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Proteomic Profiling of Ovarian Cancer Models Using TMT-LC-MS/MS

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Ovarian Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1049))

Abstract

Herein, we have utilized two cellular models of epithelial ovarian cancer (EOC), where transfer of normal chromosome 18 material into the EOC cell lines TOV-112D and TOV-21G induced in vitro and in vivo suppression of tumorigenic phenotype in derived hybrid clones. Two-dimensional-liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) with tandem mass tagging (TMT) was then employed to profile the whole cell, secreted and crude membrane proteomes of the parental and hybrid cell models to identify differentially expressed proteins as potential markers of ovarian tumor suppression. Protein changes of interest were confirmed by immunoblotting in additional hybrid and revertant cell lines. This method afforded quantitative coverage of around 1,000 unique proteins and is applicable to the analysis of any cell model, tissue or biofluid.

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References

  1. Cox J, Mann M (2007) Is proteomics the new genomics? Cell 130:395–398

    Article  PubMed  CAS  Google Scholar 

  2. Timms JF, Cutillas PR (2010) Overview of quantitative LC-MS techniques for proteomics and activitomics. Methods Mol Biol 658:19–45

    Article  PubMed  CAS  Google Scholar 

  3. 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

    Article  PubMed  CAS  Google Scholar 

  4. Choe L, D'Ascenzo M, Relkin NR et al (2007) 8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease. Proteomics 7:3651–3660

    Article  PubMed  CAS  Google Scholar 

  5. Pierce A, Unwin RD, Evans CA et al (2008) Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases. Mol Cell Proteomics 7:853–863

    Article  PubMed  CAS  Google Scholar 

  6. Thompson A, Schafer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904

    Article  PubMed  CAS  Google Scholar 

  7. Dayon L, Hainard A, Licker V et al (2008) Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal Chem 80:2921–2931

    Article  PubMed  CAS  Google Scholar 

  8. Sinclair J, Metodieva G, Dafou D et al (2011) Profiling signatures of ovarian cancer tumour suppression using 2D-DIGE and 2D-LC-MS/MS with tandem mass tagging. J Proteomics 74:451–465

    Article  PubMed  CAS  Google Scholar 

  9. Sinclair J, Timms JF (2011) Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS. Methods 54:361–369

    Article  PubMed  CAS  Google Scholar 

  10. Bantscheff M, Boesche M, Eberhard D et al (2008) Robust and sensitive iTRAQ quantification on an LTQ Orbitrap mass spectrometer. Mol Cell Proteomics 7:1702–1713

    Article  PubMed  CAS  Google Scholar 

  11. Griffin TJ, Xie H, Bandhakavi S et al (2007) iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer. J Proteome Res 6:4200–4209

    Article  PubMed  CAS  Google Scholar 

  12. Guo T, Gan CS, Zhang H et al (2008) Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation. J Proteome Res 7:4831–4840

    Article  PubMed  CAS  Google Scholar 

  13. Ramus SJ, Pharoah PD, Harrington P et al (2003) BRCA1/2 mutation status influences somatic genetic progression in inherited and sporadic epithelial ovarian cancer cases. Cancer Res 63:417–423

    PubMed  CAS  Google Scholar 

  14. Dafou D, Grun B, Sinclair S et al. (2010) Microcell mediated chromosome transfer identifies EPB41L3 as a functional suppressor of epithelial ovarian cancers. Neoplasia 12:579–589

    Google Scholar 

  15. Dafou D, Ramus SJ, Choi K et al (2009) Chromosomes 6 and 18 induce neoplastic suppression in epithelial ovarian cancer cells. Int J Cancer 124:1037–1044

    Article  PubMed  CAS  Google Scholar 

  16. Ting L, Rad R, Gygi SP et al (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8:937–940

    Article  PubMed  CAS  Google Scholar 

  17. Wenger CD, Lee MV, Hebert AS et al (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8:933–935

    Article  PubMed  CAS  Google Scholar 

  18. van Ulsen P, Kuhn K, Prinz T et al (2009) Identification of proteins of Neisseria meningitidis induced under iron-limiting conditions using the isobaric tandem mass tag (TMT) labeling approach. Proteomics 9:1771–1781

    Article  PubMed  Google Scholar 

  19. Stella R, Cifani P, Peggion C et al (2011) Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons. J Proteome Res 11:523–536

    Article  PubMed  Google Scholar 

  20. Byers HL, Campbell J, van Ulsen P et al (2009) Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring. J Proteomics 73:231–239

    Article  PubMed  CAS  Google Scholar 

  21. Viner RI, Zhang T, Second T et al (2009) Quantification of post-translationally modified peptides of bovine alpha-crystallin using tandem mass tags and electron transfer dissociation. J Proteomics 72:874–885

    Article  PubMed  CAS  Google Scholar 

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Sinclair, J., Timms, J.F. (2013). Proteomic Profiling of Ovarian Cancer Models Using TMT-LC-MS/MS. In: Malek, A., Tchernitsa, O. (eds) Ovarian Cancer. Methods in Molecular Biology, vol 1049. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-547-7_20

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  • DOI: https://doi.org/10.1007/978-1-62703-547-7_20

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-546-0

  • Online ISBN: 978-1-62703-547-7

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