肺癌是世界癌症常見死因之ㄧ，五年存活率僅13%，癌細胞的轉移更是造成肺癌難以治療、死亡率偏高的主因，分泌蛋白質於癌症發展的過程中在浸潤及轉移上扮演著重要角色，同時它亦具有能夠進入血液循環的特性，因此可被應用於癌症的診斷及監測上。蛋白質體的分析方法能有助於新的癌症生物指標發現及抗癌藥物的研究，因此本研究目的便是利用定量蛋白質體分析方法比較不同轉移能力的非小肺癌細胞株CL1-5、CL1-0之分泌蛋白體 (secretome) ，找出在兩者間有差異表現之蛋白質，並從中找出可能與轉移有關之蛋白質。在分泌蛋白質收集過程為避免培養液中血清干擾分泌蛋白質的分析，將肺癌細胞株置換到不含血清的培養液靜置24小時，收集CL1-5、CL1-0上清液，將上清液進行濃縮、stacking SDS-PAGE gel、膠內水解等步驟，將水解後的胜肽樣本與iTRAQ (Isobaric Tags for Relative and Absolute Quantification) 反應，CL1-5標定iTRAQ標記 117，CL1-0標定iTRAQ標記114。本研究為了增加鑑定蛋白質的數目，利用強陽離子交換管柱對樣本進行分離，共分成28個區段 (fractionation) ，並分別除去樣本中鹽類，以液相層析串聯質譜儀 (LC-MS/MS) 來進行定性及定量的分析，共鑑定出540個蛋白質，由定量軟體Multi-Q進行分析，最後有511個蛋白質可供定量，在CL1-5、CL1-0中表現約2倍差異之蛋白質，在CL1-5中有13種蛋白質表現量高於CL1-0，而在CL1-0中則有11種蛋白質表現量高於CL1-5，從中挑選10個蛋白質以西方墨點法確認蛋白質的表現趨勢與定量蛋白質體分析的結果一致。本研究所找到分泌蛋白質，期許有助於了解肺癌轉移的機制，並提供抑制肺癌轉移蛋白質治療藥物之發展及可能肺癌生物指標之開發。

Lung cancer is the most common cause of cancer death in the world. The 5 year survival rate of lung cancer is about 13% after treatment. Tumor invasion and metastasis are the most common causes of death in cancer patients. It is known that involves multiple steps, including angiogenesis, cell attachment, invasion and cell proliferation. However, the molecular mechanisms of metastasis in lung cancer are not well understood. Secreted proteins play critical roles in metastasis processes and therefore, it seems reasonable to investigate the relationship between secreted protein expression and cancer metastasis. In this study we hypothesize is that differences in metastatic potential can be explained by differential proteins expression levels. The aim of this study was to use quantitative proteomic analysis to compare protein expression in NSCLC cell lines (CL1-5 and CL1-0) with different metastatic potential. The CL1-0 and CL1-5 cells cultured in 15 cm culture dishes were incubated in serum-free medium for 24 h and the CM (conditioned medium) were harvested. Equal amounts of secretome samples of CL1-0 and CL1-5 cell CM were run on stacking SDS-PAGE gel, followe by in-gel digestion with trypsin. The peptides from CL1-5 and CL1-0 were tagged respectively with iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) labeling tag 117 and 114, and were then pooled. The pooled sample was fractionated by strong cation exchange (SCX) chromatography before nano-LC-ESI-MS/MS analysis. The protein identification of secretome samples was carried out by using nano-LC-ESI-MS/MS in addition to the MASCOT database search engine. A total of 540 proteins from the secretome sample were identified. Protein quantification was determined by the Multi-Q program to evaluate changes in the protein level in CL1-5 CM as compared to CL1-0. A total of 13 proteins were over-expressed (over 2.06 fold) in CL1-5 CM as compared to CL1-0. In addition, 11 proteins were under-expressed (less than 0.509 fold) in CL1-0 cell compared to CL1-5. The biological functions associated with metastasis and invasion of differentially expressed proteins were searched by bioinformatics. The proteins that had differential expression level according to quantitative analysis were validated by Western blot. The results obtained from this study may provide useful information to better understand the mechanisms involved in the process of lung cancer metastasis.

Adkins JN, Varnum SM, Auberry KJ, Moore RJ, Angell NH, Smith RD, et al., 2002. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry. Mol Cell Proteomics 1(12): 947-955.

