Abstract
Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and influence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the profiling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted from the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identification of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome profiling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical difficulties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical workflow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.
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References
Pandey A, Mann M (2000) Proteomics to study genes and genomes. Nature 405:837–846
Tyers M, Mann M (2003) From genomics to proteomics. Nature 422:193–197
May M (2009) From cells, secrets of the secretome leak out. Nat Med 15:828
Walter P, Gilmore R, Blobel G (1984) Protein translocation across the endoplasmic reticulum. Cell 38:5–8
Nickel W (2003) The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. Eur J Biochem 270:2109–2119
Karagiannis GS, Pavlou MP, Diamandis EP (2010) Cancer secretomics reveal pathophysiological pathways in cancer molecular oncology. Mol Oncol 4:496–510
Makridakis M, Vlahou A (2010) Secretome proteomics for discovery of cancer biomarkers. J Proteomics 73:2291–2305
Xue H, Lu B, Lai M (2008) The cancer secretome: a reservoir of biomarkers. J Transl Med 6:1–12
Pavlou MP, Diamandis EP (2010) The cancer cell secretome: a good source for discovering biomarkers? J Proteomics 73:1896–1906
Stastna M, Van Eyk JE (2012) Secreted proteins as a fundamental source for biomarker discovery. Proteomics 12:1–14
Dowling P, Clynes M (2011) Conditioned media from cell lines: a complementary model to clinical specimens for the discovery of disease-specific biomarkers. Proteomics 11:794–804
Skalnikova H, Motlik J, Gadher SJ, Kovarova H (2011) Mapping of the secretome of primary isolates of mammalian cells, stem cells and derived cell lines. Proteomics 11:691–708
Veenstra TD, Conrads TP, Hood BL, Avellino AM, Ellenbogen RG, Morrison RS (2005) Biomarkers: mining the biofluid proteome. Mol Cell Proteomics 4:409–418
Mischak H, Allmaier G, Apweiler R et al (2010) Recommendations for biomarker identification and qualification in clinical proteomics. Sci Transl Med 2:46ps42
Lim JWE, Bodnar A (2002) Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics 2:1187–1203
Lee MJ, Kim J, Kim MY et al (2010) Proteomic analysis of tumor necrosis factor-alpha-induced secretome of human adipose tissue-derived mesenchymal stem cells. J Proteome Res 9:1754–1762
Rocco M, Malorni L, Cozzolino R et al (2011) Proteomic profiling of human melanoma metastatic cell line secretomes. J Proteome Res 10:4703–4714
Farina A, D’Aniello C, Severino V et al (2011) Temporal proteomic profiling of embryonic stem cell secretome during cardiac and neural differentiation. Proteomics 11:3972–3982
Klein-Scory S, Kubler S, Diehl H et al (2010) Immunoscreening of the extracellular proteome of colorectal cancer cells. BMC Cancer 70:1–19
Srisomsap C, Sawangareetrakul P, Subhasitanont P et al (2010) Proteomic studies of cholangiocarcinoma and hepatocellular carcinoma cell secretomes. J Biomed Biotechnol 2010:437143
Tang CE, Guan YJ, Yi B et al (2010) Identification of the amyloid beta-protein precursor and cystatin C as novel epidermal growth factor receptor regulated secretory proteins in nasopharyngeal carcinoma by proteomics. J Proteome Res 9:6101–6111
Del Galdo F, Shaw MA, Jimenez SA (2010) Proteomic analysis identification of a pattern of shared alterations in the secretome of dermal fibroblasts from systemic sclerosis and nephrogenic systemic fibrosis. Am J Pathol 177:1638–1646
Roca-Rivada A, Alonso J, Al-Massadi O et al (2011) Secretome analysis of rat adipose tissues shows location-specific roles for each depot type. J Proteomics 74:1068–1079
Mathias RA, Wang B, Ji H et al (2009) Secretome-based proteomic profiling of Ras-transformed MDCK cells reveals extracellular modulators of epithelial-mesenchymal transition. J Proteome Res 8:2827–2837
De la Fuente A, Mateos J, Lesende-Rodriguez I et al (2012) Proteome analysis during chondrocyte differentiation in a new chondrogenesis model using human umbilical cord stroma mesenchymal stem cells. Mol Cell Proteomics 11:M111.010496
Weiss W, Gorg A (2009) High-resolution two-dimensional electrophoresis. Methods Mol Biol 564:13–32
Mathias RA, Ji H, Simpson RJ (2012) Proteomic profiling of the epithelial-mesenchymal transition using 2D DIGE. Methods Mol Biol 854:269–286
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
Bendtsen JD, Jensen LJ, Blom N, Von Heijne G, Brunak S (2004) Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des Sel 17:349–356
Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795
Bantscheff M, Schirle M, Sweetman G, Rick J, Kuster B (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389:1017–1031
Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386
May C, Brosseron F, Chartowski P, Schumbrutzki C, Schoenebeck B, Marcus K (2011) Instruments and methods in proteomics. Methods Mol Biol 696:3–26
Brewis IA, Brennan P (2010) Proteomics technologies for the global identification and quantification of proteins. Adv Protein Chem Struct Biol 80:1–44
Mann M (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7:952–958
Chen CY, Chi LM, Chi HC et al (2012) Stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics study of a thyroid hormone-regulated secretome in human hepatoma cells. Mol Cell Proteomics 11:M111.011270
Kashyap MK, Harsha HC, Renuse S et al (2010) SILAC-based quantitative proteomic approach to identify potential biomarkers from the esophageal squamous cell carcinoma secretome. Cancer Biol Ther 10:796–810
Gronborg M, Kristiansen TZ, Iwahori A et al (2006) Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach. Mol Cell Proteomics 5:157–171
Formolo CA, Williams R, Gordish-Dressman H, MacDonald TJ, Lee NH, Hathout Y (2011) Secretome signature of invasive glioblastoma multiforme. J Proteome Res 10:3149–3159
Calamia V, Rocha B, Mateos J, Fernandez-Puente P, Ruiz-Romero C, Blanco FJ (2011) Metabolic labeling of chondrocytes for the quantitative analysis of the interleukin-1-beta-mediated modulation of their intracellular and extracellular proteomes. J Proteome Res 10:3701–3711
Greco TM, Seeholzer SH, Mak A, Spruce L, Ischiropoulos H (2010) Quantitative mass spectrometry-based proteomics reveals the dynamic range of primary mouse astrocyte protein secretion. J Proteome Res 9:2764–2774
Chan CY, Masui O, Krakovska O et al (2011) Identification of differentially regulated secretome components during skeletal myogenesis. Mol Cell Proteomics 10:M110.004804
Henningsen J, Rigbolt KT, Blagoev B, Pedersen BK, Kratchmarova I (2010) Dynamics of the skeletal muscle secretome during myoblast differentiation. Mol Cell Proteomics 9:2482–2496
Ross PL, Huang YN, Marchese JN et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169
Chenau J, Michelland S, de Fraipont F et al (2009) The cell line secretome, a suitable tool for investigating proteins released in vivo by tumors: application to the study of p53-modulated proteins secreted in lung cancer cells. J Proteome Res 8:4579–4591
Chiu KH, Chang YH, Wu YS, Lee SH, Liao PC (2011) Quantitative secretome analysis reveals that COL6A1 is a metastasis-associated protein using stacking gel-aided purification combined with iTRAQ labeling. J Proteome Res 10:1110–1125
Lietzen N, Ohman T, Rintahaka J et al (2011) Quantitative subcellular proteome and secretome profiling of influenza A virus-infected human primary macrophages. PLoS Pathog 7:e1001340
Zhong J, Krawczyk SA, Chaerkady R et al (2010) Temporal profiling of the secretome during adipogenesis in humans. J Proteome Res 9:5228–5238
Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999
Khwaja FW, Svoboda P, Reed M, Pohl J, Pyrzynska B, Van Meir EG (2006) Proteomic identification of the wt-p53-regulated tumor cell secretome. Oncogene 25:7650–7661
Pocsfalvi G, Votta G, De Vincenzo A et al (2011) Analysis of secretome changes uncovers an autocrine/paracrine component in the modulation of cell proliferation and motility by c-Myc. J Proteome Res 10:5326–5337
Bonzon-Kulichenko E, Martinez-Martinez S, Trevisan-Herraz M, Navarro P, Redondo JM, Vazquez J (2011) Quantitative in-depth analysis of the dynamic secretome of activated Jurkat T-cells. J Proteomics 75:561–571
Roelofsen H, Dijkstra M, Weening D, de Vries MP, Hoek A, Vonk RJ (2009) Comparison of isotope-labeled amino acid incorporation rates (CILAIR) provides a quantitative method to study tissue secretomes. Mol Cell Proteomics 8:316–324
Bondarenko PV, Chelius D, Shaler TA (2002) Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry. Anal Chem 74:4741–4749
Chelius D, Bondarenko PV (2002) Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry. J Proteome Res 1:317–323
Wang W, Zhou H, Lin H et al (2003) Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal Chem 75:4818–4826
Ishihama Y, Oda Y, Tabata T et al (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4:1265–1272
Lu P, Vogel C, Wang R, Yao X, Marcotte EM (2007) Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nat Biotechnol 25:117–124
Malard V, Chardan L, Roussi S et al (2012) Analytical constraints for the analysis of human cell line secretomes by shotgun proteomics. J Proteomics 75:1043–1054
Xue H, Lu B, Zhang J et al (2010) Identification of serum biomarkers for colorectal cancer metastasis using a differential secretome approach. J Proteome Res 9:545–555
Gao BB, Stuart L, Feener EP (2008) Label-free quantitative analysis of one-dimensional PAGE LC/MS/MS proteome: application on angiotensin II-stimulated smooth muscle cells secretome. Mol Cell Proteomics 7:2399–2409
