫外光吸收劑之最佳化條件 …………………..34 表2-2 電噴灑游離法於正負模式下偵測紫外光吸收劑之定性與定量離子.....35 表2-3 Fe3O4/SiO2結合液相層析串聯質譜儀偵測尿液中紫外光吸收劑之最佳化條件………………………………………………………………….....54 表2-4 Fe3O4/SiO2萃取結合液相層析串聯質譜儀偵測尿液中紫外光吸收劑之線性範圍、線性相關係數、偵測極限、定量極限與精密度(n=3)…..…57 表2-5 Fe3O4/SiO2萃取結合液相層析串聯質譜儀於尿液樣品分析之結果…...59 表3-1 比較不同磁性奈米粒子輔助電噴灑游離法分析紫外光吸收劑之離子強度 ………………………………………………………………………..83 表3-2 比較多層奈米碳管輔助電噴灑游離法分析紫外光吸收劑之離子強度 ………………………………………………………………………..86 表4-1 利用電噴灑游離法結合高解析質譜儀分析泛素蛋白質+5價至+10價之最強離子訊號…………………………………………………………....116   圖目錄 圖 1- 1 (a)電噴灑游離法與(b)正離子模式下離子形成過程之示意圖 ………...8 圖 1- 2四極矩之構造圖 ………………………………………………………..10 圖 1- 3串聯質譜技術四種掃描模式 ……………………………………………..12 圖 1- 4離子阱構造圖 ……………………………………………………………..14 圖 1- 5二維離子阱質量分析器構造圖 ………………………………………..16 圖 1- 6線性離子阱上DC、RF與AC電位施予之示意圖 …………………..17 圖 1- 7 Thermo Finnigan設計之LTQ離子偵測器示意圖 ………………………..18 圖 2- 1十一種紫外光吸收劑分析物之結構 …………………………………..31 圖 2- 2樣品前處理流程圖 ………………………………………………………..38 圖 2- 3合成磁性奈米材料之FT-IR分析圖 (紅色: Fe3O4；黃綠色: Fe3O4/SiO2) ……………………………………………………………………………..40 圖 2- 4合成磁性奈米材料之ESCA分析圖 (黑色實線: Fe3O4；紅色虛線: Fe3O4/SiO2) ……………………………………………………………..41 圖 2- 5合成磁性奈米材料之HR-XRD分析圖 (黑色實線: Fe3O4；紅色虛線: Fe3O4/SiO2) ……………………………………………………………..42 圖 2- 6合成磁性奈米材料之TEM分析圖 ………………………………………..43 圖 2- 7 Fe3O4/SiO2添加量對紫外光吸收劑萃取效率之影響 …………………..45 圖 2- 8溶液pH值對紫外光吸收劑萃取效率之影響 ………………………..46 圖 2- 9萃取時間對紫外光吸收劑萃取效率之影響 ……………………………..47 圖 2- 10脫附溶劑種類對紫外光吸收劑脫附效果之影響 ………………………..49 圖 2- 11脫附溶劑體積對紫外光吸收劑脫附效率之影響 ………………………..50 圖 2- 12脫附時間對紫外光吸收劑脫附效率之影響 ……………………………..52 圖 2- 13緩衝溶液添加量對紫外光吸收劑萃取效率之影響 …………………..53 圖 2- 14濃度為100 ng mL-1 紫外光吸收劑添加於尿液中，經Fe3O4/SiO2萃取結合LC-MS/MS分析之層析質譜圖. (a) HBP; (b) BP-7(c) BP-1; (d) BP-9; (e) BP-3; (f) BP-8; (g) BP-2 ; (h) BP-6 ;(i) 4-MBC; (j) OD-PABA; (k) BP-12. …………………………………………………………………..55 圖 2- 15 Fe3O4/SiO2萃取結合液相層析串聯質譜儀於S5尿液樣品分析所得之層析質譜圖. (a) HBP; (b) BP-7(c) BP-1; (d) BP-9; (e) BP-3; (f) BP-8; (g) BP-2 ; (h) BP-6 ;(i) 4-MBC; (j) OD-PABA; (k) BP-12. ……………..60 圖 3- 1 實驗基材製備流程圖 …………………………………………………..70 圖 3- 2 實驗流程圖 ……………………………………………………………..72 圖 3- 3 一滴溶液結合電噴灑分析之側視圖 …………………………………..74 圖 3- 4 液珠直接電噴灑。