Akiyama SK, Olden K, Yamada KM. 1995. Fibronectin and integrins in invasion and metastasis. Cancer metastasis reviews 14(3): 173-189.

Albelda SM, Mette SA, Elder DE, Stewart R, Damjanovich L, Herlyn M, et al., 1990. Integrin distribution in malignant melanoma: association of the beta 3 subunit with tumor progression. Cancer research 50(20): 6757-6764.

Ayala I, Giacchetti G, Caldieri G, Attanasio F, Mariggio S, Tete S, et al., 2009. Faciogenital dysplasia protein Fgd1 regulates invadopodia biogenesis and extracellular matrix degradation and is up-regulated in prostate and breast cancer. Cancer research 69(3): 747-752.

Balasubramani M, Day BW, Schoen RE, Getzenberg RH. 2006. Altered expression and localization of creatine kinase B, heterogeneous nuclear ribonucleoprotein F, and high mobility group box 1 protein in the nuclear matrix associated with colon cancer. Cancer research 66(2): 763-769.

Beckles MA, Spiro SG, Colice GL, Rudd RM. 2003. Initial evaluation of the patient with lung cancer: symptoms, signs, laboratory tests, and paraneoplastic syndromes. Chest 123(1 Suppl): 97S-104S.

Begley LA, MacDonald JW, Day ML, Macoska JA. 2007. CXCL12 activates a robust transcriptional response in human prostate epithelial cells. The Journal of biological chemistry 282(37): 26767-26774.

Bergers G, Benjamin LE. 2003. Tumorigenesis and the angiogenic switch. Nature reviews 3(6): 401-410.

Bernacka K, Kuryliszyn-Moskal A, Sierakowski S. 1988. The levels of alpha 1-antitrypsin and alpha 1-antichymotrypsin in the sera of patients with gastrointestinal cancers during diagnosis. Cancer 62(6): 1188-1193.

Bertin N, Clezardin P, Kubiak R, Frappart L. 1997. Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia. Cancer research 57(3): 396-399.

Blobel G. 2000. Protein targeting (Nobel lecture). Chembiochem 1(2): 86-102.
Walter P, Blobel G. 1982. Translocation of proteins across the endoplasmic reticulum. Oncodev Biol Med 4(1-2): 137-146.

Brandt DT, Baarlink C, Kitzing TM, Kremmer E, Ivaska J, Nollau P, et al., 2009. SCAI acts as a suppressor of cancer cell invasion through the transcriptional control of beta1-integrin. Nature cell biology 11(5): 557-568.

Butler CA, Darragh KM, Currie GP, Anderson WJ. 2006. Variation in lung cancer survival rates between countries: do differences in data reporting contribute? Respiratory medicine 100(9): 1642-1646.

Cai G, Zhang X, Hong Q, Shao F, Shang X, Fu B, et al., 2008. Tissue inhibitor of metalloproteinase-1 exacerbated renal interstitial fibrosis through enhancing inflammation. Nephrol Dial Transplant 23(6): 1861-1875.

Campbell SC, Volpert OV, Ivanovich M, Bouck NP. 1998. Molecular mediators of angiogenesis in bladder cancer. Cancer research 58(6): 1298-1304.

Chanin TD, Merrick DT, Franklin WA, Hirsch FR. 2004. Recent developments in biomarkers for the early detection of lung cancer: perspectives based on publications 2003 to present. Current opinion in pulmonary medicine 10(4): 242-247.

Chen G, Gharib TG, Huang CC, Thomas DG, Shedden KA, Taylor JM, et al 2002. Proteomic analysis of lung adenocarcinoma: identification of a highly expressed set of proteins in tumors. Clin Cancer Res 8(7): 2298-2305.

Chen JJ, Peck K, Hong TM, Yang SC, Sher YP, Shih JY, et al., 2001. Global analysis of gene expression in invasion by a lung cancer model. Cancer research 61(13): 5223-5230.

Chen ZJ, Le HB, Zhang YK, Qian LY, Li WD. 2009. Microvessel density and expression of thrombospondin-1 in non-small cell lung cancer and their correlation with clinicopathological features. The Journal of international medical research 37(2): 551-556.

Christensen L. 1990. Fibronectin: a discrimination marker between small invasive carcinomas and benign proliferative lesions of the breast. Apmis 98(7): 615-623.

Chu YW, Yang PC, Yang SC, Shyu YC, Hendrix MJ, Wu R, et al., 1997. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. American journal of respiratory cell and molecular biology 17(3): 353-360.

