發展奈米探針用以快速和專一性純化硫基亞硝基化胜肽

Development of Affinity Nano Probes for Rapid and Specific Enrichment of S-nitrosylated Peptides

楊牧軒

指導教授 ： 林俊成

國立清華大學/理學院/化學系/碩士(2013年)

摘要

一氧化氮分子藉由與半胱胺酸上的硫基形成可逆共價鍵結，可調節蛋白質的活性和功能進而影響多種生理機制。本論文的目標是發展便捷的方式從複雜樣品中純化硫基亞硝基胜肽。我們將文獻中兩種硫酯基磷探針與兩種固相載體磁性奈米粒子及珠狀瓊脂糖結合，經由追蹤式還原接合亞硝基化硫基反應，將一級RSNO轉換成相對穩定的雙硫鍵產物，應用於純化和鑑定RSNOs。將亞硝基化標準胜肽PTP1B與BSA複雜樣品混合後，探針可成功的純化出PTP1B，並得知珠狀瓊脂糖在複雜樣品中能夠減少分專一性吸附。使用2nd_TEP@agarose探針，已成功從COS-7細胞間質液中純化出能被亞硝基化的胜肽。硫酯基磷探針在研究亞硝基化蛋白上具有極大的潛力，因此我們接著改變探針結構，合成出另一種硫酯基磷探針，探討結構對RSNO反應性的影響。

關鍵字

硫基亞硝基化胜肽 ； 奈米探針 ； 親合性探針 ； 磁性奈米粒子

並列摘要

Nitric oxide (NO) is covalently attached to cysteine thiols of proteins resulting in the formation of S-nitrosothiols (RSNOs), and regulation of protein activity and function in a wide range of physiological processes. The objectives of this thesis are development of probes for the enrichment of S-nitrosylated peptides. We conjugated two types of thioester phosphine ligand to either magnetic nanoparticle or agarose bead, respectively for identification and purification of RSNOs. Furthermore, through traceless reductive ligation of S-nitrosothiol mechanism the unstable primary RSNOs were converted to stable disulfide-iminophosphorane products. The S-nitrosylated PTP1B peptide mixed with tryptic BSA mixture was successfully captured by developed probe and identified by MALDI-TOF. In addition, the agarose beads show less non-specific interaction to tryptic BSA peptide. The enrichment of S-nitrosylated peptides from COS-7 cell lysate was achieved by 2nd_TEP@agarose. Thus, This strategy is a potential tool for investigate of S-nitrosylation proteins. Furthermore, we synthesized other type of thioester phosphine ligand by changing the original structure and investigate the effect of probe structure on the reactivity to RSNO.

並列關鍵字

S-nitrosylated Peptides ； Nano Probes ； Affinity probes ； magnetic nanoparticle

參考文獻

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