(a) 電灑電壓未施加前；(a) 電灑電壓施加後 …..75 圖 3- 5 3.5 μL 0.1%甲酸水溶液液珠直接電灑之質譜圖 ………………………..76 圖 3- 6 層層堆疊模式之側視圖 ……………………………………………..78 圖 3- 7 堆疊式樣品製備法結合電噴灑質譜法分析含1 μg mL-1紫外光吸收劑之尿液與Fe3O4/SiO2磁奈米粒子混合溶液所得之(a) TIC圖與選擇離子層析圖(b) 4-MBC, m/z 255；(c) OD-PABA, m/z 278與(d) BP-12, m/z 327。 ………………………………………………………………………..79 圖 3- 8 均勻混合模式之側視圖 ……………………………………………..80 圖 3- 9 均勻混合式樣品製備法結合電噴灑質譜法分析含1 μg mL-1紫外光吸收劑之尿液與Fe3O4/SiO2磁奈米粒子混合溶液所得之選擇離子層析圖。(a) 4-MBC, m/z 255；(b) OD-PABA, m/z 278與(c) BP-12, m/z 327。 …..81 圖 3- 10 均勻混合式樣品製備法結合電噴灑質譜法分析含1 μg mL-1紫外光吸收劑之尿液與Fe3O4磁奈米粒子混合溶液所得之選擇離子層析圖。(a) 4-MBC, m/z 255；(b) OD-PABA, m/z 278與(c) BP-12, m/z 327。 …..82 圖 3- 11 (a)噴灑前、(b)噴灑後基材表面CCD圖。 ……………………………..85 圖 4-1 血紅素基團(heme group)結構 ………………………………………..92 圖 4-2 細胞色素C之結構圖 …………………………………………………..94 圖 4-3 氧分子於肌紅蛋白中heme group鍵結示意圖 ……………….……….95 圖 4-4 泛素蛋白質之結構圖 ………………………………………….……….97 圖 4-5 合成奈米粒子之TEM圖 ………………………………………107 圖 4-6 細胞色素C於不同比例20 nm金奈米粒子溶液之ESI-MS質譜圖 …108 圖 4-7 細胞色素C之+8價離子訊號於不同體積比例金奈米粒子溶液之高解析ESI-MS質譜圖 ………………………………………………………110 圖 4-8 細胞色素C之+7價離子訊號於不同體積比例金奈米粒子溶液之ESI-MS高解析質譜圖 …………………………………………………111 圖 4-9 泛素於不同體積比例10 nm金奈米粒子溶液之ESI-MS質譜圖 ....112 圖 4-10 泛素+6價離子訊號峰於不同體積比例金奈米粒子(10 nm)溶液之ESI-MS質譜圖 ………………………………………………………113 圖 4-11 泛素於不同體積比例金奈米粒子(10 nm) 溶液之ESI-MS高解析質譜圖 ………………………………………………………………………115 圖 4-12 泛素於不同體積比例金奈米粒子(50 nm)溶液之ESI-MS質譜圖 …117 圖 4-13 泛素於不同體積比例金奈米粒子(50 nm)溶液之ESI-MS高解析質譜圖 ………………………………………………………………………118 圖 4-14 肌紅蛋白於不同體積比例金奈米粒子(10 nm)溶液之ESI-MS質譜圖 ………………………………………………………………………120 圖 4-15 金奈米粒子(棕色線)、肌紅蛋白(橘色線)及其混合溶液(黃綠色線)之拉曼光譜圖 ……………………………………………………………121 圖 4-16 肌紅蛋白於不同比例二氧化鈦奈米粒子溶液之ESI-MS質譜圖 ....123 圖 4-17 肌紅蛋白於不同比例空白溶液稀釋之ESI-MS質譜圖 …………....124 圖 4-18 肌紅蛋白+13價至+19價離子訊號之質譜圖 ………………………12

