Abstract
Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell lines and then confirmed in blood. However, there has been little experi-mental evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.
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References
Amin, S.A., Huang, C.C., Reierstad, S., Lin, Z., Arbieva, Z., Wiley, E., Saborian, H., Haynes, B., Cotterill, H., Dowsett, M. et al. (2006). Paracrine-stimulated gene expression profile favors estradiol production in breast tumors. Mol. Cell. Endocrinol. 253, 44–55.
Apweiler, R., Hermjakob, H., and Sharon, N. (1999). On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta 1473, 4–8.
Bause, E. (1983). Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem. J. 209, 331–336.
Blobel, G. (2000). Protein targeting (Nobel lecture). Chembiochem 1, 86–102.
Chang, J.W., Kang, U.B., Kim, D.H., Yi, J.K., Lee, J.W., Noh, D.Y., Lee, C., and Yu, M.H. (2008). Identification of circulating endorepellin LG3 fragment:Potential use as a serological biomarker for breast cancer. Proteomics Clin. Appl. 2, 23–32.
Chen, Y., Zhang, Y., Yin, Y., Gao, G., Li, S., Jiang, Y., Gu, X., and Luo, J. (2005). SPD—a web-based secreted protein database. Nucleic Acids Res. 33, D169–173.
Csiszar, K. (2001). Lysyl oxidases: a novel multifunctional amine oxidase family. Prog. Nucleic Acid Res. Mol. Biol. 70, 1–32.
Goo, Y.A., Liu, A.Y., Ryu, S., Shaffer, S.A., Malmstrom, L., Page, L., Nguyen, L.T., Doneanu, C.E., and Goodlett, D.R. (2009). Identification of secreted glycoproteins of human prostate and bladder stromal cells by comparative quantitative proteomics. Prostate 69, 49–61.
Goulet, B., Sansregret, L., Leduy, L., Bogyo, M., Weber, E., Chauhan, S.S., and Nepveu, A. (2007). Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation. Mol. Cancer Res. 5, 899–907.
Guerrier, L., Righetti, P.G., and Boschetti, E. (2008). Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library. Nat. Protoc. 3, 883–890.
Hakomori, S. (2001). Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv. Exp. Med. Biol. 491, 369–402.
Hanash, S.M., Pitteri, S.J., and Faca, V.M. (2008). Mining the plasma proteome for cancer biomarkers. Nature 452, 571–579.
Hiraiwa, M., O’Brien, J.S., Kishimoto, Y., Galdzicka, M., Fluharty, A.L., Ginns, E.I., and Martin, B.M. (1993). Isolation, characterization, and proteolysis of human prosaposin, the precursor of saposins (sphingolipid activator proteins). Arch. Biochem. Biophys. 304, 110–116.
Huang, L.J., Chen, S.X., Huang, Y., Luo, W.J., Jiang, H.H., Hu, Q.H., Zhang, P.F., and Yi, H. (2006). Proteomics-based identification of secreted protein dihydrodiol dehydrogenase as a novel serum markers of non-small cell lung cancer. Lung Cancer 54, 87–94.
Keller, A., Eng, J., Zhang, N., Li, X.J., and Aebersold, R. (2005). A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol. Syst. Biol. 1, 2005 0017.
Kim, Y.J., and Varki, A. (1997). Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconj. J. 14, 569–576.
Kim, H.J., Kang, H.J., Lee, H., Lee, S.T., Yu, M.H., Kim, H., and Lee, C. (2009). Identification of S100A8 and S100A9 as serological markers for colorectal cancer. J. Proteome Res. 8, 1368–1379.
Klee, E.W., Finlay, J.A., McDonald, C., Attewell, J.R., Hebrink, D., Dyer, R., Love, B., Vasmatzis, G., Li, T.M., Beechem, J.M., et al. (2006). Bioinformatics methods for prioritizing serum biomarker candidates. Clin. Chem. 52, 2162–2164.
