利用表面化學改質之奈米層狀結構矽基材晶片與四氧化三鐵磁性奈米粒子以提升磷酸化胜肽於質譜儀檢測之靈敏度之研究

近年來，透過組織工程，許多研究團隊於再生醫學新技術有了重大突破，以幹細胞修復受損組織成功的案例也逐年增加。然而幹細胞是否能應用於臨床治療，取決於適當的細胞調控，其和信號傳遞過程中伴隨的蛋白質磷酸化有著密不可分的關係。不幸的是，磷酸化蛋白質在細胞樣本內含量極低，分析前複雜的純化程序往往也在所難免。質譜儀可對未知生物分子提供珍貴的質荷比之定性與定量資訊，向來是蛋白質體學實驗及生物分析領域上一大利器。近年研究發現奈米結構多孔矽具有吸收紫外光雷射能力，作為輔助脫附游離的介質具有高靈敏度的表現，且在分析小分子樣本時不受基質所干擾。本研究成功研發出以多孔矽改良之奈米層狀結構矽（nanofilament silicon, nSi）質譜分析晶片作為基材，並以特定官能基如羧基、胺基、苯環或金屬親合等接枝於表面，直接分離樣本胜肽。再結合Fe3O4/TiO2 與Fe3O4/ZnO 核/殼結構奈米粒子，藉其表面與磷酸根之專一性吸附更可辨識出β-casein digest中的磷酸化胜肽，且偵測極限可達10-9 M以下。此法可簡化複雜的磷酸化胜肽純化程序並增加質譜分析的效能與選擇性，將可使幹細胞與再生醫學研究效率大幅提升。

關鍵字

表面化學改質；奈米層狀結構矽基材晶片；表面輔助雷射脫附質譜儀；磁性奈米粒子；磷酸化胜肽

並列摘要

In recent year, many research groups have made breakthrough in regenerative medicine through tissue engineering. The number of successful cases using stem cell to repair the damage organ also increases year by year. A appropriate cell regulations facilitate stem cell for possible application to clinical treatment, and the regulation deeply related to protein phosphorylation in cell transduction pathway. Unfortunately, low abundance of phosphorylated peptides and proteins in cell makes identification of phosphorylation sites difficult; hence a complicated purification process prior to analysis is usually unavoidable. Mass spectrometry (MS) is a useful tool to identify unknown biomolecules qualitatively and quantitatively in proteomics and other related bioanalysis field because it provides valuable mass to charge (m/z) information. In recent researches, silicon-based nanostructure are used as UV absorbing media to ionize biomolecules and show high sensitivity on low molecular weight biological samples without matrix background. In this study, we used nanofilament silicon (nSi) chips as a base which graft the different functional groups onto the chip surface such as carboxyl, amino, phenyl or immobilized metal affinity. We found those modification could separate sample peptides specificly. Phosphopeptide derived from β-casein digest has also been detected by nSi combining with Fe3O4/TiO2 and Fe3O4/ZnO core/shell nanoparticles, and the detection limit could reach 10-9 M. These approaches enable us to simplify the purification process of phosphorylated peptides and proteins, increase the selectivity and sensitivity of MS analysis, and make stem cell and regenerative medicine research more efficient.

並列關鍵字

nanofilament silicon (nSi) chip ； SALDI-MS ； magnetic nanoparticles ； phosphopeptide ； surface modification

參考文獻

1. Mason, C.; Dunnill, P., A brief definition of regenerative medicine. Regen Med 2008, 3 (1), 1-5.

2. Mason, C.; Manzotti, E., ''The Little Purple Book'': BSI Glossary of Regenerative Medicine. Regen Med 2009, 4 (4), 483-484.

3. Thomson, J. A.; Swaney, D. L.; Wenger, C. D.; Coon, J. J., Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry. P Natl Acad Sci USA 2009, 106 (4), 995-1000.

4. Blagoev, B.; Olsen, J. V.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M., Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127 (3), 635-648.

5. Hunter, T., Signaling - 2000 and beyond. Cell 2000, 100 (1), 113-127.

國際替代計量

利用表面化學改質之奈米層狀結構矽基材晶片與四氧化三鐵磁性奈米粒子以提升磷酸化胜肽於質譜儀檢測之靈敏度之研究

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