A novel method of ultrasound-assisted dispersive liquid-liquid microextraction coupled to liquid chromatography-mass spectrometry for the determination of trace organoarsenic compounds in edible oil

A novel approach, ultrasound-assisted dispersive liquid-liquid microextraction combined with liquid chromatography-mass spectrometry (UA-DLLME with LC-MS) is demonstrated to be quite useful for the determination of trace amounts of organoarsenic compounds in edible oil. The organoarsenic compounds studied include dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and 3-nitro-4-hydroxyphenyl arsenic acid (Roxarsone). Orthogonal array experimental design (OAD) was utilized to investigate the parameter space of conditions for UA-DLLME. The optimum conditions were found to be 4 min of ultrasonic extraction using 1.25 mL of mixed solvent with 50 mu L of buffer solution. Under these optimal conditions, the linear range was from 10 ng g(-1) tc 500 ng g(-1) for DMA and Roxarsone, from 25 ng g(-1) to 500 ng g(-1) for MMA. Limits of detection of DMA, MMA and Roxarsone were 1.0 ng g(-1), 3.0 ng g(-1) and 5.8 ng g(-1), respectively. The precisions and recoveries also were investigated by spiking 3-level concentrations in edible oil. The recoveries obtained were over 89.9% with relative standard deviation (RSD) of 9.6%. The new approach was utilized to successfully detect trace amounts of organoarsenic compounds in various edible oil samples. (C) 2011 Elsevier B.V. All rights reserved

Polo-like kinase 2 regulates angiogenic sprouting and blood vessel development.

Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates

Preparation and characterization of antibody-drug conjugates acting on HER2-positive cancer cells.

Two systems of antibody-drug conjugates (ADCs), noncleavable H32-DM1 and cleavable H32-VCMMAE, were developed by using different linkers and drugs attached to the anti-HER2 antibody H32, which is capable of cell internalization. Activated functional groups, including an N-hydroxysuccinimidyl (NHS) ester and a maleimide, were utilized to make the ADCs. Mass spectrometry, hydrophobic interaction chromatography, polyacrylamide gel electrophoresis, and in vitro cell assays were performed to analyze and optimize the ADCs. Several H32-VCMMAE ADCs were established with higher DARs and greater synthetic yields without compromising potency. The anticancer efficacy of H32-DM1 was 2- to 8-fold greater than that of Kadcyla®. The efficacy of H32-VCMMAE was in turn better than that of H32-DM1. The anticancer efficacy of these ADCs against N87, SK-BR-3 and BT474 cells was in the following order: H32-VCMMAE series > H32-DM1 series > Kadcyla®. The optimal DAR for H32-VCMMAE was found to be 6.6, with desirable attributes including good cell penetration, a releasable payload in cancer cells, and high potency. Our results demonstrated the potential of H32-VCMMAE as a good ADC candidate

Nε-carboxymethyllysine-mediated endoplasmic reticulum stress promotes endothelial cell injury through Nox4/MKP-3 interaction

N(ε)-carboxymethyllysine (CML) is an important driver of diabetic vascular complications and endothelial cell dysfunction. However, how CML dictates specific cellular responses and the roles of protein tyrosine phosphatases and ERK phosphorylation remain unclear. We examined whether endoplasmic reticulum (ER) localization of MAPK phosphatase-3 (MKP-3) is critical in regulating ERK inactivation and promoting NADPH oxidase-4 (Nox4) activation in CML-induced endothelial cell injury. We demonstrated that serum CML levels were significantly increased in type 2 diabetes patients and diabetic animals. CML induced ER stress and apoptosis, reduced ERK activation, and increased MKP-3 protein activity in HUVECs and SVECs. MKP-3 siRNA transfection, but not that of MKP-1 or MKP-2, abolished the effects of CML on HUVECs. Nox4-mediated activation of MKP-3 regulated the switch to ERK dephosphorylation. CML also increased the integration of MKP-3 with ERK, which was blocked by silencing MKP-3. Exposure of antioxidants abolished CML-increased MKP-3 activity and protein expression. Furthermore, immunohistochemical staining of both MKP-3 and CML was increased, but phospho-ERK staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. Our results indicate that an MKP-3 pathway might regulate ERK dephosphorylation through Nox4 during CML-triggered endothelial cell dysfunction/injury, suggesting that therapeutic strategies targeting the Nox4/MKP-3 interaction or MKP-3 activation may have clinical implications for diabetic vascular complications