Daniel H. 2002. Genomics and proteomics: importance for the future of nutrition research. The British journal of nutrition 87 Suppl 2: S305-311.

de Jong D, Jansen RW, Pijnenburg YA, van Geel WJ, Borm GF, Kremer HP, et al., 2007. CSF neurofilament proteins in the differential diagnosis of dementia. Journal of neurology, neurosurgery, and psychiatry 78(9): 936-938.

Dean RA, Overall CM. 2007. Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome. Mol Cell Proteomics 6(4): 611-623.

Dean RA, Smith D, Overall CM. 2007. Proteomic identification of cellular protease substrates using isobaric tags for relative and absolute quantification (iTRAQ). Current protocols in protein science / editorial board, John E Coligan [et al Chapter 21: Unit 21 18.

Demidov VP, Iakubovskaia RI, Karmakova TA, Avdeev GI, Komissarov AB, Sorokin EE, et al., 1990. [alpha 1-proteinase inhibitor in breast cancer]. Voprosy onkologii 36(1): 23-29.

Field JK, Smith DL, Duffy S, Cassidy A. 2005. The Liverpool Lung Project research protocol. International journal of oncology 27(6): 1633-1645.

Foekens JA, Kos J, Peters HA, Krasovec M, Look MP, Cimerman N, et al., 1998. Prognostic significance of cathepsins B and L in primary human breast cancer. J Clin Oncol 16(3): 1013-1021.

Fong KM, Kida Y, Zimmerman PV, Smith PJ. 1996. TIMP1 and adverse prognosis in non-small cell lung cancer. Clin Cancer Res 2(8): 1369-1372.

Fujita A, Sato JR, Festa F, Gomes LR, Oba-Shinjo SM, Marie SK, et al., 2008. Identification of COL6A1 as a differentially expressed gene in human astrocytomas. Genet Mol Res 7(2): 371-378.

Geho DH, Bandle RW, Clair T, Liotta LA. 2005. Physiological mechanisms of tumor-cell invasion and migration. Physiology (Bethesda, Md 20: 194-200.

Ghosal R, Kloer P, Lewis KE. 2009. A review of novel biological tools used in screening for the early detection of lung cancer. Postgraduate medical journal 85(1005): 358-363.

Goufman EI, Moshkovskii SA, Tikhonova OV, Lokhov PG, Zgoda VG, Serebryakova MV, et al., 2006. Two-dimensional electrophoretic proteome study of serum thermostable fraction from patients with various tumor conditions. Biochemistry 71(4): 354-360.

Gygi SP, Rist B, Griffin TJ, Eng J, Aebersold R. 2002. Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags. Journal of proteome research 1(1): 47-54.

Hattori H, Kaneda T, Lokeshwar B, Laszlo A, Ohtsuka K. 1993. A stress-inducible 40 kDa protein (hsp40): purification by modified two-dimensional gel electrophoresis and co-localization with hsc70(p73) in heat-shocked HeLa cells. Journal of cell science 104 ( Pt 3): 629-638.

He P, He HZ, Dai J, Wang Y, Sheng QH, Zhou LP, et al., 2005. The human plasma proteome: analysis of Chinese serum using shotgun strategy. Proteomics 5(13): 3442-3453.

Hedstrom J, Haglund C, Kemppainen E, Leinimaa M, Leinonen J, Stenman UH. 1999. Time-resolved immunofluorometric assay of trypsin-1 complexed with alpha(1)-antitrypsin in serum: increased immunoreactivity in patients with biliary tract cancer. Clinical chemistry 45(10): 1768-1773.

Hegele A, Heidenreich A, Kropf J, von Knobloch R, Varga Z, Hofmann R, et al., 2004. Plasma levels of cellular fibronectin in patients with localized and metastatic renal cell carcinoma. Tumour Biol 25(3): 111-116.

Helleman J, Jansen MP, Ruigrok-Ritstier K, van Staveren IL, Look MP, Meijer-van Gelder ME, et al., 2008. Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response. Clin Cancer Res 14(17): 5555-5564.

Han CL, Chien CW, Chen WC, Chen YR, Wu CP, Li H, et al., 2008. A multiplexed quantitative strategy for membrane proteomics: opportunities for mining therapeutic targets for autosomal dominant polycystic kidney disease. Mol Cell Proteomics 7(10): 1983-1997.

