- 學位論文
利用質譜分析技術自鱉蛋水解物中篩選具有抑制酪胺酸磷酸酶活性之磷酸化胜肽
Screening of protein tyrosine phosphatase 1B inhibitory phosphopeptides derived from enzymatic hydrolysates of Pelodiscus sinensis using mass spectrometry
摘要
糖尿病為一種常見的新陳代謝疾病，尤以胰島素阻抗為表徵的第二型糖尿病為主，酪胺酸磷酸酶(PTPT1B)已被證實為影響胰島素訊息傳遞之負向調控因子。因此，酪胺酸磷酸酶之抑制劑，已逐漸成為治療第二 型糖尿病之新藥標的。 鱉(Pelodiscus sinensis)，俗稱甲魚，富含豐富的營養成分具有提高人體免疫力以及促進新陳代謝之功效。根據文獻記載鱉蛋水解物中的短鏈胜肽，具備有效的降低血壓之保健功能。然而，鱉蛋蛋白水解物中磷酸 化胜肽之活性則尚未被驗證。 本研究之目的在於建立鱉蛋磷酸化胜肽最佳純化方法並篩選製備具抑制酪胺酸磷酸酶(PTP1B)活性之磷酸化胜肽。在本研究中利用嗜熱菌蛋白酶(Thermolysin)及胰蛋白酶(Trypsin)兩種不同酵素將鱉蛋蛋白進行水解後，透過45μm、5μm及14nm三種不同粒徑的二氧化鈦(TiO2)純化製備取得磷酸化胜肽，進而評估其PTP1B抑制活性。結果顯示，兩種酵素水解物，經由45μm二氧化鈦的純化下具有較高的PTP1B抑制活性。將水解物用質譜儀鑑定出其磷酸化胜肽後，挑選出源自於鱉蛋主要蛋白vitellogenin-1-like具有相同序列但磷酸化位點不同之胜肽，並利用固相胜肽合成儀合成出標準胜肽加以進行身分驗證，並進行PTP1B活性測試，結果顯示，具兩個磷酸根之胜肽FK10-PP具有較佳之IC50值83.41µM±5.26µM。酵素抑制動力學研究，FK10-PP為非競爭型抑制，且本研究利用分子模擬對接(Molecular docking)模擬FK10-PP與PTP1B之作用，結果顯示，FK10-PP所對接之位點非PTP1B之活性中心，進一步證實FK10-PP為非競爭型抑制劑。本研究結果證實，鱉蛋經嗜熱菌蛋白酶水解所生成之磷酸化胜肽具有抑制PTP1B之活性，有利於未來開發降血糖功效之保健食品。
並列摘要
Diabetes is one of the common metabolic diseases. Insulin resistance is the major factor responsible for type 2 diabetes, and protein tyrosine phosphatase 1B (PTPT1B) has been shown to be a negative regulator of insulin resistance. Therefore, the inhibitors of protein tyrosine phosphatase 1B can be considered as a therapeutic target of type 2 diabetes. Pelodiscus sinensis, commonly known as the soft-shelled turtle, is rich in nutrients that enhance the body's immunity and promote metabolism. According to previous study, peptides derived from soft-shelled turtle hydrolysate showed health benifits for lowering blood pressure. However, the activity of phosphorylated peptides derived from soft-shelled turtle egg has not yet been reported. The aim of this study was to establish an optimal method for the purification of phosphorylated peptides from the hydrolysate of soft-shelled turtle yolk (SSY) proteins and to screen and prepare phosphorylated peptides that can inhibit the activity of protein tyrosine phosphatase 1B. The SSY proteins were hydrolyzed using thermolysin or trypsin, and then purified using titanium dioxide beads (TiO2) of 5 μm, 45 μm and 14 nm in diameter, respectively. The activities of phosphorylated peptides were then evaluated for in vitro PTP1B inhibitory activity. The results indicated that both enzymatic hydrolysates after TiO2 (45μm) enrichment showed the best PTP1B inhibitory activity.The hydrolysate was identified by a mass spectrometer as a phosphorylated peptide. Pick out peptides from the Soft shell turtle eggs vitellogenin-1-like that have the same sequence but different phosphorylation sites, and synthesize standard peptides to perform PTP1B activity tests. The phosphate peptide FK10-PP has a preferred IC50 value of 83.41 μM ± 5.26 μM. The results of enzyme inhibition kinetics showed that FK10-PP was a non-competitive type. In this study, molecular docking was used to simulate the interaction between FK10-PP and PTP1B. The results showed that the docking site of FK10-PP was not in PTP1B active site. It was further confirmed that FK10-PP is a non-competitive inhibitor. In this study, we concluded that thermolysin hydrolysate of SSTE contained the phosphorylated peptides which is potential for food development.
參考文獻
延伸閱讀
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