Profiling signatures of ovarian cancer tumour suppression using 2D-DIGE and 2D-LC-MS/MS with tandem mass tagging John Sinclair, Gergana Metodieva, Dimitra Dafou, Simon A. Gayther, John F. Timms ⁎ Cancer Proteomics Laboratory, EGA Institute for Women's Health, University College London UCL, United Kingdom ARTICLE INFO ABSTRACT Article history: Received 16 November 2010 Accepted 22 December 2010 Available online 13 January 2011 Epithelial ovarian cancer (EOC) is the most common form of gynaecological malignancy in the developed world and has a poor prognosis due to its late detection. Identifying molecular markers of the disease may provide novel approaches to screening and could enable targeted treatment and the design of novel therapies. Although blood is recognized as a highly important source of disease-related biomarkers, the complexity and dynamic range of protein abundance in body fluids has hampered proteomic biomarker discovery and alternative approaches using cell models may be more successful. Herein, we have utilized two cellular models of 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 their tumourigenic phenotype. A combination of quantitative two-dimensional difference gel electrophoresis (2D-DIGE) and two-dimensional- liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) with tandem mass tagging (TMT) was employed to examine the whole cell, secreted and crude membrane proteomes of the parental and hybrid cell models to identify differentially expressed proteins as potential markers of tumour suppression. Protein changes of interest were confirmed by immunoblotting in additional hybrid and revertant cell lines where incorporated chromosome 18 material was lost. One candidate marker was also tested in sera from a set of ovarian cancer cases and controls. We have identified a list of promising candidate biomarkers for further testing and functional characterization. © 2011 Elsevier B.V. All rights reserved. Keywords: Proteomic profiling Ovarian cancer Tumour suppression 2D-DIGE Mass spectrometry 2D-LC-MS/MS Tandem mass tagging Biomarkers 1. Introduction Epithelial ovarian cancer (EOC) is the most lethal form of gynaecological malignancy in the western world. Every year there are over 200,000 new cases of ovarian cancer worldwide and overall incidence rates are increasing [1]. Whilst patients diagnosed with early stage disease have a good prognosis (5 year survival rate >80%), overall survival rates are as low as 30% mainly due to its late detection, when the disease has spread beyond the pelvis (FIGO stage III) [2,3]. Thus, there is an urgent need for early detection methods to reduce mortality. At present, there is no screening test that is reliable enough for EOC detection in the general population. The tumour marker serum CA125 is only approved for use in monitoring disease recurrence [4,5]. Indeed, whilst 80% of women presenting with advanced stage disease have raised CA125, it is elevated in only 50–60% of women with early stage disease and can be elevated in non-cancerous gynaecological complications such as benign ovarian neoplasms, JOURNAL OF PROTEOMICS 74 (2011) 451 – 465 Abbreviations: EOC, epithelial ovarian cancer; Ch18, chromosome 18; MMCT, microcell-mediated chromosomal transfer; 2D-DIGE, two- dimensional difference gel electrophoresis; MS, mass spectrometry; 2D-LC, two-dimensional liquid chromatography; TMT, tandem mass tagging; WCL, whole cell lysate; NOSE, normal ovarian surface epithelium; CID, collision-induced dissociation; HCD, higher energy collision-induced dissociation; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; SCX, strong cation exchange; UKOPS, United Kingdom Ovarian Cancer Population Study ⁎ Corresponding author. Cancer Proteomics Laboratory, EGA Institute for Women's Health, Cruciform Building, Gower St, London, WC1E 6BT, United Kingdom. E-mail address: jtimms@wibr.ucl.ac.uk (J.F. Timms). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2010.12.009 available at www.sciencedirect.com www.elsevier.com/locate/jprot