Henry SC, Daniell XG, Burroughs AR, Indaram M, Howell DN, Coers J, et al., 2009. Balance of Irgm protein activities determines IFN-gamma-induced host defense. Journal of leukocyte biology 85(5): 877-885.

Helling S, Schmitt E, Joppich C, Schulenborg T, Mullner S, Felske-Muller S, et al., 2006. 2-D differential membrane proteome analysis of scarce protein samples. Proteomics 6(16): 4506-4513.

Herszenyi L, Farinati F, Cardin R, Istvan G, Molnar LD, Hritz I, et al., 2008. Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer. BMC cancer 8: 194.

Hofmann UB, Eggert AA, Blass K, Brocker EB, Becker JC. 2003. Expression of matrix metalloproteinases in the microenvironment of spontaneous and experimental melanoma metastases reflects the requirements for tumor formation. Cancer research 63(23): 8221-8225.

Hsu PI, Chen CH, Hsieh CS, Chang WC, Lai KH, Lo GH, et al., 2007. Alpha1-antitrypsin precursor in gastric juice is a novel biomarker for gastric cancer and ulcer. Clin Cancer Res 13(3): 876-883.

Jacot W, Lhermitte L, Dossat N, Pujol JL, Molinari N, Daures JP, et al., 2008. Serum proteomic profiling of lung cancer in high-risk groups and determination of clinical outcomes. J Thorac Oncol 3(8): 840-850.

Jeftinija S. 2006. The story of cell secretion: events leading to the discovery of the 'porosome' - the universal secretory machinery in cells. Journal of cellular and molecular medicine 10(2): 273-279.

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al., 2008. Cancer statistics, 2008. CA: a cancer journal for clinicians 58(2): 71-96.

Jin J, Park J, Kim K, Kang Y, Park SG, Kim JH, et al., 2009. Detection of differential proteomes of human beta-cells during islet-like differentiation using iTRAQ labeling. Journal of proteome research 8(3): 1393-1403.

Jonsson AP, Aissouni Y, Palmberg C, Percipalle P, Nordling E, Daneholt B, et al., 2001. Recovery of gel-separated proteins for in-solution digestion and mass spectrometry. Analytical chemistry 73(22): 5370-5377.

John AS, Hu X, Rothman VL, Tuszynski GP. 2009. Thrombospondin-1 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human tumor cells: exploring the functional significance in tumor cell invasion. Experimental and molecular pathology 87(3): 184-188.

Joyce JA, Baruch A, Chehade K, Meyer-Morse N, Giraudo E, Tsai FY, et al., 2004. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer cell 5(5): 443-453.

Jung K, Nowak L, Lein M, Priem F, Schnorr D, Loening SA. 1997. Matrix metalloproteinases 1 and 3, tissue inhibitor of metalloproteinase-1 and the complex of metalloproteinase-1/tissue inhibitor in plasma of patients with prostate cancer. International journal of cancer 74(2): 220-223.

Karameris A, Panagou P, Tsilalis T, Bouros D. 1997. Association of expression of metalloproteinases and their inhibitors with the metastatic potential of squamous-cell lung carcinomas. A molecular and immunohistochemical study. American journal of respiratory and critical care medicine 156(6): 1930-1936.

Kleivi K, Lind GE, Diep CB, Meling GI, Brandal LT, Nesland JM, et al., 2007. Gene expression profiles of primary colorectal carcinomas, liver metastases, and carcinomatoses. Molecular cancer 6: 2.

Kuvaja P, Talvensaari-Mattila A, Paakko P, Turpeenniemi-Hujanen T. 2005. The absence of immunoreactivity for tissue inhibitor of metalloproteinase-1 (TIMP-1), but not for TIMP-2, protein is associated with a favorable prognosis in aggressive breast carcinoma. Oncology 68(2-3): 196-203.

Ladunga I. 2000. Large-scale predictions of secretory proteins from mammalian genomic and EST sequences. Current opinion in biotechnology 11(1): 13-18.

Lawlor K, Nazarian A, Lacomis L, Tempst P, Villanueva J. 2009. Pathway-based biomarker search by high-throughput proteomics profiling of secretomes. Journal of proteome research 8(3): 1489-1503.

Lee YS, Chen PW, Tsai PJ, Su SH, Liao PC. 2006. Proteomics analysis revealed changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure. Proteomics 6(7): 2236-2250.

Lerner I, Baraz L, Pikarsky E, Meirovitz A, Edovitsky E, Peretz T, et al., 2008. Function of heparanase in prostate tumorigenesis: potential for therapy. Clin Cancer Res 14(3): 668-676.

