應用二氧化鈦及氧化鋁奈米材料於生物質譜分析及磷酸化蛋白質體分析方法的發展

Abstract

基質輔助雷射脫附游離質譜法(Matrix-Assisted Laser Desorption/Ionization, MALDI）已被廣泛地使用於各類型樣品的分析，然而MALDI基質和分析物之間的互溶性及形成共結晶化的性質是決定分析結果好壞的關鍵因素。本論文的第一部份是以溶膠凝膠法製備出具有Anatase晶型及中孔洞奈米結構的二氧化鈦薄膜，利用其具有吸收氮氣雷射能量的特性，而發展出二氧化鈦-表面輔助雷射脫附游離質譜法(TiO2-Surface Assisted Laser Desorption/Ionization mass spectrometry, TiO2-SALDI-MS)。此質譜法不需要外加基質和樣品互溶及進行共結晶化，因此在進行分析時可以降低離子抑制的效應，並且減少「訊號集中點」的產生，成功地改善MALDI基質使用上的缺點。而在樣品溶液中添加高濃度的檸檬酸銨緩衝溶液，能夠提供分析物質子化的來源，並且提升此質譜法之靈敏度及可偵測之質量範圍。目前此質譜法的質量偵測上限約為24 kDa，在分析胜肽時的靈敏度約為50 fmol，而偵測insulin時其靈敏度約為900 amol，並且可應用於含有高濃度鹽類樣品的去鹽及分析。 質譜法已經成為目前磷酸化蛋白質體研究的核心技術，然而由於蛋白質體的樣品十分複雜，再加上磷酸化胜肽的含量低且游離效率較差，因此在進行質譜分析前，需要適當的濃縮技術進行純化，而常用的親和層析法卻往往受限於專一性不高以及沖提過程中樣品損失或被稀釋的問題。本論文的第二部份是以溶膠凝膠法製備出具有核-殼結構的二氧化鈦及氧化鋁磁性奈米探針，利用探針表面包覆之二氧化鈦及氧化鋁與磷酸根之間的親和力，可以成功地濃縮複雜樣品中的磷酸化蛋白質或胜肽，並且直接以TiO2-SALDI-MS或是MALDI-MS/MS進行分析。而應用奈米探針本身的超順磁性，可以在抓取目標物後以外加磁場進行快速分離，有效地簡化操作的程序並且縮短時間。氧化鋁磁性奈米探針對於磷酸化胜肽的專一性要優於二氧化鈦磁性奈米探針，幾乎沒有非特異性吸附的現象產生，具有樣品體積需求量少、專一性高、靈敏度高及快速分析的優點，並且已經成功地應用於牛奶、蛋白液、細胞溶解物、人類血清等複雜樣品中微量磷酸化胜肽的濃縮萃取。基質的選擇對於磷酸化胜肽的分析結果有顯著的影響，若以CHCA做為MALDI基質，可以推測出胜肽序列中含有的磷酸化位置數目，而使用雙基質系統可以成功地將此方法的偵測極限降低至約2.5 fmol。 磷酸化蛋白質體的相對定量分析方法，目前是以同位素標記法結合質譜分析做為主要的工具，然而同位素標記的價格昂貴，並且可能會有反應不完全的問題。本論文的第三部份，是利用氧化鋁磁性奈米探針對於磷酸化物質具有的專一親和性，成功地發展出一種磷酸化蛋白質及胜肽的相對定量分析方法，當樣品溶液中存在磷酸化蛋白質、胜肽或小分子時，會與氧化鋁磁性奈米探針親和而與核黃素磷酸鈉螢光分子產生競爭，進而將核黃素磷酸鈉取代出來，因此可以利用溶液的螢光強度高低進行磷酸化樣品的相對定量分析。以磷酸化胜肽為例，此方法可相對定量的範圍約在10-8~10-6 M之間，並且具有良好的選擇性，而由於核黃素磷酸鈉螢光分子的修飾及取代步驟皆可應用微波輔助來加速反應的進行，可以有效地縮短實驗操作所需的時間，而分離後的奈米探針可再進一步以質譜進行定性分析，是一種簡單、快速、高專一性且不需標記的方法，並有潛力發展與癌症有關的磷酸化生物分子的快速篩檢。

