臺灣博碩士論文加值系統

本論文設計及合成出一種以奈米鑽石為固相載體，表面修飾上鋯金屬離子（Zr4+）及聚精胺酸高分子（polyarginine）的新型多功能性複合奈米粒子（zirconium ion and polyarginine functionalized nanodiamonds，PA-ND@PSS-Zr4+）。我們以奈米鑽石當為載體，修飾上帶有正電胍基團的聚精胺酸高分子，合成出聚精氨酸奈米鑽石（PA-ND），再利用聚精氨酸表面帶有正電胍基團與苯乙烯磺酸表面帶有負電磺酸基團的多價庫倫靜電結合作用力，將聚（4-苯乙烯磺酸鈉）（Poly（sodium 4-styrenesulfonate））修飾到聚精氨酸奈米鑽石（PA-ND）上，合成出表面具有聚苯乙烯磺酸鈉-聚精氨酸共修飾的奈米鑽石探針（PA-ND@PSS），最後利用聚苯乙烯磺酸對金屬離子的高親和性吸附能力，將帶正電的鋯金屬離子（Zr4+）修飾到聚苯乙烯磺酸鈉-聚精氨酸奈米鑽石（PA-ND@PSS）上，合成出鋯離子-聚苯乙烯磺酸鈉-聚精氨酸奈米鑽石（PA-ND@PSS-Zr4+），並將其應用於濃縮及選擇性分離水溶液中磷酸化胜肽。我們使用粒徑儀、界面電位儀、元素分析儀、紫外光/可見光光譜儀、金屬離子EDTA滴定法，來量測所合成的新型複合奈米粒子的表面及結構特性。
我們將所合成的粒子（PA-ND@PSS-Zr4+）應用於萃取β-酪蛋白（β-casein）酵素水解溶液中的磷酸化胜肽。由實驗結果顯示，針對β-酪蛋白酵素水解溶液進行萃取時，使用基質輔助雷射脫附游離-飛行式質譜儀（MALDI-TOF MS）進行樣品分析，磷酸化胜肽的質譜訊號的最低偵測極限可以達到50 fmol。 在β-casein 和牛血清白蛋白（Bovine serum Albumin，BSA） 的蛋白質混合物的酵素水解溶液的混和物實驗中。實驗結果顯示，即使 β-casein : BSA 的莫爾比例達到1 : 1000倍的基質複雜環境中，質譜結果顯示奈米粒子仍能選擇性的針對樣品中的磷酸化胜肽進行分離。在針對真實樣品（低脂牛奶）的酵素水解溶液進行分析時，可以看到 3組來自β-酪蛋白中磷酸化胜肽片段的訊號。我們所合成的複合奈米粒子表面具有雙重官能基，可以利用不同的表面官能基對磷酸化胜肽片結合能力具有差異性的特點，使用不同離子強度的洗提緩衝溶液（elution buffer）， 可以將結合能力不同的磷酸化胜肽片段，分批次脫附下來，在使用第一組洗提緩衝溶液（12.5 % NH4OH）時，可以看到4組鍵結能力較弱的單磷酸化胜肽的訊號，再使用第二組洗提緩衝溶液（0.2 M DHB in 67% ACN/5% Phosphoric acid ）進行脫附時，可以看到2組鍵結能力較強的多磷酸化胜肽的訊號，相較於單純使用DHB 當洗提緩衝溶液時，能夠偵測到數量更多的磷酸化胜肽片段的訊號。
我們所合成的雙官能基奈米複合材料（PA-ND@PSS-Zr4+），具有製備方法簡單、快速，並且將來能輕易置換不同類型的金屬離子，可以做為一個具多功能性的奈米探針，應用於在磷酸化胜肽的分離及濃縮，有助於磷酸化蛋白質體學的研究。

In this work, we have developed a facile method for efficient production of metal-ion and polyarginine co-functionalized nanodiamonds （denotes as PA-ND@PSS-Mn+） as a novel IMAC platform for phosphoproteomic analysis. Polyarginine-coated nanodiamonds （PA-ND） were mixed with poly-styrenesulfonate（PSS） via multivalent electrostatic interactions. Immobilization of zirconium ion on the surface of poly-arginine/poly-styrenesulfonate（PSS） functionalized nanodiamonds was done by a 30-min incubation in ZrCl4 solution at room temperature. The PA-ND@PSS-Zr4+　was further characterized using particle size and zeta-potential analyzer, energy dispersive spectroscopy, and EDTA titration, and its performance for selective enrichment of phosphorpeptides was evaluated using the tryptic digests of a complex BSA/β-casein protein mixture in a molar ratio of 1000:1. Experimental results showed that PA-ND@PSS-Zr4+ can be applied for selective identification of singly phosphorylated peptides as well as multiply phosphorylated peptides via a two-step elution process.　 The obtained zirconium ion and polyarginine co-functionalized nanodiamonds were successfully applied to the selective enrichment of both standard protein digests and real samples.