Levin Y, Schwarz E, Wang L, Leweke FM, Bahn S. 2007. Label-free LC-MS/MS quantitative proteomics for large-scale biomarker discovery in complex samples. Journal of separation science 30(14): 2198-2203.

Liu W, Cheng S, Asa SL, Ezzat S. 2008. The melanoma-associated antigen A3 mediates fibronectin-controlled cancer progression and metastasis. Cancer research 68(19): 8104-8112.

Lu X, Zhu H. 2005. Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins. Mol Cell Proteomics 4(12): 1948-1958.

Lu, Y.-T.; Han, C.-L.; Wu, C.-L.; Yu, T.-M.; Chien, C.-W.; Liu, C.-L.; Chen, Y.-J. Proteomic Profiles of Bronchoalveolar Lavage Fluid from Patients with Ventilator-associated Pneumonia by Gel-assisted Digestion and 2DLC/MS/MS. Proteomics 2008, 2, 1208.

Luo LY, Katsaros D, Scorilas A, Fracchioli S, Bellino R, van Gramberen M, et al., 2003. The serum concentration of human kallikrein 10 represents a novel biomarker for ovarian cancer diagnosis and prognosis. Cancer research 63(4): 807-811.

Macabeo-Ong M, Shiboski CH, Silverman S, Ginzinger DG, Dekker N, Wong DT, et al., 2003. Quantitative analysis of cathepsin L mRNA and protein expression during oral cancer progression. Oral oncology 39(7): 638-647.

Magi B, Bini L, Perari MG, Fossi A, Sanchez JC, Hochstrasser D, et al., 2002. Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study. Electrophoresis 23(19): 3434-3444.

Martyniuk CJ, Alvarez S, McClung S, Villeneuve DL, Ankley GT, Denslow ND. 2009. Quantitative proteomic profiles of androgen receptor signaling in the liver of fathead minnows (Pimephales promelas). Journal of proteome research 8(5): 2186-2200.

Mbeunkui F, Fodstad O, Pannell LK. 2006. Secretory protein enrichment and analysis: an optimized approach applied on cancer cell lines using 2D LC-MS/MS. Journal of proteome research 5(4): 899-906.

Mbeunkui F, Metge BJ, Shevde LA, Pannell LK. 2007. Identification of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines representing a progression of breast cancer. Journal of proteome research 6(8): 2993-3002.

McElroy MK, Kaushal S, Tran Cao HS, Moossa AR, Talamini MA, Hoffman RM, et al., 2009. Upregulation of thrombospondin-1 and angiogenesis in an aggressive human pancreatic cancer cell line selected for high metastasis. Molecular cancer therapeutics 8(7): 1779-1786.

Menendez V, Fernandez-Suarez A, Galan JA, Perez M, Garcia-Lopez F. 2005. Diagnosis of bladder cancer by analysis of urinary fibronectin. Urology 65(2): 284-289.

Meredith JE, Jr Schwartz MA. 1997. Integrins, adhesion and apoptosis. Trends in cell biology 7(4): 146-150.

Moreno CS, Matyunina L, Dickerson EB, Schubert N, Bowen NJ, Logani S, et al., 2007. Evidence that p53-mediated cell-cycle-arrest inhibits chemotherapeutic treatment of ovarian carcinomas. PloS one 2(5): e441.

Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, et al., 2001. Involvement of chemokine receptors in breast cancer metastasis. Nature 410(6824): 50-56.

Nagai H, Isemura M, Arai H, Abe T, Shimoda S, Motomiya M, et al., 1986. Pattern of fibronectin distribution in human lung cancer. Journal of cancer research and clinical oncology 112(1): 1-5.

Nagatsuka H, Han PP, Tsujigiwa H, Siar CH, Gunduz M, Sugahara T, et al., 2005. Heparanase gene and protein expression in ameloblastoma: possible role in local invasion of tumor cells. Oral oncology 41(5): 542-548.

Nakopoulou L, Giannopoulou I, Lazaris A, Alexandrou P, Tsirmpa I, Markaki S, et al., 2003. The favorable prognostic impact of tissue inhibitor of matrix metalloproteinases-1 protein overexpression in breast cancer cells. Apmis 111(11): 1027-1036.

Nguyen DX, Massague J. 2007. Genetic determinants of cancer metastasis. Nat Rev Genet 8(5): 341-352.