Lawlor, K., Nazarian, A., Lacomis, L., Tempst, P., and Villanueva, J. (2009). Pathway-based biomarker search by high-throughput proteomics profiling of secretomes. J. Proteome Res. 8, 1489–1503.
Lee, H., Yi, E.C., Wen, B., Reily, T.P., Pohl, L., Nelson, S., Aebersold, R., and Goodlett, D.R. (2004). Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 803, 101–110.
Liotta, L.A., Ferrari, M., and Petricoin, E. (2003). Clinical proteomics: written in blood. Nature 425, 905.
Liu, T., Qian, W.J., Gritsenko, M.A., Camp, D.G., 2nd, Monroe, M.E., Moore, R.J., and Smith, R.D. (2005). Human plasma Nglycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. J. Proteome Res. 4, 2070–2080.
Mann, M. (2006). Functional and quantitative proteomics using SILAC. Nat. Rev. Mol. Cell Biol. 7, 952–958.
Meijer, D., Jansen, M.P., Look, M.P., Ruigrok-Ritstier, K., van Staveren, I.L., Sieuwerts, A.M., van Agthoven, T., Foekens, J.A., Dorssers, L.C., and Berns, E.M. (2009). TSC22D1 and PSAP predict clinical outcome of tamoxifen treatment in patients with recurrent breast cancer. Breast Cancer Res. Treat. 113, 253–260.
Oh, Y., Muller, H.L., Lamson, G., and Rosenfeld, R.G. (1993). Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition. J. Biol. Chem. 268, 14964–14971.
Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., et al. (2004). Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169.
Roth, J. (2002). Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control, and cell interactions. Chem. Rev. 102, 285–303.
Siriwardana, G., Bradford, A., Coy, D., and Zeitler, P. (2006). Autocrine/paracrine regulation of breast cancer cell proliferation by growth hormone releasing hormone via Ras, Raf, and mitogen-activated protein kinase. Mol. Endocrinol. 20, 2010–2019.
States, D.J., Omenn, G.S., Blackwell, T.W., Fermin, D., Eng, J., Speicher, D.W., and Hanash, S.M. (2006). Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study. Nat. Biotechnol. 24, 333–338.
Touab, M., Villena, J., Barranco, C., Arumi-Uria, M., and Bassols, A. (2002). Versican is differentially expressed in human melanoma and may play a role in tumor development. Am. J. Pathol. 160, 549–557.
Tsukuba, T., Okamoto, K., Yasuda, Y., Morikawa, W., Nakanishi, H., and Yamamoto, K. (2000). New functional aspects of cathepsin D and cathepsin E. Mol. Cells 10, 601–611.
Wu, C.C., Chien, K.Y., Tsang, N.M., Chang, K.P., Hao, S.P., Tsao, C.H., Chang, Y.S., and Yu, J.S. (2005). Cancer cell-secreted proteomes as a basis for searching potential tumor markers: nasopharyngeal carcinoma as a model. Proteomics 5, 3173–3182.
Yi, E.C., Marelli, M., Lee, H., Purvine, S.O., Aebersold, R., Aitchison, J.D., and Goodlett, D.R. (2002). Approaching complete peroxisome characterization by gas-phase fractionation. Electrophoresis 23, 3205–3216.
Zhang, H., Li, X.J., Martin, D.B., and Aebersold, R. (2003). Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21, 660–666.
Zhang, H., Yi, E.C., Li, X.J., Mallick, P., Kelly-Spratt, K.S., Masselon, C.D., Camp, D.G., 2nd, Smith, R.D., Kemp, C.J., and Aebersold, R. (2005). High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry. Mol. Cell. Proteomics 4, 144–155.
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Ahn, Y., Kang, UB., Kim, J. et al. Mining of serum glycoproteins by an indirect approach using cell line secretome. Mol Cells 29, 123–130 (2010). https://doi.org/10.1007/s10059-010-0008-0
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DOI: https://doi.org/10.1007/s10059-010-0008-0