Abstract
Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.
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Welsh J B, Sapinoso L M, Kern S G, et al. Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum. Proc Natl Acad Sci USA, 2003, 100: 3410–3415, 1:CAS:528:DC%2BD3sXisVCnur8%3D, 10.1073/pnas.0530278100, 12624183
Huang C M, Ananthaswamy H N, Barnes S, et al. Mass spectrometric proteomics profiles of in vivo tumor secretomes: Capillary ultrafiltration sampling of regressive tumor masses. Proteomics, 2006, 6: 6107–6116, 1:CAS:528:DC%2BD28XhtlWgtLrM, 10.1002/pmic.200600287, 17051643
Mbeunkui F, Metge B J, Shevde L A, et al. Identification of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines representing a progression of breast cancer. J Proteome Res, 2007, 6: 2993–3002, 1:CAS:528:DC%2BD2sXnsV2rtrk%3D, 10.1021/pr060629m, 17608509
Ye Q H, Qin L X, Forgues M, et al. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med, 2003, 9: 416–423, 1:CAS:528:DC%2BD3sXisVOmu7o%3D, 10.1038/nm843, 12640447
Gupta G P, Massagué J. Cancer metastasis: Building a framework. Cell, 2006, 127: 679–695, 1:CAS:528:DC%2BD28Xht1yqs7rL, 10.1016/j.cell.2006.11.001, 17110329
Tang Z Y, Ye S L, Liu Y K, et al. A decade’s studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol, 2004, 130: 187–196, 10.1007/s00432-003-0511-1, 14685850
Diehl H C, Stuhler K, Volmer M W, et al. A catalogue of proteins released by colorectal cancer cells in vitro as an alternative source for biomarker discovery. Proteomics Clin Appl, 2007, 1: 47–61, 1:CAS:528:DC%2BD2sXjtFCjs7c%3D, 10.1002/prca.200600491, 21136611
Pellitteri-Hahn M C, Warren M C, Didier D N, et al. Improved mass spectrometric proteomic profiling of the secretome of rat vascular endothelial cells. J Proteome Res, 2006, 5: 2861–2864, 1:CAS:528:DC%2BD28XpsF2hsbY%3D, 10.1021/pr060287k, 17022658
Yamashita R, Fujiwara Y, Ikari K, et al. Extracellular proteome of human hepatoma cell, HepG2 analyzed using two-dimensional liquid chromatography coupled with tandem mass spectrometry. Mol Cell Biochem, 2007, 298: 83–92, 1:CAS:528:DC%2BD2sXkt1egurk%3D, 10.1007/s11010-006-9354-9, 17109077
Zwickl H, Traxler E, Staettner S, et al. A novel technique to specifically analyze the secretome of cells and tissues. Electrophoresis, 2005, 26: 2779–2785, 1:CAS:528:DC%2BD2MXotFamsro%3D, 10.1002/elps.200410387, 15966010
Trost M, Wehmhöner D D, Kärst U, et al. Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenic Listeria species. Proteomics, 2005, 5: 1544–1557, 1:CAS:528:DC%2BD2MXktVWjs7c%3D, 10.1002/pmic.200401024, 15838904
Chan X C, McDermott J C, Siu K W. Identification of secreted proteins during skeletal muscle development. J Proteome Res, 2007, 6: 698–710, 1:CAS:528:DC%2BD2sXktFSmsg%3D%3D, 10.1021/pr060448k, 17269726
Sardana G, Marshall J, Diamandis E P. Discovery of candidate tumor markers for prostate cancer via proteomic analysis of cell culture-conditioned medium. Clin Chem, 2007, 53: 429–437, 1:CAS:528:DC%2BD2sXis1Wnsrk%3D, 10.1373/clinchem.2006.077370, 17259234
Lou X M, Xiao T, Zhao K, et al. Cathepsin D is secreted from M-BE cells: Its potential role as a biomarker of lung cancer. J Proteome Res, 2007, 6: 1083–1092, 1:CAS:528:DC%2BD2sXhtlGltLc%3D, 10.1021/pr060422t, 17284061
Wu C C, Cheng H C, Chen S J, et al. Identification of collapsin response mediator protein-2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes. Proteomics, 2008, 8: 316–332, 1:CAS:528:DC%2BD1cXhvVSqtLY%3D, 10.1002/pmic.200700819, 18203259
