Alpha-1-antitrypsin (AAT)甲一型抗胰蛋白酵素
在胃癌血漿中轉譯後修飾的表現

癌症在國人的十大死因連續三十一年排名第一,在台灣胃癌的死亡率是居癌症死因的第六位，然而當胃癌在癌症早期就被診斷出來時，其生存率高達95%以上，故一個能早期診斷胃癌的生物標記是必要的。鑑於此理由, 開發出新的診斷方法能提高診斷胃癌的特異性和敏感度,是迫切之急的任務。本篇研究主要在探討Alpha-1-Antitrypsin(AAT)甲一型抗胰蛋白酵素在血漿中轉譯後修飾的表現，以蛋白質體分析策略，找出有意義的轉譯後修飾拿來做為開發檢測胃癌的生物標記。透過比較健康人及胃癌患者血漿中，蛋白質量（表現量）及質（轉譯後修飾）的變化，找出可以運用到臨床的生物標記。在本研究中，實驗材料是收集正常人46 位和胃癌病人46 位的血漿檢體。先進行免疫沉澱法，確認抗體有抓到目標蛋白Alpha-1-Antitrypsin(AAT)，再使用一維凝膠電泳 (SDS-PAGE)和西方墨點法(Western blot)探討AAT 在胃癌血漿中的蛋白質表現量；之後,再利用膠體內水解(In-Gel digestion)的方式.將我們需要的蛋白質區塊在一維凝膠電泳(SDS-PAGE)上切下, 打入奈米級液相層析串連質譜儀(nano-LC/MS/MS)。分析蛋白質以trypsin 水解脢降解成胜肽碎片所得到的數據，推測胜肽的氨基酸序列。收集質譜資料後加以分析，資料庫比對後找出可能發生的修飾然後鑑定出轉譯後修飾的位置。實驗結果顯示AAT 的蛋白質表現量在胃癌早期與正常人相比沒有明顯的差異(P=0.186)，且在胃癌晚期與正常人相比也無明顯差異(P=0.091)。此外，AAT 的 ROC curve AUC 只有 0.660，靈敏度和特異性分別為 71.74%和63.04%，得知利用血漿中的蛋白質表現量無法分辨胃癌早晚期和正常人的差異,沒有明顯的變化。因此改為探討 AAT 的轉譯後修飾表現。發現 AAT 的胺基酸上有許多新穎性的修飾且倍數差異在三倍以上包含有methylation(Glu-370、Glu-378、Glu -387 Lys-367)、Hydroxylation(Asn-271、Pro-279、 Asp-280、Lys-283)、Dimethylation (Proline-385)和n-Decanoate(Serine -325)。所以結論是我們可以利用這些蛋白質轉譯後修飾的差異特性當作診斷胃癌的生物標記並且加以量化比較。

關鍵字

甲一型抗胰蛋白酵素；胃癌；轉譯後修飾

並列摘要

The leading cause of death is contributing to cancer in our country ranked over thirty-one consecutive years .Gastric cancer is the sixth leading cause of cancer deaths in Taiwan, yet survival rates are over 95% when it is diagnosed at an early stage, the need for biomarkers for early diagnosis can’t be overemphasized. So, to issue a new diagnostic method can improve the specificity and sensitivity of diagnosis of gastric cancer is very important. This study was to investigate the translational modifications (PTM) in plasma performance of Alpha-1-Antitrypsin (AAT). So we utilize proteomic analysis strategy, by comparing healthy people and patients with gastric cancer plasma protein quality and qualitative changes can be applied to identify clinical biomarkers. In the present study, we used some approaches including Immunoprecipitation ，nano-LC-MS/MS ， western blot to screen AAT biomarker in plasma samples which were obtained from 46patients with gastric cancer and 46 healthy volunteers. Then we study those statistics and PTMs in order to find out useful diagnostic tools. Experimental results indicate that neither early stage gastric cancer group nor late stage gastric cancer group show no significant differences compared with Normal group. Thus, to use the plasma protein expression can’t distinguish between cancer and normal differences between early and late cancer stage. So we change the side to observe the post-translational modifications of AAT performance. The PTMs data showed there were a lot of PTMs in gastric cancer，including methylation(Glu-370、Glu-378、Glu -387 Lys-367)、Hydroxylation(Asn-271、Pro-279、Asp-280、Lys-283)、HexHexNAc(Threonine-369)、Dimethylation (Proline-385)and n-Decanoate(Serine -325)。The ROC curve shows that those modifications are perfect tools to identify gastric cancer。In conclusion，those PTMs will be good biomarkers that we can use these proteins characteristic differences to screen cancer and quantify it。