臺灣博碩士論文加值系統

現今利用質譜來分析磷酸化蛋白質或胜肽仍然存在著困難度，所以市面上有很多
已經販售的商品(例如:IMAC、TiO2)，能夠在質譜分析前，將磷酸化蛋白質及磷酸化
胜肽做一個濃縮萃取的前置效果，以利於後面質譜在鑑定磷酸化位置的便利。
本篇以上述目標為方針，利用EDC試劑把表面充滿著羧基的奈米鑽石和聚精胺酸
上的胺基連結形成共價鍵，藉由聚精氨酸與磷酸化蛋白質及磷酸化胜肽之間所具有的
高專一性結合特性，使覆蓋精氨酸的奈米鑽石成為能夠選擇性的濃縮萃取磷酸化蛋白
質及磷酸化胜肽的功能性探針。為了證明功能性粒子對於磷酸化蛋白質及磷酸化胜肽
的高選擇性和專一性，我們利用了經過蛋白酶切割作用後的α-casein、β-casein和牛奶
的胜肽溶液，加入20 μg的功能性探針粒子到溶液中作濃縮萃取，來證實之。
實驗結果顯示覆蓋著聚精胺酸的奈米鑽石粒子不但對於磷酸化胜肽有很好的專一
選擇性，也能在極稀薄的溶液中還是可以抓取到磷酸化胜肽(50 fmol) ，且由結果發現聚精胺酸@奈米鑽石粒子有個偏好，喜歡去抓有多個位置磷酸化的胜肽，這個喜愛是跟一般市售產品有很大的不同。
由實驗證實了利用覆蓋著聚精胺酸的奈米鑽石粒子具有對磷酸化白質及磷酸化胜
肽的專一性、選擇性、靈敏性。且抓取”多”磷酸化胜肽的能力也比一般市售的產品
來的好。這不但有助於研究磷酸化蛋白質體學多了一個新的濃縮萃取策略，也希望能
夠更廣泛運用在磷酸化蛋白質體學上。

Mass spectrometric identification of phosphoproteins and their phosphorylation sites is challenging , due to their low abundance in nature. Therefore, it is essential to perform an enrichment strategy prior to MS analysis.
In this work, we employed polyarginine (PA) - coated diamond nanoparticles (nominal size 100 nm) as high affinity probes to selectivity capture phosphoproteins / phosphopeptides from a complex solution of tryptic digested proteins. Compared to IMAC and MOAC methods for phosphopeptide enrichment, The polyarginine-coated nanodiamonds show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 μL) of tryptic digests of proteins such as α-casein、β-casein and non-fat milk at a concentration as low as 1 × 10-9 M. The concentration is much lower than that can be achieved by using other commercial technologies kits.
This work presents a new strategy that not only effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. The new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in both complex and diluted environments.

I. 中文摘要
II. 英文摘要
壹、緒論
1.1 磷酸化的重要性．．．．．．．．．．．．．．．．．．．．．．．．．．1
1.2 鑑定磷酸化的方法．．．．．．．．．．．．．．．．．．．．．．．．．3
1.3 利用基質輔助雷射脫附法(MALDI)質譜法分析磷酸化蛋白質上的問題．．．4
1.4 純化磷酸化胜肽或磷酸化蛋白質的方法．．．．．．．．．．．．．．．．5
1.5 奈米鑽石近年發展與應用．．．．．．．．．．．．．．．．．．．．．．8
1.6 磷酸基團與精胺酸之間的作用力．．．．．．．．．．．．．．．．．．．10
貳、實驗目的．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 12
参、實驗
3.1 儀器裝置．．．．．．．．．．．．．．．．．．．．．．．．．．．．．13
3.2 實驗藥品．．．．．．．．．．．．．．．．．．．．．．．．．．．．．15
3.3 羧基-奈米鑽石的製備 ．．．．．．．．．．．．．．．．．．．．．．．16
3.4 聚精胺酸-羧基奈米鑽石的製備 ．．．．．．．．．．．．．．．．．．．18
3.5 α-casein、β-casein的酵素消化步驟．．．．．．．．．．．．．．．．19
3.6 牛奶的酵素消化流程．．．．．．．．．．．．．．．．．．．．．．．．20
3.7 利用覆蓋聚精胺酸的羧基奈米鑽石來濃縮萃取已用酵素消化的α-casein、β- casein、牛奶的步驟 ．．．．．．．．．．．．．．．．．．．．． ．21
3.8 基質溶液的配製 ．．．．．．．．．．．．．．．．．．．．．．．．．22
3.9 多磷酸化胜肽喜好實驗步驟．．．．．．．．．．．．．．．．．．．．．23
3.10 Bruker Daltonics產品(#221865) Phosphopeptides Capturing Kit
MB-IMAC Fe來濃縮萃取已用酵素消化的β-casein的步驟． ．．．．．．24
3.11 Perkin-Elmer 產品(Cat.#PRT301001KT) Phos-trap Phosphopeptide
Enrichment Kit來濃縮萃取已用酵素消化的β-casein的步驟 ．．．．26
3.12 回收率測定的實驗步驟．．．．．．．．．．．．．．．．．．．．．．．27
3.13 磷酸化蛋白質濃縮萃取的製備與步驟．．．．．．．．．．．．．．．．．29
肆、結果與討論
4.1 羧基奈米鑽石與聚精胺酸-羧基奈米鑽石的結果討論．．．．．．．．．．．31
4.2 利用覆蓋聚精胺酸的羧基奈米鑽石來濃縮萃取已用酵素消化的α-casein
、β-casein、牛奶的結果．．．．．．．．．．．．．．．．．．．．．．33
4.3 證明聚精胺酸@羧基奈米鑽石在高濃度的變性試劑、鹽類和非磷酸化蛋白質
也能濃縮萃取磷酸化蛋白質的結果 ．．．．．．．．．．．．．．．．．．43
4.4 聚精胺酸@羧基奈米鑽石來濃縮萃取已用酵素消化的β-casein，在不同廠牌
MALDI-TOF MS 上分析出來的偵測極限比較 ．．．．．．．．．．．．．46
4.5 空白實驗結果 ．．．．．．．．．．．．．．．．．．．．．．．．．．．48
4.6 聚精胺酸@羧基奈米鑽石對於多磷酸化胜肽喜好實驗的結果．．．．．．．．51
4.7 以酵素消化過的 1 pmol β-casein 為範例，比較市售的IMAC、TiO2和我們的
聚精胺酸@羧基奈米鑽石粒子濃縮萃取同樣品效果的差異結果．．．．．．．59
4.8 回收率測定的結果 ．．．．．．．．．．．．．．．．．．．．．．．．．61
4.9 聚精胺酸@羧基奈米鑽石對於磷酸化蛋白質的飽和吸附力之測定的結果．．．66
4.10 磷酸化蛋白質濃縮萃取的結果 ．．．．．．．．．．．．．．．．．．．．67
伍、結論 ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．71
陸、未來與展望 ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．72
柒、參考資料 ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．73
捌、附錄 ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．76

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