應用官能化磁性奈米粒子為含組胺酸標記及磷酸化蛋白質/胜肽之親和探針

Abstract

氧化鐵磁性奈米粒子因為本身具有高表面積／體積比例且具有超順磁性的特性，已被用為親和探針進行特定分析物之親和濃縮。在親和濃縮後，可藉由外加磁場快速將特定分析物從樣品溶液中分離出來，達到快速萃取濃縮目標物的目的。本論文已成功製作出兩種奈米探針，製作方法為先將金屬螯合劑－晴基三乙酸修飾在氧化鐵磁性奈米粒子的表面，再各螯合鎳離子(Ⅱ)與鋯離子(Ⅳ)於其上，可分別用作純化含組胺酸標記之重組蛋白質/胜肽以及含磷酸化胜肽之探針。 萃取方法為將螯合有金屬離子的磁性奈米探針 (30 μg) 混以50 μL的樣品溶液，利用微量吸管在樣品瓶中上下來回快速吸抽此混合物30秒後，即可以基質輔助雷射脫附游離質譜法進行分析。實驗結果顯示含鎳離子(Ⅱ)之奈米探針可成功地親和萃取樣品中含有組氨酸標記之蛋白質 (C192S) 與胜肽，偵測極限分別為10-7 M (50 μL) 與10-9 M (50 μL)。而含鋯離子(Ⅳ)之奈米探針可成功地親和萃取α-酪蛋白、β-酪蛋白、及牛奶的胰蛋白酵素消化產物中含磷酸化的胜肽片段，此方法對α-酪蛋白及β-酪蛋白消化產物之偵測極限約在10-9 M (50 μL) 左右。

On the basis of the high surface area to volume ratio and the superparamagnetic property, iron oxide magnetic nanoparticles have been widely used as the affinity probes for specific analytes. After trapping process, the affinity probes-target species conjugates can be readily isolated from the sample solution by employing an external magnetic field. The whole analysis time used in concentration and isolation is therefore dramatically reduced. In this dissertation, two types of metal ions immobilized iron oxide magnetic nanoparticles for enriching histidine (his)-tagged proteins and phosphorylated proteins and peptides were generated. Nitriacetic acid (NTA) was first immobilized on the surfaces of magnetic nanoparticles. NTA immobilized magnetic nanoparticles were capable of chelating metal ions onto their surfaces. Ni(II) and Zr(IV) ions were selected to be chelated by the NTA immobilized magnetic nanoparticles. Ni(II) immobilized magnetic nanoparticles were successfully employed to selectively enrich his-tagged proteins and peptides from complex samples such as cell lysates. Pipeting the sample solution in and out of a tip in a sample vial for only 30 sec could enrich sufficient target species for matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) analysis. The detection limit for a his-tagged peptide (6×his) is 10-9 M (50 μL), while the detection limit for a his-tagged protein (C192S) is 10-7 M (50 μL). Additionally, Zr(IV) ions immobilized magnetic nanoparticles have been demonstrated to have the capacity of enriching phosphoproteins and phosphopeptides such as α&β-caseins, milk samples, and their tryptic digestion products. The sample solution was enriched by pipeting the sample solution in and out of a tip in a sample vial for 30 sec. The results indicated that the detection limit for α&β-caseins is 10-9 M (50 μL).