Selective Enrichment of Multiply Phosphorylated Peptides Using Polyarginine-coated Nanodiamonds Prior to Liquid Chromatography Mass Spectrometric Analysis.

當今利用質譜來分析磷酸化胜肽仍存在著困難度，所以目前市面上有很多商品（例如：TiO2、IMAC），能夠分離純化磷酸化蛋白，進而提高後面質譜在鑑定磷酸化蛋白位置的效率，但目前卻多著重於單磷酸胜肽的鑑定，而本實驗室之前所發展出覆蓋著聚精胺酸的奈米鑽石探針，可藉由聚精氨酸與多磷酸化胜肽之間的高專一性結合特性，使其能夠選擇性的單一濃縮多磷酸化胜肽，但為了使之能夠應用於分析真實複雜系統上，我們必須條件化其分析環境及提供一個能有效率沖提其所濃縮的分析物的沖提試劑，以利於之後更廣泛的運用，所以本篇首要目標，即以提高上述探針之性能為中旨。 首先，為了提高奈米鑽石與覆蓋在上之聚精氨酸的鍵結效率，我們在緩衝溶液中進行鍵結反應，使反應效率達到最高值，次之，利用改變探針的萃取濃縮試劑之比例及環境的酸鹼值，來找出探針的最佳分析環境，再者，我們開發出一種高效能的沖提試劑，能有效率把分析物從探針上沖提下來的，並能適用於液相層析串聯質譜儀，有助於日後在磷酸化蛋白質體界中多磷酸化這塊領域的探討。 根據實驗結果顯示，我們已成功的把聚精氨酸覆蓋在奈米鑽石探針表面（平均粒徑86.9 nm），而覆蓋聚精氨酸後的奈米鑽石在去離子水下的表面電荷也由- 27.6 mV提升到+ 43.29 mV，並可發現在緩衝溶液下所合成的探針，其表面電位也比原本方法提升了許多（原本~ 0 mV），且，在不改變分析物的情況下，利用改變探針分析環境的條件，我們發現待測物在含有40 %乙腈及1 %的三氟乙酸的情況下，探針能在高複雜環境下（汙染物≧5000倍分析物），還能對分析物（多磷酸胜肽）有高度專一性的選擇，再者，我們也找到可利用含有12.5 %的氨水下的0.5 M 磷酸氫二鉀沖提劑，可輕易的在三十分鐘內有效率的把分析物從探針下沖提下來，並成功的利用探針把一含有三個磷酸的胰島素受器之激酶胜肽從高複雜環境中（酵素消化後牛血清白蛋白胜肽）濃縮萃取出來，且可利用液相層析質譜儀來檢視探針之萃取結果。 綜合以上，我們已成功的發展出一個對多磷酸化胜肽更具有高度專一性、選擇性且能應用高複雜系統中的奈米探針，並能提升以往較少應用於液相層析串聯式質譜儀（LC-MSMS）上辨別多磷酸胜肽序列的效率。Identification of multi-phosphorylated peptides by liquid chromatography-tandem mass spectrometry（LC-MS/MS）remains a challenging task due to the poor ionization of the species. For this reason, various kits to selectively enrich phosphopeptides prior to MS analysis have been developed and commercialized, but most of them are selective only for singly phosphorylated peptides. Interestingly, poly-arginine-coated nanodiamond（PA-ND）shows a high selectivity for multi-phosphorylated peptides and this has been demonstrated by Chang et al. For further applications in real sample analysis with LC-MS/MS, an effective elution buffer to separate the target molecules from the PA-ND substrate has yet to be found. In this work, we have successfully synthesized a much highly selective probe by coating PA on ND（average diameter: 86.9 nm → 140.6 nm）in 0.1 M MES buffer. The zeta potential in deionized water increased from -27.6 mV to +43.29 mV, better than the previous one, ~0 mV. Furthermore, we found a better enrichment condition, with 40 % acetonitrile containing 1 % trifluoroacetic acid, that allows us to selectively probe multi-phosphorylated peptides in very complex samples（with impurities ≥5000 higher than of the target analyte）. Finally, we also found that 12.5 % ammonium water containing 0.5 M potassium phosphate can easily elute the enriched substance from the probe. LC-MS/MS can therefore successfully identify a triply phosphorylated peptide extracted from a highly mixture by the newly synthesized PA-ND particles and with the newly developed protocols