Rifai N, Gillette MA, Carr SA (2006) Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol 24:971–983
Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730
Kitteringham NR, Jenkins RE, Lane CS, Elliott VL, Park BK (2009) Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics. J Chromatogr B Analyt Technol Biomed Life Sci 877:1229–1239
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. J Proteome Res 5:899–906
Yao L, Zhang Y, Chen K, Hu X, Xu LX (2011) Discovery of IL-18 as a novel secreted protein contributing to doxorubicin resistance by comparative secretome analysis of MCF-7 and MCF-7/Dox. PLoS One 6:e24684
Schiarea S, Solinas G, Allavena P et al (2010) Secretome analysis of multiple pancreatic cancer cell lines reveals perturbations of key functional networks. J Proteome Res 9:4376–4392
Pellitteri-Hahn MC, Warren MC, Didier DN et al (2006) Improved mass spectrometric proteomic profiling of the secretome of rat vascular endothelial cells. J Proteome Res 5:2861–2864
Alvarez-Llamas G, Szalowska E, de Vries MP et al (2007) Characterization of the human visceral adipose tissue secretome. Mol Cell Proteomics 6:589–600
Stastna M, Chimenti I, Marban E, Van Eyk JE (2010) Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes. Proteomics 10:245–253
Pirkmajer S, Chibalin AV (2011) Serum starvation: caveat emptor. Am J Physiol Cell Physiol 301:C272–C279
Hasan NM, Adams GE, Joiner MC (1999) Effect of serum starvation on expression and phosphorylation of PKC-alpha and p53 in V79 cells: implications for cell death. Int J Cancer 80:400–405
Keene SD, Greco TM, Parastatidis I et al (2009) Mass spectrometric and computational analysis of cytokine-induced alterations in the astrocyte secretome. Proteomics 9:768–782
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63
Phillips HJ, Terryberry JE (1957) Counting actively metabolizing tissue cultured cells. Exp Cell Res 13:341–347
Weng LP, Wu CC, Hsu BL et al (2008) Secretome-based identification of Mac-2 binding protein as a potential oral cancer marker involved in cell growth and motility. J Proteome Res 7:3765–3775
Dowell JA, Johnson JA, Li L (2009) Identification of astrocyte secreted proteins with a combination of shotgun proteomics and bioinformatics. J Proteome Res 8:4135–4143
Polisetty RV, Gupta MK, Nair SC et al (2011) Glioblastoma cell secretome: analysis of three glioblastoma cell lines reveal 148 non-redundant proteins. J Proteomics 74:1918–1925
Wu CC, Hsu CW, Chen CD et al (2010) Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas. Mol Cell Proteomics 9:1100–1117
Caccia D, Zanetti Domingues L, Micciche F et al (2011) Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways. J Proteome Res 10:4196–4207
Yoon JH, Yea K, Kim J et al (2009) Comparative proteomic analysis of the insulin-induced L6 myotube secretome. Proteomics 9:51–60
Canals F, Colome N, Ferrer C, Plaza-Calonge Mdel C, Rodriguez-Manzaneque JC (2006) Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis. Proteomics 6(Suppl 1):S28–S35
Chevallet M, Diemer H, Luche S, van Dorsselaer A, Rabilloud T, Leize-Wagner E (2006) Improved mass spectrometry compatibility is afforded by ammoniacal silver staining. Proteomics 6:2350–2354
Kashat L, So AK, Masui O et al (2010) Secretome-based identification and characterization of potential biomarkers in thyroid cancer. J Proteome Res 9:5757–5769
Ralhan R, Masui O, Desouza LV, Matta A, Macha M, Siu KW (2011) Identification of proteins secreted by head and neck cancer cell lines using LC-MS/MS: strategy for discovery of candidate serological biomarkers. Proteomics 11:2363–2376
Luo X, Liu Y, Wang R, Hu H, Zeng R, Chen H (2011) A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer. J Proteomics 74:528–538
Wang CL, Wang CI, Liao PC et al (2009) Discovery of retinoblastoma-associated binding protein 46 as a novel prognostic marker for distant metastasis in nonsmall cell lung cancer by combined analysis of cancer cell secretome and pleural effusion proteome. J Proteome Res 8:4428–4440
Chang KP, Wu CC, Chen HC et al (2010) Identification of candidate nasopharyngeal carcinoma serum biomarkers by cancer cell secretome and tissue transcriptome analysis: potential usage of cystatin A for predicting nodal stage and poor prognosis. Proteomics 10:2644–2660
Acknowledgments
We gratefully acknowledge Dr. Menotti Ruvo (IBB, CNR of Naples) and Dr. Carla Pasquarello Mosimann (Geneva Proteomics Core Facility) for their useful suggestions and support.
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Severino, V., Farina, A., Chambery, A. (2013). Analysis of Secreted Proteins. In: Zhou, M., Veenstra, T. (eds) Proteomics for Biomarker Discovery. Methods in Molecular Biology, vol 1002. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-360-2_4
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DOI: https://doi.org/10.1007/978-1-62703-360-2_4
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