利用磁性奈米複合材料作為樣品前處理及蛋白質消化以應用於質譜分析

Abstract

隨著蛋白質體學的興起，蛋白質身分鑑定的重要性與日漸增，為了更有效的確定不同種類的蛋白質，將蛋白質消化成為胜肽後送入資料庫比對是目前最常用的蛋白質身分鑑定方法，傳統上利用蛋白質水解酶水解的方法步驟冗長且繁複，完成整個流程需要超過數小時，如此費時又費力的蛋白質消化步驟限制了大量的蛋白質體分析作業，因此越來越多的研究著重於發展快速且方便的蛋白質消化方法。 使用磁性奈米粒子來加速蛋白質消化為近幾年來新興的方法，磁性奈米粒子所含的金屬性具有良好吸熱及傳導熱能的功效，為了加速水解酶水解蛋白質的速率必須要提高能量以克服水解酶反應的活化能。微波輔助具有瞬間能提高能量釋出熱能的特性，因此結合微波及磁性奈米粒子已被證實為可有效加速蛋白質消化的方法。為了更有效的提升水解酶的催化速率，本方法加入甲殼素包覆之磁性奈米粒子並於表面進行金還原反應，如此可更有效的增進奈米金的包覆形成金奈米磁性粒子。在原先的氧化鐵奈米粒子上增加一層金薄膜可提高奈米粒子的金屬性，接著再利用金硫的鍵結修飾上11-mercapto undecanoic acid及水解酶。將此磁性奈米複合材料加入蛋白質溶液中使用微波輔助加熱，將上層液取出後利用液相層析-電灑游離質譜分析消化的胜肽，發現金奈米磁性粒子修飾水解酶的材料微波一分鐘的消化效率與傳統消化方法效率比較有更佳的趨勢，再與先前研究提出氧化鐵磁性奈米粒子上直接修飾水解酶的材料在相同微波條件及時間下相比，消化效率有顯著的提升，如此可證明金薄膜的金屬性確實有發揮其吸收微波迅速加溫的功效以提升消化效率。

此種將水解酶修飾在磁性奈米粒的的材料具有可直接使用磁鐵分離的特性，如此不僅可減少水解酶在質譜分析上的干擾訊號，更可達成回收再利用的特性。 另外我們也提出一種利用磷酸基在氧化鐵磁性奈米粒子上修飾長碳鏈的複合材料，此材料具有比用矽烷基修飾的粒子水溶性高且不易水解的特性，修飾上的長碳鏈官能基能與非極性蛋白質形成物理吸附作用，接著用水清洗掉內含的極性鹽類，如此可降低鹽類訊號在質譜儀分析的干擾訊號以提高蛋白質在質譜儀上分析的訊號，如此可更有效的提高蛋白質的分析。

With the acceleration of proteome research, the technology for protein identification become more important. In order to identify the different kind of protein effectively, using proteolytic digestion of protein and sequence blast of database is the main method recently. However, The conventional practice of protein digestion in solution is time-consuming and labor-intensive. The whole protocol would spend over sixteen hours, limiting the large-scale proteome analysis. Thus, several approaches have been developed for rapid and efficient protein digestion. One popular approach is the use of magnetic nanoparticle to accelerate the protein digestion. The metal property of magnetic nanoparticle contain the good ability of heat absorbance and heat conductivity. microwave-assisted could be further accelerated by magnetite beads ,which had been proven to be excellent absorbers of microwave radiation. Thus, considering the advantages of immobilization of enzyme on magnetic spherical particles, as well as the convenience and rapid-fashion of microwave-assisted digestion approach, it is feasible to develop a highly efficient digestion method by the combination of both. In order to overcome the activation energy of protease effectively, we synthesis the Au core-shell magnetic nanoparticle to erase the metal property. Using this magnetic nanocomposites materials to combine with microwave-assisted to digest the protein, and using the Liquid-chromatograph-electrospray ionization tandom mass to analyze the sample, we find the digestion efficiency of trpsin-immobilized coreshell nanoparticle is better than only trypsin digestion. Comparing with the previous research which using the trypsin-immobilized nanoparticle material in the same microwave condition and time, the digestion efficiency increase a lot. This material which immobilized trypsin after using can use the magnet to separate, it not only can reduce the disturbance singnal of trypsin but also can be used recyclablely. On the other hand, we also develope a magnetic nanocomposites materials which modify the n-alkyl group by using the phosphonic acid. This material is characterized with high solubility and stability. The n-alkyl modification can bind to nonpolar protein by hydrophobic interaction and exclude the polar salt. This alkyl-functionalized magnetic nanoparticle can reduce the salt signal and enhance the protein analysis.