Using functional iron oxide magnetic nanoparticles as the affinity probes to selectively enrich histidine-tagged and phosphorylated proteins and peptides

• Main Authors: Yi-Cheng Li, 李翊誠
• Other Authors: Yu-Chie Chen
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/10901443085236382173

碩士 === 國立交通大學 === 應用化學系所 === 94 === 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).