Nickel W. 2005. Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells. Traffic (Copenhagen, Denmark) 6(8): 607-614.

Ocak S, Chaurand P, Massion PP. 2009. Mass spectrometry-based proteomic profiling of lung cancer. Proceedings of the American Thoracic Society 6(2): 159-170.

Omenn GS, States DJ, Adamski M, Blackwell TW, Menon R, Hermjakob H, et al., 2005. Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Proteomics 5(13): 3226-3245.

Ornstein DK, Rayford W, Fusaro VA, Conrads TP, Ross SJ, Hitt BA, et al., 2004. Serum proteomic profiling can discriminate prostate cancer from benign prostates in men with total prostate specific antigen levels between 2.5 and 15.0 ng/ml. The Journal of urology 172(4 Pt 1): 1302-1305.

Otterbein LE, Choi AM. 2000. Heme oxygenase: colors of defense against cellular stress. American journal of physiology 279(6): L1029-1037.

Pan TC, Zhang RZ, Sudano DG, Marie SK, Bonnemann CG, Chu ML. 2003. New molecular mechanism for Ullrich congenital muscular dystrophy: a heterozygous in-frame deletion in the COL6A1 gene causes a severe phenotype. American journal of human genetics 73(2): 355-369.

Pepe G, Giusti B, Bertini E, Brunelli T, Saitta B, Comeglio P, et al., 1999. A heterozygous splice site mutation in COL6A1 leading to an in-frame deletion of the alpha1(VI) collagen chain in an italian family affected by bethlem myopathy. Biochemical and biophysical research communications 258(3): 802-807.

Phadke ND, Molloy MP, Steinhoff SA, Ulintz PJ, Andrews PC, Maddock JR. 2001. Analysis of the outer membrane proteome of Caulobacter crescentus by two-dimensional electrophoresis and mass spectrometry. Proteomics 1(5): 705-720.

Pieper R, Gatlin CL, Makusky AJ, Russo PS, Schatz CR, Miller SS, et al., 2003. The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3(7): 1345-1364.

Pieper R, Gatlin CL, McGrath AM, Makusky AJ, Mondal M, Seonarain M, et al., 2004. Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 4(4): 1159-1174.

Planque C, Kulasingam V, Smith CR, Reckamp K, Goodglick L, Diamandis EP. 2009. Identification of five candidate lung cancer biomarkers by proteomics analysis of conditioned media of four lung cancer cell lines. Mol Cell Proteomics 8(12): 2746-2758.

Polakis P. 2000. Wnt signaling and cancer. Genes & development 14(15): 1837-1851.

Powell WC, Knox JD, Navre M, Grogan TM, Kittelson J, Nagle RB, et al., 1993. Expression of the metalloproteinase matrilysin in DU-145 cells increases their invasive potential in severe combined immunodeficient mice. Cancer research 53(2): 417-422.

Qian X, Rothman VL, Nicosia RF, Tuszynski GP. 2001. Expression of thrombospondin-1 in human pancreatic adenocarcinomas: role in matrix metalloproteinase-9 production. Pathol Oncol Res 7(4): 251-259.

Ramaswamy S, Ross KN, Lander ES, Golub TR. 2003. A molecular signature of metastasis in primary solid tumors. Nature genetics 33(1): 49-54.

Righetti PG, Castagna A, Antonucci F, Piubelli C, Cecconi D, Campostrini N, et al., 2004. Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches. Journal of chromatography 1051(1-2): 3-17.

Rousselet N, Mills L, Jean D, Tellez C, Bar-Eli M, Frade R. 2004. Inhibition of tumorigenicity and metastasis of human melanoma cells by anti-cathepsin L single chain variable fragment. Cancer research 64(1): 146-151.

Ru QC, Zhu LA, Katenhusen RA, Silberman J, Brzeski H, Liebman M, et al., 2006. Exploring human plasma proteome strategies: high efficiency in-solution digestion protocol for multi-dimensional protein identification technology. Journal of chromatography 1111(2): 175-191.

Sabounchi-Schutt F, Astrom J, Hellman U, Eklund A, Grunewald J. 2003. Changes in bronchoalveolar lavage fluid proteins in sarcoidosis: a proteomics approach. Eur Respir J 21(3): 414-420.

Sawaya R, Zuccarello M, Highsmith R. 1987. Alpha-1-antitrypsin in human brain tumors. Journal of neurosurgery 67(2): 258-262.

