功能性的奈米碳包鐵粒子作為濃縮磷酸化胜肽與磷酸化蛋白質的親和性探針

摘要碳包覆磁性材料有很廣泛的應用，例如核磁共振的成像示劑、記憶的磁性材料、以及腫瘤熱療法。然而，比起在這類核殼式材料的製備合成方面上，本文的著重於這類核殼材料表面的官能基化的策略。在這篇論文中，我們分別所製備的修飾聚精胺酸與二氧化鈦在磁性奈米碳包鐵粒子，對於濃縮與分離磷酸化蛋白質或被酵素消化後的磷酸化胜肽能夠作為一個很好的親和性探針，這些樣品例如?-casein、?-casein和牛奶。聚丙烯酸修飾奈米碳包鐵，是一個很容易接枝其他的親和性分子的一個配位體，例如分別經由via carbodiimide化學方式修飾聚精胺酸與sol-gel化學方式修飾二氧化鈦，使用親和性探針在萃取磷酸化胜肽，只要短短10分鐘的時間，就能夠充分得到準確的資訊。磷酸化胜肽被親和性探針所萃取時，可以使用外部的磁場移除一些非特定吸附的胜肽與過程中所洗淨所使用一些的溶劑。最後，被吸附在粒子表面的磷酸化胜肽使用含 5% 磷酸的2,5-dihydroxybenzoic acid沖提下來後置入樣品盤，接著進基質輔助雷射脫附游離質譜儀分析。在粒子萃取後，大量非磷酸胜肽離子的訊號被抑制了，而藉由基質輔助雷射脫附游離質譜儀分析能夠靈敏性地偵測磷酸化胜肽，並且偵測的樣品濃度極限範圍為低 femtomol。在奈米碳包鐵表面這修飾聚精胺酸與二氧化鈦這兩種親和性配位體，對磷酸化胜肽萃取有一個很好的專一性與靈敏性。藉由基質輔助雷射脫附游離質譜儀快速地分析，由於這兩種型式的親和性探針所萃取到的磷酸化胜肽有一個互補的特性，所以在磷酸化蛋白質體學中可以得到更豐富的資訊。

關鍵字

奈米碳包鐵；聚精胺酸；二氧化鈦；磷酸化蛋白質體學

並列摘要

Abstract Carbon encapsulated magnetic nanoparticles have wide applications such as, serving as contrast reagents for magnetic resonance imaging (MRI), magnetic data storage medium, and magnetic hyperthermia. However, compared to the intense studies in the synthetic strategies for fabricating such core/shell materials, little effort has been devoted to the surface functionalziation strategies. In the report, we produced polyarginine-immobilized and titanium dioxide-immobilized carbon encapsulated magnetic iron nanoparticles (Fe@CNPs) as high affinity probes to extract and enrich phosphopeptides and phosphoprotein from a number of enzymatic digested products including α- and β-casein and milk sample. The poly(arcylic acid)-coated Fe@CNPs can be easily grafted with affinity ligands such as polyarginine (PA) or titanium dioxide via carbodiimide chemistry or sol-gel chemistry, respectively. For the capture of phosphopeptides, only a 10-s incubation time with affinity probes is sufficient to prove faithful information. The affinity probes with trapped phosphopeptides are then isolated by magnetic separation, cleaned with washing solvents to remove nonspecifically bound peptides. Finally, the trapped phosphopeptides were eluted with 2,5-dihydroxybenzoic acid (2,5-DHB) containing 5% phosphoric acid and deposited onto the MALDI target plate for analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). After enrichment, the ion suppression caused by abundant nonphosphopeptides is eliminated, and the detection sensitivity of phosphopeptides by MALDI MS analysis is in the low femtomol range Both polyarginine and TiO2-coated Fe@CNPs exhibits good specificity and sensitivity toward phosphopeptides. These two types of affinity probes process complementary properties for phosphopeptide mapping by MALID MS and can be combined to provide rich information for phosphoproteomic analysis.