Li Y, Tang Z Y, Ye S L, et al. Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray. J Cancer Res Clin Oncol, 2003, 129: 43–51, 1:CAS:528:DC%2BD3sXjsl2nsrs%3D, 10.1007/s00432-003-0493-z, 12618900
Mbeunkui F, Fodstad O, Pannell L K. Secretory protein enrichment and analysis: An optimized approach applied on cancer cell lines using 2D LC-MS/MS. J Proteome Res, 2006, 5: 899–906, 1:CAS:528:DC%2BD28XitFOiur4%3D, 10.1021/pr050375p, 16602697
Chevallet M, Diemer H, Dorssealer A V, et al. Toward a better analysis of secreted proteins: The example of the myeloid cells secretome. Proteomics, 2007, 7: 1757–1770, 1:CAS:528:DC%2BD2sXntVSisLw%3D, 10.1002/pmic.200601024, 17464941
Pan T T, Fang C, Gang Y Z, et al. Subproteomic analysis of the cellular proteins associated with the 3′ untranslated region of the hepatitis C virus genome in human liver cells. Biochem Biophys Res Commun, 2006, 347: 683, 1:CAS:528:DC%2BD28Xntl2jsb4%3D, 10.1016/j.bbrc.2006.05.214
Callister S J, Dominguez M A, Nicora C D, et al. Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from rhodobacter sphaeroides 2.4.1. aerobic and photosynthetic cell cultures. J Proteome Res, 2006, 5: 1940–1947, 1:CAS:528:DC%2BD28Xmtlalu7o%3D, 10.1021/pr060050o, 16889416
Chen M, Ying W T, Song Y P, et al. Analysis of human liver proteome using replicate shotgun strategy. Proteomics, 2007, 7: 2479, 1:CAS:528:DC%2BD2sXos1Ghtbg%3D, 10.1002/pmic.200600338, 17623305
Bendtsen J D, Jensen L J, Blom N, et al. Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des Sel, 2004, 17: 349–356, 1:CAS:528:DC%2BD2cXntFagtrY%3D, 10.1093/protein/gzh037, 15115854
Krogh A, Larsson B, Heijne G, et al. Predicting transmembrane protein topology with a hidden markov model: Application to complete genomes. J Mol Biol, 2001, 305: 567–580, 1:CAS:528:DC%2BD3MXisFCguw%3D%3D, 10.1006/jmbi.2000.4315, 11152613
Ho E, Hayen A, Wilkins M R. Characterisation of organellar proteomes: A guide to subcellular proteomic fractionation and analysis. Proteomics, 2006, 6: 5746, 1:CAS:528:DC%2BD28Xht1yqsbnI, 10.1002/pmic.200600241, 17068763
Antelmann H, Tjalsma H, Voigt B, et al. A proteomic view on genomebased signal peptide predictions. Genome Res, 2001, 11: 1484–1502, 1:CAS:528:DC%2BD3MXmvV2mtrw%3D, 10.1101/gr.182801, 11544192
Zhang Z, Henzel W J. Signal peptide prediction based on analysis of experimentally. Protein Sci, 2004, 13: 2819–2824, 1:CAS:528:DC%2BD2cXotVegtLY%3D, 10.1110/ps.04682504, 15340161
Cao J, Shen C, Wang H, et al. Identification of N-Glycosylation sites on secreted proteins of human hepatocellular carcinoma cells with a complementary proteomics approach. J Proteome Res, 2009, 8: 662–672, 1:CAS:528:DC%2BD1MXlt1Snsg%3D%3D, 10.1021/pr800826u, 19196183
Cao J, Hu Y, Shen C, et al. Zeolite LTL nanocrystal-driving high efficient enrichment of secretory proteins in human hepatocellular carcinoma cells. Proteomics, 2009, 9: 4881–4888, 1:CAS:528:DC%2BD1MXhtleqtbbM, 10.1002/pmic.200800877, 19743415
Chen Y J, Zhang Y, Yin Y, et al. SPD: A web-based secreted protein database. Nucleic Acids Res, 2005, 33: 169–173, 10.1093/nar/gki093
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Cao, J., Shen, C., Zhang, J. et al. Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells. Sci. China Life Sci. 54, 34–38 (2011). https://doi.org/10.1007/s11427-010-4122-1
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DOI: https://doi.org/10.1007/s11427-010-4122-1