Schafer T, Zentgraf H, Zehe C, Brugger B, Bernhagen J, Nickel W. 2004. Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells. The Journal of biological chemistry 279(8): 6244-6251.

Shinyo Y, Kodama J, Hongo A, Yoshinouchi M, Hiramatsu Y. 2003. Heparanase expression is an independent prognostic factor in patients with invasive cervical cancer. Ann Oncol 14(10): 1505-1510.

Sieuwerts AM, Usher PA, Meijer-van Gelder ME, Timmermans M, Martens JW, Brunner N, et al., 2007. Concentrations of TIMP1 mRNA splice variants and TIMP-1 protein are differentially associated with prognosis in primary breast cancer. Clinical chemistry 53(7): 1280-1288.

Soltermann A, Tischler V, Arbogast S, Braun J, Probst-Hensch N, Weder W, et al., 2008. Prognostic significance of epithelial-mesenchymal and mesenchymal-epithelial transition protein expression in non-small cell lung cancer. Clin Cancer Res 14(22): 7430-7437.

Su LP, Zhang JP, Xu HB, Chen J, Wang Y, Xiong SD. 2005. [The role of CXCR4 in lung cancer metastasis and its possible mechanism]. Zhonghua yi xue za zhi 85(17): 1190-1194.

Sunamura M, Duda DG, Ghattas MH, Lozonschi L, Motoi F, Yamauchi J, et al., 2003. Heme oxygenase-1 accelerates tumor angiogenesis of human pancreatic cancer. Angiogenesis 6(1): 15-24.

Sung HJ, Cho JY. 2008. Biomarkers for the lung cancer diagnosis and their advances in proteomics. BMB reports 41(9): 615-625.

Thon JN, Schubert P, Duguay M, Serrano K, Lin S, Kast J, et al., 2008. Comprehensive proteomic analysis of protein changes during platelet storage requires complementary proteomic approaches. Transfusion 48(3): 425-435.

Tirumalai RS, Chan KC, Prieto DA, Issaq HJ, Conrads TP, Veenstra TD. 2003. Characterization of the low molecular weight human serum proteome. Mol Cell Proteomics 2(10): 1096-1103.

Tomlinson J, Barsky SH, Nelson S, Singer S, Pezeshki B, Lee MC, et al., 1999. Different patterns of angiogenesis in sarcomas and carcinomas. Clin Cancer Res 5(11): 3516-3522.

Topic AS, Jelic-Ivanovic ZD, Spasojevic-Kalimanovska VV, Spasic SM. 2006. Association of moderate alpha-1 antitrypsin deficiency with lung cancer in the Serbian population. Archives of medical research 37(7): 866-870.

Trachte AL, Suthers SE, Lerner MR, Hanas JS, Jupe ER, Sienko AE, et al., 2002. Increased expression of alpha-1-antitrypsin, glutathione S-transferase pi and vascular endothelial growth factor in human pancreatic adenocarcinoma. American journal of surgery 184(6): 642-647; discussion 647-648.

Tsai MF, Wang CC, Chang GC, Chen CY, Chen HY, Cheng CL, et al., 2006. A new tumor suppressor DnaJ-like heat shock protein, HLJ1, and survival of patients with non-small-cell lung carcinoma. Journal of the National Cancer Institute 98(12): 825-838.

Tyan YC, Wu HY, Lai WW, Su WC, Liao PC. 2005. Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry. Journal of proteome research 4(4): 1274-1286.

Urbich C, Heeschen C, Aicher A, Sasaki K, Bruhl T, Farhadi MR, et al., 2005. Cathepsin L is required for endothelial progenitor cell-induced neovascularization. Nature medicine 11(2): 206-213.

Vanegas OC, Zhang RZ, Sabatelli P, Lattanzi G, Bencivenga P, Giusti B, et al., 2002. Novel COL6A1 splicing mutation in a family affected by mild Bethlem myopathy. Muscle & nerve 25(4): 513-519.

Van Molle W, Libert C, Fiers W, Brouckaert P. 1997. Alpha 1-acid glycoprotein and alpha 1-antitrypsin inhibit TNF-induced but not anti-Fas-induced apoptosis of hepatocytes in mice. J Immunol 159(7): 3555-3564.

Verdecchia A, Francisci S, Brenner H, Gatta G, Micheli A, Mangone L, et al., 2007. Recent cancer survival in Europe: a 2000-02 period analysis of EUROCARE-4 data. The lancet oncology 8(9): 784-796.

