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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cox J, Mann M (2007) Is proteomics the new genomics? Cell 130:395–398
Timms JF, Cutillas PR (2010) Overview of quantitative LC-MS techniques for proteomics and activitomics. Methods Mol Biol 658:19–45
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
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
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
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
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
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
Sinclair J, Timms JF (2011) Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS. Methods 54:361–369
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
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
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
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
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
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
Ting L, Rad R, Gygi SP et al (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8:937–940
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
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
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
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
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
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-62703-547-7_20
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-546-0
Online ISBN: 978-1-62703-547-7
eBook Packages: Springer Protocols