目次
謝誌 I
摘要 II
Abstract IV
目次 V
圖目次 VII
表目次 X
第一章 緒論 1
1.1 前言 1
1.2 後轉譯修飾 1
1.3 蛋白質磷酸化的重要性 2
1.4 質譜儀應用於蛋白質體分析 3
1.5 基質輔助雷射脫附離子化飛行時間質譜儀（Matrix Assisted Laser Desorption / Ionization-Time of Flight Mass Spectrometer） 5
1.6 使用質譜儀分析磷酸化蛋白質所遇到的問題 7
1.7 純化磷酸化蛋白質與胜肽的方法 7
1.8 聚精胺酸修飾奈米鑽石 11
1.9聚精胺酸奈米鑽石對磺酸基團的鍵結 12
1.10 鋯金屬離子固定在奈米粒子上 13
第二章 實驗目的 14
第三章 實驗方法 15
3.1 實驗藥品 15
3.2 儀器裝置 17
3.3 Microwave-Assisted Surface Functionalization of Nanodiamonds （ND）. 20
3.4 Preparation of Zr4+- immobilized nanodiamonds （ND- Zr4+） . 21
3.5 Preparation of Polyarginine-Coated Nanodiamonds （PA-ND） . 21
3.6 Preparation of Poly（sodium 4-styrenesulfonate）/Polyarginine co-functionalized Nanodiamonds （PA-ND@PSS）. 22
3.7 Preparation of PA-ND@PSS-Zr4+. 22
3.8 Titrimetric determination of zirconium 22
3.9 Tryptic Digestion of Proteins 23
3.10 Phosphopeptide enrichment using ND-Zr4+ and PA-ND@PSS-Zr4+. 23
3.11 Mass spectrometric analysis. 24
第四章 結果與討論 25
4.1粒子的粒徑與界面電位量測 25
4.2粒子的能量散射光譜儀（EDS）測定 28
4.3聚苯乙烯磺酸鈉-聚精氨酸奈米鑽石對鋯離子的吸附量 29
4.4蛋白質標準品中磷酸化胜肽的質譜分析 30
4.5粒子對磷酸化胜肽的偵測靈敏度 32
4.6 利用粒子萃取複雜環境中磷酸化胜肽的質譜分析 33
4.7粒子對磷酸化胜肽的飽和吸附量估計 34
4.8 利用粒子萃取真實樣品（低脂牛奶）中磷酸化胜肽的質譜分析 37
4.9 比較鋯離子-羧基奈米鑽石（ND-Zr4+）和鋯離子-聚苯乙烯磺酸鈉-聚精氨酸奈米鑽石（PA-ND@PSS-Zr4+ ）的表徵以及對萃取標準品的效果 40
4.10 PA-ND@PSS-Zr4+ 奈米粒子修飾上的官能基對萃取標準品中磷酸化胜肽的影響 42
4.11 PA-ND@PSS-Zr4+ 奈米粒子萃取標準品中磷酸化胜肽利用不同的脫附溶液進行兩次脫附 44
4.12 PA-ND@PSS-Zr4+ 奈米粒子萃取真實樣品（低脂牛奶）中磷酸化胜肽利用不同的脫附溶液進行兩次脫附 46
第五章 結論 50
參考文獻 51

