A1-抗胰凝乳蛋白酵素在胃癌和大腸直腸癌血漿中轉譯後修飾的表現

世界衛生組織2012 年統計資料中，全球大腸直腸癌發生率排名第三位，而胃癌發生率排名第六位。目前大腸直腸癌的血液腫瘤標記常見的是CEA，胃癌常用的血液腫瘤標記為CEA、CA 19-9 和CA 72-4，但這些生物標記的靈敏度和特異性並不是很好。有文獻探討Alpha-1- antichymotrypsin (ACT) 在胃癌和大腸直腸癌血漿中蛋白質濃度來當作診斷工具，但比較少的研究探討轉譯後修飾。本實驗主要探討ACT 在這兩癌症血漿中轉譯後修飾的表現，希望找到有意義的轉譯後修飾可以開發為胃癌和大腸直腸癌之免疫檢測或質譜檢測平台的生物標記。本實驗材料是收集正常人和癌症的血漿檢體，先進行一維凝膠電泳 (SDS-PAGE)和西方墨點法(Western blot)，觀察ACT 在血漿中的蛋白質表現量。接著進行免疫沈澱法，經由SDS-PAGE 確認抗體抓到A1-抗胰凝乳蛋白酵素的位置，之後將ACT band 切下來利用膠體內水解(In-Gel digestion)將要測定的蛋白質分離出來，再用奈米級液相層析串連質譜儀(nano-LC/MS/MS)分析並尋找可能發生的修飾然後鑑定出轉譯後修飾的位置，並加以量化比較。實驗結果發現ACT 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，但在胃癌晚期與正常人相比有明顯差異，然而ACT 在大腸直腸癌早期有明顯差異。接著觀察ACT 的轉譯後修飾表現，ACT在胃癌血漿中有三個修飾分別是Trimethylation (Asp-194)、n-Octanoate (Thr-269)和Palmitoylation (Lys-177)，其中Trimethylation 修飾的差異有三倍多。而在大腸直腸癌血漿中有三個修飾分別是Hydroxylation (Asn-323)、Methylation-2 (Glu-334)和4-Hydroxy-2-nonenal (Arg-298)，其中Hydroxylation 修飾的差異有兩倍多。本實驗結論是利用串聯質譜鑑定出這些轉譯後修飾的位置並且量化比較，可當作胃癌和大腸直腸癌的生物標記。

關鍵字

A1-抗胰凝乳蛋白酵素；轉譯後修飾

並列摘要

According to the report of World Health Organization in 2012, colorectal cancer is the third prevalent cancer and gastric cancer is the sixth prevalent cancer. Carcinoembryonic antigen (CEA) is the most use of tumor markers of colorectal cancer. The major biomarkers of gastric cancer are CEA、CA 19-9 and CA 72-4. However, the specificity and sensitivity of those markers are not good . Until now, some researches have explored the protein level of Alpha-1- antichymotrypsin (ACT) in the plasma of gastric and colorectal cancer, but few researches show the post-translational modification (PTM) of ACT in both cancers. In this study, we use proteomic approaches such as western blot, immunoprecipitation, and nano- LC/MS/MS to analyze the plasma samples from normal and cancer groups. Then we identify PTMs between those samples in order to find out the useful PTM sites of ACT to be a diagnostic tool. As our results, there was no significance of protein level between the early stage of gastric cancer group and normal group. However, there was significance of that in the early stage of colorectal cancer group. The PTM data showed that there were three PTMs in gastric cancer, including Trimethylation (Asp-194), n-Octanoate (Thr-269) and Palmitoylation (Lys-177). There were three PTMs in colorectal cancer, including Hydroxylation (Asn-323), Methylation-2 (Glu-334) and 4-Hydroxy-2-nonenal (Arg-298). There was significance of those PTMs expression between the cancer group and normal group. In conclusion, those PTMs will be good biomarkers for gastric cancer and colorectal cancer by using tandem mass to identify and quantify them.