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been widely used in the analysis for various types of analytes. The solubility and co-crystallization between analytes and matrices are the major concerns in MALDI analysis. In the first part of this thesis, we generated mesoporous titania sol-gel thin films with anatase nanocrystalline, which have good absorption capability at the wavelength of 337 nm and suitable to be used as the substrate in laser desorption mass spectrometric analysis. Therefore, an approach called TiO2 surface-assisted laser desorption/ionization mass spectrometry (TiO2-SALDI-MS) was proposed. Because the solubility and co-crystallization between analytes with matrices are not the concerns in this approach, ion suppression effects and sweet spot problems observed in conventional MALDI analysis are avoided. It was found that adding ammonium citric salts as the proton source in TiO2-SALDI samples can improve the sensitivity and the detectable mass range. Furthermore, ammonium citrate salts also play the role as the de-satling agents during TiO2-SALDI analysis. The upper detectable mass is about 24 kDa, and the detection limits for peptides and insulin are ca. 50 fmol and ca. 900 amol, respectively. Mass spectrometry has become the most powerful technique for phosphoproteomics analysis. However, because of the low abundance of phosphoproteins found in biological samples and ion suppression effect observed in mass spectrometric analysis for phosphopeptides, it is of significance to selectively enrich phosphopeptides prior to MS analysis. Affinity based chromatography has been employed to enrich phosphopeptides, but non-specific binding and sample loss during enrichment and elution steps remain as problems. In the second part of this thesis, titania and alumina coated magnetic nanoparticles (Fe3O4@TiO2 NPs and Fe3O4@Al2O3 NPs) are generated and used as the affinity probes for phosphoproteins and phosphopeptides. The target analytes trapped by the affinity probes can be characterized directly by TiO2-SALDI-MS or MALDI-MS/MS analysis. Based on the superparamagnetic properties of the affinity probes, the analyte-conjugated probes can be rapidly isolated by the external magnetic field. Additionally, it was found that the trapping performance of Fe3O4@Al2O3 NPs for phosphopeptides is superior to that of Fe3O4@TiO2 NPs. The specificity of Fe3O4@Al2O3 NPs for phosphopeptides is quite good. The results show that this approach can be used to enrich phosphorylated peptides/proteins from complex samples such as non-fat milk, egg white, cell lysate and human serum with high specificity and high sensitivity. The selection of the MALDI matrix is also crucial for the analysis of phosphopeptides. When using α-cyano-4-hydroxycinnamic acid (CHCA) as the MALDI matrix, the number of phosphorylation sites of phosphopeptides can be estimated based on their fragment ions. Furthermore, using a mixture of 2,5-DHB (dihydroxybenzoic acid) and CHCA as the matrix can lower the detection limit of phosphopeptides to ca. 2.5 fmol. Stable isotope labeling for quantitative phosphoproteins is generally used in mass spectrometric analysis. However, the cost of isotopes is high, and incomplete reactions may arise during isotope labeling. Thus, in the last part of this thesis, a method for quantitative phosphoproteomics analysis was developed. That is, a fluorescent reporter probes was fabricated by immobilizing riboflavin 5’-monophosphate, which is a dye with strong fluorescence, onto the surfaces of Fe3O4@Al2O3 NPs (RF-Fe3O4@Al2O3 NPs) via phosphate-alumina chelating. When RF-Fe3O4@Al2O3 NPs were added to a solution containing phosphorylated species, the competition binding between the RF molecules on the NPs and free phosphorylated species in the solution take places. As a consequence, RF molecules were replaced from the nanoparticles by the phosphorylated species and dissolved into the solution. On the basis of the fluorescence intensity that RF molecules contribute in the solution, the amount of phosphorylated proteins/peptides can be estimated. The dynamic linear range is in 0.01 ~1 μM. To shorten the analysis time, microwave-heating was used for the modification of RF molecules on the Fe3O4@Al2O3 NPs and dye-replacement by phosphorylated species. Additionally, qualitative analysis by MALDI MS also can be carried out by detecting the RF-Fe3O4@Al2O3 NPs immobilizing with phosphorylated species. This label-free approach is simple, fast, and cost-effective. In conclusion, the methods developed in this thesis have the potentials to be employed for phosphoproteomics analysis after further studies.