圖目次
Figure 1 Protein kinases and protein phosphatases6. 2
Figure 2 Reversible protein phosphorylation regulates most aspects of cell life. 3
Figure 3 Mass Spectrometry Instrument Design. 4
Figure 4 Matrix-Assisted Laser Desorption Ionization - Time of Flight Process11. 5
Figure 5 MALDI analyses with a time of flight mass analyzer12. 6
Figure 6 Chemically replace these phosphates by biotinylated moieties18. 8
Figure 7 Phosphoramidate chemistry19. 8
Figure 8 Typical methods for Phosphopeptide enrichment（a）Immobilized metal ion affinity chromotography （IMAC） （b）Metal oxide affinity chromotography （MOAC）25. 10
Figure 9 Structure of arginine28. 11
Figure 10 Arrangement of phosphate groups about the guanidine group to form an “arginine fork”28. 11
Figure 11 The structure of methyl sulfonate with guanidine28. 12
Figure 12 Functionalization of ND surfaces using oxidative methods34. 20
Figure 13 Synthesis of Polyarginine-Coated Nanodiamonds （PA-ND）26. 21
Figure 14 Hydrodynamic diameters of ND、ND-Zr4+ 、PA-ND、 PA-ND@PSS、 PA-ND@PSS-Zr4+. 26
Figure 15 Zeta-potential measurements of ND、ND-Zr4+、PA-ND、PA-ND@PSS、PA-ND@PSS-Zr4+. 27
Figure 16 Energy dispersive X-ray （EDX） analysis of the ND-Zr4+ . 28
Figure 17 Energy dispersive X-ray （EDX） analysis of the PA-ND@PSS-Zr4+. 29
Figure 18 MALDI-TOF mass spectra of the tryptic digest of β-casein （1 pmol）. After enrichment by （a） ND and （b） ND-Zr4+ and （c） PA-ND@PSS-Zr4+. 31
Figure 19 MALDI-TOF mass spectra of the tryptic digest of β-casein After enrichment by （a） ND-Zr4+ （300 fmol ） and （b） PA-ND@PSS-Zr4+（200 fmol ）and （c） PA-ND@PSS-Zr4+（50 fmol ）. 32
Figure 20 MALDI-TOF mass spectra of the tryptic digest mixture of β-casein （1 pmol） and BSA. （a） Direct analysis of the peptide mixture at a molar ratio of 1 : 100; after enrichment by ND-Zr4+ at molar ratios of （b） 1 : 200; after enrichment by PA-ND@PSS-Zr4+ at molar ratios of （c） 1 : 500, and （d） 1 : 1000. 34
Figure 21 Sequential enrichment of phosphopeptides from tryptic β-casein digest by ND-Zr4+ （20 μg per extraction）. 35
Figure 22 Sequential enrichment of phosphopeptides from tryptic β-casein digest by PA-ND@PSS-Zr4+（20 μg per extraction）. 36
Figure 23 MALDI-TOF mass spectra of tryptic digests of the nonfat milk （a） Direct analysis; after enrichment by （b） ND-Zr4+ （c）PA-ND@PSS-Zr4+. 38
Figure 24 MALDI-TOF mass spectra of tryptic digests of β-casein （1 pmol）; after enrichment by （a）PA-ND （b）PA-ND@PSS （c）PA-ND@PSS-Zr4+. 43
Figure 25 MALDI-TOF mass spectra of tryptic digests of β-casein （1 pmol）, obtained with enrichment using PA-ND@PSS-Zr4+ followed by （a） only DHB elution and （b） first-step NH4OH elution and （c） second-step DHB elution. 45
Figure 26 MALDI-TOF mass spectra of tryptic digests of low-fat milk; （a） direct analsis, obtained with enrichment using PA-ND@PSS-Zr4+ followed by （b） only DHB elution and （c） first-step NH4OH elution and （d） second-step DHB elution. 47
Figure 27 Comparison of the peak intensities of identified phosphopeptides from tryptic milk protein digests obtained with enrichment followed by only DHB elution or stepwise elution with NH4OH and DHB. 49


















表目次
Table 1 Some common and important post-translational modifications2. 1
Table 2 Reported and Calculated Values for Acid Dissociation Constants （pKa） of Methyl Acids with Various Acid Functional Unitsa28. 13
Table 3 Identified phosphopeptides from -casein digest by MALDI-MS analysis. 31
Table 4 List of phosphorylated peptides identified from the low-fat milk digests in the MALDI-TOF mass spectra using ND-Zr4+ and PA-ND@PSS-Zr4+. The phosphorylation sites are indicated in bold italic text. The phosphorylated peptides were derived by tyrptic digestion of a-casein S1 （α-S1） and S2 （α-S2） andβ-casein （b）. 39
Table 5 Characterization of the ND-Zr4+ and PA-ND@PSS-Zr4+ affinity absorbents 41
Table 6 List of phosphopeptides from tryptic digest of non-fat milk sequentially eluted from PA-ND@PSS-Zr4+ 48

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