標題: 高通量酵素連結免疫吸附分析法(ELISA)測定蛋白質酪氨酸亞硫酸化
High-throughput enzyme-linked immunosorbent assay (ELISA) for the determination of protein tyrosine sulfation
作者: 林芝綺
Lin, Chih-Chi
楊裕雄
Yang, Yuh-Shyong
生物科技學系
關鍵字: 酵素聯結免疫吸附分析法;蛋白質酪氨酸亞硫酸化;轉譯後修飾作用;ELISA;Protein sulfation;Post-translational modification
公開日期: 2010
摘要: 蛋白質酪氨酸亞硫酸化作用,是被蛋白質酪氨酸亞硫酸化酵素所催化,這是一個普遍的轉譯後修飾作用(PTM)。大多數會被輸送到高基氏體網路的分泌及穿膜蛋白,都有可能被硫酸化。蛋白質亞硫酸化作用藉由改變蛋白質間的作用力進而調節許多生理與病理的反應,包括止血作用,白血球的運送,以及病毒的感染…等。因此,開發一個容易使用的平台,對於能夠廣泛的偵測TPST的酵素活性,以及檢測被亞硫酸化的蛋白質是非常重要的。傳統被用來偵測蛋白質亞硫酸化作用的方法包括繁瑣的放射性標記與質譜儀分析;然而,同位素的來源昂貴,以及在酸性條件下,酪氨酸殘基上的亞硫酸基團非常不穩定,這些都是對於一個高通量的分析法所需要令人擔心的因素。在這項研究中,我在酵素連結免疫吸附分析法(ELISA)上結合了PAPSS與TPST催化反應,利用GST-PSGL-1當作受質。此方法在最佳化的條件下,提供了高靈敏性、高通量、以及節省時間與花費的優點。利用ELISA為基礎的TPST分析系統,能夠被廣泛的用來尋找潛在的TPST受質以及抑制劑甚至是蛋白質間相互作用力之分析。此外,我們更能整合此系統到蛋白質晶片上,將有助於未來蛋白質體學的研究。
Protein tyrosine sulfation, catalyzed by tyrosylprotein sulfotransferase (TPST), is a prevalent post-translational modification (PTM). Most secreted and transmembrane spanning proteins, which transit a trans-Golgi network, are likely to be sulfated. Protein sulfation regulates numerous physiological and pathological processes including hemostasis regulation, leukocyte trafficking, and viral infection by changing the strength of protein-protein interaction. Accordingly, it is crucial to develop a facile platform to comprehensively determine TPST enzyme activity and examine the sulfated protein. Traditional protocols for detecting of protein tyrosine sulfation involve tedious radioactive labeling and mass spectrum. However, the costly isotope source and even the extremely labile sulfate group on the tyrosine residue under acidic condition are the concerns for high-throughput analysis. In this study, I combined PAPSS-coupled TPST catalyzed reaction with an enzyme-linked immunosorbent assay (ELISA) using GST-PSGL-1 as a substrate and developed a novel method for the detection of protein tyrosine sulfation. This method offered high sensitivity, high throughput, and savings in time and costs under the optimal condition. We are employing this assay system to discover TPST substrates, inhibitors and analyze protein-protein interaction. Furthermore, we may integrate our system to the protein chip experiments for the proteomics study in the future.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079828517
http://hdl.handle.net/11536/47726
Appears in Collections:Thesis