肺癌是一種常見且易致死的疾病，其治癒率不超過13%。早期的癌細胞轉移是造成治療失敗與病人死亡的主要原因。有鑒於成功的手術與化學治療技術往往受限於轉移現象的發生，許多研究致力於開發能夠抑制癌細胞轉移的治療方法，但目前僅找到非常少數與轉移機制相關的標的物。所以若是能了解更多關於調控腫瘤細胞的轉移機制，對於設計一個新型的癌症治療是非常重要的。在近期的癌症研究當中，有許多證據證實了在惡性腫瘤的發展過程中，酪胺酸磷酸化蛋白質參與腫瘤細胞浸潤及轉移的調控。根據這些研究結果，此篇研究的目的即是以磷酸化蛋白質體比較分析方法來分析具有不同轉移能力的肺癌細胞株(CL1-0和CL1-5)中，鑑定出與調控肺癌轉移相關的酪胺酸磷酸化蛋白質。首先我們將CL1-0 和 CL1-5兩株肺癌細胞先進行免疫沉澱法，將細胞中的酪胺酸磷酸化蛋白質純化下來，接著利用膠體電泳分離(stacking gel)將CL1-0和CL1-5兩株肺癌細胞進行蛋白質回收，再以本實驗室發展出的分析策略來分析。研究策略為使用鹼性磷酸酶將其中一部分樣本中的磷酸化去除，經過液相層析質譜分析(LC-MS)與另一部分去除磷酸化的樣品比較，在圖譜中找出質量相差79.966Da的訊號，針對這些訊號進行串聯式質譜分析得到酪胺酸磷酸化蛋白質體。接著再以非標記定量方法，對這些酪胺酸磷酸化蛋白質在CL1-0 和 CL1-5兩組數據中做相對量上的比較。在本研究當中，總共鑑定到335個的酪胺酸位置，而其中有302個不重覆的酪胺酸磷酸化胜肽，分別對應到276個不重覆蛋白質上，其中我們發現了有30個酪胺酸磷酸化蛋白質在CL1-5肺癌細胞中表現量較高；另外6個酪胺酸磷酸化則在CL1-0的肺癌細胞中表現量較高。因此我們進一步針對這36個有顯著性差異的酪胺酸磷酸化蛋白利用磷酸化預測分析軟體比對，分別對應到6個與轉移相關的磷酸激酶。最後再將這36個酪胺酸磷酸化蛋白質利用MetaCore軟體進行交互作用分析，發現其中11個蛋白質ErbB4, SCYL1, GRB7, TXK, FGD2, FAK, CRMP4, SYTL4, CTNNB1 , ERBB2 與 DPEP2與過去文獻中指出和癌症轉移有相關的EGFR、MMP-2、ErbB2、ErbB4、 β-catenin、PI3K、c-Src、FAK等蛋白質的激酶有交互作用。本研究從中找出與肺癌轉移相關的酪胺酸磷酸化蛋白質目標，相亯在未來可以提供在肺癌轉移上的研究，用來做為發展及設計藥物標靶的對象來阻止癌症轉移的發生。

Lung cancer is a lethal disease, and early metastasis is the major cause of treatment failure and cancer-related death. Tyrosine phosphorylated (P-Tyr) proteins are involved in the invasive and metastatic behaviors of lung cancer. Identification of tyrosine phosphorylated protein targets are associated with lung cancer metastasis by performing comparative phosphoproteome analysis on lung cancer cells with differential invasive abilities (CL1-0 and CL1-5). Tyrosine phosphorylated proteins were immunoprecipitated from CL1-0 and CL1-5 cells and resolved on a stacking gel. The comprehensive characterization of both CL1-0 and CL1-5 cell tyrosine phosphoproteome was achieved by our previously developed Signal Mining Strategy. Label-free quantitative analysis of those defined tyrosine phosphoproteins was used to screen for potential protein targets associated with lung cancer metastasis. We have identified 335 tyrosine phosphorylated sites from 302 unique tyrosine phosphorylated peptides, corresponding to 276 tyrosine phosphorylated proteins. Among them, 30 tyrosine phosphorylation proteins were with higher levels in CL1-5 cell while 6 with higher levels in CL1-0 cell. Those 36 differentially expressed tyrosine phosphorylation proteins converge toward six phosphorylation motifs extracted by use of the Motif-X algorithm. Only tyrosine-phosphorylated peptides with altered levels between CL1-0 and CL1-5 cell lines were included. After interactome analysis by MetaCore software, ErbB4, SCYL1, GRB7, TXK, FGD2, FAK, CRMP4, SYTL4, CTNNB1 , ERBB2 and DPEP2 appear in one interaction network which contains EGFR, MMP-2, ErbB2, ErbB4, β-catenin, PI3K, c-Src, and FAK pathways that have been reported highly associated with cancer metastasis. We have identified 36 tyrosine phosphorylated proteins associated with lung cancer metastasis. Eleven of them will be subjected for further validation for their roles in metastasis. Those phosphorylated protein targets identified in this study may provide novel information to not only mechanistic aspects of lung cancer metastasis but also drug targets for hampering the cancer metastatic processes.

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