Volmer MW, Stuhler K, Zapatka M, Schoneck A, Klein-Scory S, Schmiegel W, et al., 2005. Differential proteome analysis of conditioned media to detect Smad4 regulated secreted biomarkers in colon cancer. Proteomics 5(10): 2587-2601.

Ward AM, Cooper EH, Houghton AL. 1977. Acute phase reactant proteins in prostatic cancer. British journal of urology 49(5): 411-418.

Washburn MP, Wolters D, Yates JR, 3rd. 2001. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature biotechnology 19(3): 242-247.

Walter P, Blobel G. 1982. Translocation of proteins across the endoplasmic reticulum. Oncodev Biol Med 4(1-2): 137-146.

Webb MG, O'Donnell MP, Draper RJ, Horner B, Phillips JP. 1984. Brain-type creatine phosphokinase serum levels before and after ECT. Br J Psychiatry 144: 525-528.

Weng LP, Wu CC, Hsu BL, Chi LM, Liang Y, Tseng CP, et al., 2008. Secretome-based identification of Mac-2 binding protein as a potential oral cancer marker involved in cell growth and motility. Journal of proteome research 7(9): 3765-3775.

Wewer UM, Liotta LA, Jaye M, Ricca GA, Drohan WN, Claysmith AP, et al., 1986. Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin. Proceedings of the National Academy of Sciences of the United States of America 83(19): 7137-7141.

Witt S, Ziegler B, Blumentritt C, Schlosser M, Ziegler M. 1991. [Production of monoclonal antibodies in serum-free medium in dialysis tubing]. Allergie und Immunologie 37(2): 67-74.

Wu CC, Chen HC, Chen SJ, Liu HP, Hsieh YY, Yu CJ, et al., 2008. Identification of collapsin response mediator protein-2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes. Proteomics 8(2): 316-332.

Wu CC, Chien KY, Tsang NM, Chang KP, Hao SP, Tsao CH, et al., 2005. Cancer cell-secreted proteomes as a basis for searching potential tumor markers: nasopharyngeal carcinoma as a model. Proteomics 5(12): 3173-3182.

Wu MP, Young MJ, Tzeng CC, Tzeng CR, Huang KF, Wu LW, et al., 2008. A novel role of thrombospondin-1 in cervical carcinogenesis: inhibit stroma reaction by inhibiting activated fibroblasts from invading cancer. Carcinogenesis 29(6): 1115-1123.

Wu J, Kobayashi M, Sousa EA, Liu W, Cai J, Goldman SJ, et al., 2005. Differential proteomic analysis of bronchoalveolar lavage fluid in asthmatics following segmental antigen challenge. Mol Cell Proteomics 4(9): 1251-1264.

Wu WW, Wang G, Baek SJ, Shen RF. 2006. Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. Journal of proteome research 5(3): 651-658.

Xu X, Yuan G, Liu W, Zhang Y, Chen W. 2009. Expression of cathepsin L in nasopharyngeal carcinoma and its clinical significance. Experimental oncology 31(2): 102-105.

Yamao T, Kai S, Kazami A, Koizumi K, Handa T, Takemoto N, et al., 1999. Tumor markers CEA, CA19-9 and CA125 in monitoring of response to systemic chemotherapy in patients with advanced gastric cancer. Japanese journal of clinical oncology 29(11): 550-555.

Yi M, Ruoslahti E. 2001. A fibronectin fragment inhibits tumor growth, angiogenesis, and metastasis. Proceedings of the National Academy of Sciences of the United States of America 98(2): 620-624.

Yokoyama A, Ohmori T, Muramatsu T, Yokoyama T, Okuyama K, Makuuchi H, et al., 1998. Short-term follow-up after endoscopic mucosectomy of early esophageal cancer and aldehyde dehydrogenase-2 genotype in Japanese alcoholics. Cancer Epidemiol Biomarkers Prev 7(6): 473-476.

Yoshida BA, Sokoloff MM, Welch DR, Rinker-Schaeffer CW. 2000. Metastasis-suppressor genes: a review and perspective on an emerging field. Journal of the National Cancer Institute 92(21): 1717-1730.

Yu EJ, Lee Y, Rha SY, Kim TS, Chung HC, Oh BK, et al., 2008. Angiogenic factor thymidine phosphorylase increases cancer cell invasion activity in patients with gastric adenocarcinoma. Mol Cancer Res 6(10): 1554-1566.