肺癌是在全世界因各種癌症而死亡的病例人數中的第一名，雖然抽煙的人口下降有可能造成肺癌在全世界中的病例有整體下降的趨勢，但肺癌的發病率和致死率在婦女族群及發展中的都市鄉鎮中仍有明顯升高的趨勢。造成肺癌高致死率的原因有大部份是來自於肺癌容易發生早期轉移現象的關係；而癌症的轉移及浸潤是伴隨著一連串的癌症細胞和周遭環境交互作用的生化反應，在癌症的發展過程中是屬於最嚴重的階段。除了少數的癌症類型之外，癌症轉移是造成臨床治療失敗及造成癌症死亡的主要因素。所以若是能了解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，磷酸化蛋白質參與調控癌症的發展。但由於在鑑定磷酸化蛋白質時，仍有許多技術上的困難，因此此篇研究的目的即是為了要找出可信度高的磷酸化胜肽，將金屬親合性的純化方式結合液相層析串聯式質譜儀來分析肺癌細胞株CL1-5所含的磷酸化蛋白質。癌細胞裂解液將以胰蛋白酶剪切成蛋白質胜肽混合物之後，以自製的二氧化鈦管柱進行磷酸化胜肽的純化。我們參考最近所發表的一份期刊進行磷酸化胜肽的純化。接著將這些純化過後的胜肽混合物以液相層析串聯式質譜儀分析，並針對Swiss-Prot資料庫分析比對原始資料。但由於磷酸化胜肽在離子阱式的質譜儀中所難以產生完整或清晰的二次質譜圖，所以在經資料庫比對之後，容易比對到錯誤的磷酸化胜肽。因此我們將應用一套分析策略來降低在資料庫比對過程中所產生錯誤的磷酸化胜肽。由於一般在利用二次質譜圖所做的蛋白質身份鑑定所用的比對參數並不適合用來鑑定具有修飾過的胜肽，如磷酸化胜肽，因此我們在本研究當中，還加入了手動篩選磷酸化胜肽的二次質譜圖，藉以除去較低可信度的磷酸化胜肽身份鑑定。在這篇研究當中，總共找到了719個磷酸化胜肽，分別對應到274個蛋白質上，希望利用這些所鑑定到的蛋白質身份鑑定，從中找出與肺癌發展相關的磷酸化蛋白質目標，以提供在肺癌轉移研究上新的資訊，並且期望可用來做為定量分析與設計藥物標靶的對象來阻止癌症轉移的發生。

Lung cancer is one of the most common causes of cancer death in the world. Although the total annual number of cases has declined, probably due to the decreased trend in cigarette consumption, the incidence and mortality rate of lung cancer have increased at an alarming rate in the female population and in developing countries. Cancer metastasis and invasion are the most crucial steps in the cancer progress, occurring via a series of biologic activities including the interaction of tumor cells with the surrounding environment. These steps are the major causes of treatment failure and cancer deaths. Therefore, understanding the mechanisms that regulate progress of tumor cells is important for development of novel therapies to control cancer. Based on recent researches, evidences indicate that the changed protein phosphorylation result in altered invasive and metastatic properties of tumor cells. For example, Phosphorylated FAK was seen in invasive ovarian carcinoma, but not in the normal cells. Here, we used metal affinity based enrichment coupled with LC-MS/MS analysis, qualitatively but comprehensively defined the phosphoproteome of this lung cancer cell lines CL1-5. Cancer cell lysates were digested and phosphopeptides were enriched by self-packing titanium dioxide (TiO2) microcolumn. The enrichment protocol was based on a recently published method. After enriched by TiO2 microcolumn, eluted peptides were subjected to LC-MS/MS analysis and the raw data were searched against Swiss-Prot database. Although phosphopeptide enrichment has been set up, a process to reduce the false-positive phosphopeptides identification by current database search tools is needed. Commonly accepted MS/MS data filtering criteria are not suitable for data analysis with post-translational modifications such as phosphorylation. Thus, we manual validated all the MS/MS spectra to exclude low confidence identities. A total of 719 phosphorylaed peptides, assigned to 274 proteins, were identified in this study. Those identified phosphorylation events may provide novel information for not only mechanistic aspects of lung cancer but also potential drug targets for hampering the cancer metastatic processes.

Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Ville´n J, Li J., Cohn MA, Cantley LC, Gygi SP. 2004. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U. S. A. 101:12130-12135

de GM, Hensbergen P, van de WB. 2006. Phosphoproteomic analysis of cellular signaling. Electrophoresis 27(13):2676-2686.

Elisa CW, Rodrigo FC, Lance AL. 2000. General Mechanisms of Metastasis. Cancer 80: 1529-1537

Francesco C, Antonino DS, Sabrina D, Francesco R, Rita A, Giampiero LR, Silvestro ED, Francesca M. 2006. HSP60 and HSP10 Down-Regulation Predicts Bronchial Epithelial Carcinogenesis in Smokers with Chronic Obstructive Pulmonary Disease. Cancer 15: 2417-2424

Feng Y, David LS, Eric FS, Junhua W, Lianghao D, Mary SL, Matthew EM, Carrie DN, Marina AG, Keqi T, Ruihua F, Joshua NA, David GC, David JC, Richard DS. 2006. Phosphoproteome Profiling of Human Skin Fibroblast Cells in Response to Low- and High-Dose Irradiation. J Proteome Research 5:1252-1260

Grisaru GS, Salah Z, Maoz M, Pruss D, Beller U, Bar-Shavit R. 2005. Differential expression of protease activated receptor 1 (Par1) and pY397FAK in benign and malignant human ovarian tissue samples. Int J Cancer 113(3):372-8

Haiyan Y, Yu LR, Ming Y, David AL, Luanne L., Mindy L. 2006. Parallel Analysis of Transcript and Translation Profiles: Identification of Metastasis-Related Signal Pathways Differentially Regulated by Drug and Genetic Modifications Drug and Genetic Modifications. J Proteome Research 5:1555-1567

Hart IR, Fidler IJ. 1980. Role of organ selectivity in the determination of metastatic patterns of B16 melanoma. Cancer Res 40:2281-2287

Jiang W, Quazi S, Wei L, Alwin S, Maximillian TF. 2008. Global Profiling of Phosphopeptides by Titania Affinity Enrichment. J Proteome Res 6:4684-4689

Jing-Dong H, Nicholas EB, Charles SR. 1997. Molecular Characterization of a Novel A Kinase Anchor Protein from Drosophila melanogaster. J Bio Chem 272:26611-26619

Jin WH, Jie D, Hu Z, Xia QC, Zou HF, Rong Z. 2004. Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry. Rapid Commun Mass Spectrom 18: 2169-2176

Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, and Jørgensen TJD. 2005. Highly selective enrichment of phosphorylatedpeptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873-886

Lebret T, Watson RW, Molinie V. 2003. Heat shock proteins HSP27, HSP60, HSP70, and HSP90: expression in bladder carcinoma. Cancer 98:970–977

Lim Y. Mining the tumor phosphoproteome for cancer markers. 2005. Clin Cancer Res 11(9):3163-3169

Lu Z, A. Hornia YW, Jiang Q, Zang SO, DA Foster. 1997. Tumor promotion by depleting cells of protein kinase C delta. Mol Cell Biol 17:3418-3428

Michael SK, Geoffrey JC, Channing J. 1995. Tyrosine Phosphorylation Regulates the Adhesions of Ras-transformed Breast Epithelia. J Cell Biology 130: 461-471

Nicolson GL. 1988. Organ specificity of tumor metastasis: role of preferential adhesion, invasion and growth of malignant cells at specific secondary sites. Cancer Metastasis Rev 7:143-188

Rohit GS, Mark PM, Khalil B, Douglas BC, Xiaomin S. 2007. Proteomic Identification of Lynchpin Urokinase Plasminogen Activator Receptor Protein Interactions Associated with Epithelial Cancer Malignancy. J Proteome Research 6:1016-1028

Sano A, Nakamura H. 2004. Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry. Anal Sci 20:861-864

Susumu YI, Vitaly K, Saima EF, Aurelie DT, Hanna MP, Garry LC, John EE. 2007. Reference-facilitated Phosphoproteomics. Mol Cell Biol 6:1380-1391

Schremmer B, Manevich Y, Feinstein SI, Fisher AB. 2007. Peroxiredoxins in the lung with emphasis on peroxiredoxin VI. Subcell Biochem 44:317-344

Yoon PL. 2005. Mining the Tumor Phosphoproteome for Cancer Markers. Clin Cancer Res 11:3163-3169